Please find below the final 2014 Conference Booklet!
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The origin of silence: a quest to dissect heterochromatic nucleation sites of the malaria parasite
Sabine Fraschka1, Nicole Bertschi2, Till Voss2 and Richard Bartfai1 1) Department of Molecular Biology, Radboud University Nijmegen, The Netherlands 2) Gene Regulation Group, Swiss TPH, Basel, Switzerland Plasmodium falciparum is a protozoan parasite and the causative agent of the most severe form of malaria. While most parts of the parasite’s epigenome are in a transcriptionally competent, euchromatic state, distinct islands of H3K9me3 and HP1 marked heterochromatin are present at the chromosome ends and in some chromosome internal islands. Importantly, gene(families) encoded in these regions are highly relevant for parasite survival, as epigenetic memory of their expression status controls among others antigenic variation and commitment to gametocytogenesis. Although several components of the machinery that orchestrate heterochromatin formation have been identified (HP1, Sir2 A/B, HDAC2, a putative Suv39 homologue) it still remains a mystery which mechanism recruits this machinery to specific sites of the genome. In this study, we made use of a parasite line carrying HP1 fused to a destabilization domain, which enables experimental regulation of HP1 expression level. In absence of HP1 the level of H3K9me2/3 level is substantially reduced and heterochromatic genes get de-repressed providing evidence that HP1 is essential for heterochromatin (spreading)/maintenance. Despite this overall reduction, small regions within each heterochromatic domain retain H3K9me2/3 and thus might pinpoint heterochromatic nucleation sites. Intriguingly, next to these sites in the subtelomeric regions multiple binding sites for a plasmodium-specific DNA-binding protein (SIP2) are present. Preliminary results suggest that, when inserted to the euchromatic domain, these sequences are sufficient to initiate heterochromatin formation and therefore might serve as genuine nucleation sites. |
Danube Conference on Epigenetics
2014 . November 19-21
Epigenetic marks in childhood obesity
Orsolya Dóra Ács1,4, Bálint Pérerfias, Péter Hollósi", Nóra Lehotkai", Andrea Luczay", Ilona Kovalszky'', Attila Patócs3, Dóra Tőrők", András Szabó'
'Semmelweis University 2nd Department of Pediatrics
"Semmelweis University 1 st Department of Pathology and Experimentál Cancer Research 3Semmelweis University 2nd Department of Internal Medicine
4Semmelweis University School of Ph.D. Studies
Introducrion: Obesity incidence is rapidly growing worldwide, prevention is especially important in children. However, some people are more prone to gain weight than others. Several mechanisms seem to contribute to excess weight gain. There is ernerging evidence that certain epigenetic marks might contribute to obesity predisposition. Among other metabolic anomalies vitamin D deficiency is especially frequerit in overweight individuals. Vitamin D as an epigenetic regulator might be a player in the epigenetic disrurbances in obesity. Hypothesis. Low vitamin D level that is present in obesity and insuline resistance is associated with hypermethylation of CYP27B 1 gene, which leads to lower production of active vitamin D.
Objective: To investigate the correlation among obesity, metabolic paramctcrs, vitamin D levels, and methylation of certain loci.
Marérilas and merhods: We selecred a total of 83 (41 boys and 42 girls) healthy, obese (BM! pc 95<) children, age between 3 and 18 years, who were examined and followed up at the 2nd Department of Pediatrics, Semmelweis University. Anthropometric data, metabolic parameters were recorded, 24-hour blood pressure monitoring was carried out. We colleered anamnestic data focusing on perinatal issues , development and lifestyle. DNA was isolated from peripherial blood samples and treared with bisulflee . After bisulfire polimerase chain reaction (BSPCR) the rnethyl ation patrern of VDR, CYP27AI, CYP27BI, CYP2RI and CYP24AI genes was examined with Methylarion Specific High Resolution Melring Analysis (MS-HRM) in 83 cases.
Results: 63 of the 83 investigated children had at leasr one abnormality associared with metabolic syndrome, hypertension being the most frequerit (68 %). Serum vitamin D levels did not correlate with the methylation status of the investigated vitamin D related loci. However, methylation of the VDR gene was slightly different between patients with no metabolic abnormalities at all and patients with impaired glucose tolerance. Conlusion:Certain epigenetic marks might predispose to excess weight gain and obesity related metabolic disorders.
2nd Department of Pediatrics, Semmelweis University
Orsolya Dóra Ács dr [email protected]
1
Danube Conference on Epigenetics
2014 . November 19-21
HoxD9 expression is regula ted by acetylation in endothelial progenitor cells
Eugen Andrei, Florin Iordache, Constantineseu Andrei, Horia Maniu
Institute of Cellular Biology and Pathology "Nicolae Simioneseu" of Romaniari Academy, Fetal and Adult Stem Cell Therapy Department, Bucharest, Romania
The homeobox (Hox) genes encode transcription facrcrs that are involved in the morphogenesis of body. Recenr data showed that HoxD transcription factors control the cardiovascular system development, by modulation of endorhelial cell proliferadon and differentiation. So fare the role of histone acerylation in the expression of HoxD9 has not been studied to date, therefore the aim of this study was to investigate the expression of HoxD9 in endothelial progenitor cells after treatment with valproic acid (VPA), ahistone deacerylase inhibitor. Our results showed that VPA inhibirs the histon-deacetylases, leaving chromatin in an acetylated state corresponding to a decondensare conformation. qRT-PCR and western blor assays revealed that the expression of Hox09 in endorhelial progenitor cells treared with VPA was increased at both gene and protein level, suggesting that acetylation regulates Hox09 expression. Flow cytomctry analysis showed that the express ion of endorhelial specific markers such as C031, COI05, COl17, COI44, and VEGFR2 was decreásed after treatment with acetylated agent, VPA. Fúrthermore the capacity of endothelial progenitor cells to form vascular tubes on Matrigel was decteased in the presence of VPA. Investigating the role of histone acerylation in the regulation of accessibility of transcription factors to genes involved in differentiation can contribute to understanding epigenetic mechanisms underlying the differentiation of stem cells.
Institute of Cellular Biology and Pathology "Nicolae Simionescu" of Romaniari Academy, Fetal and Adult Stem Cell Therapy Department, Bucharest, Romania
Eugen Andrei [email protected]
2
Danube Conference on Epigenetics
2014 . November 19-21
The role of small RNAs in ambient temperature sensing in plants
Ivett Baksa, György Szittya ~ NARlC ~ Agricultural Biotechnology Center, Epigenetics Group, Gödöllő, Hungary
Irina Mohorianu, Tamás Dalmay ~ UEA - School of Biological Sciences, Norwich, UK
Plants continuously monitor their environment to collect and interpret the environmental elles and adjusr their developmental program to it. As sessile organisms it is absolutely essential for them that they grow and develop accordingly. Although rcmpcrarurc influences plant growth and development in a large extent, the process, how temperature is sensed and integrated inte development is largely unknown. From an earlier study we know that chromatin structure has a key role in ambient rcmpcrarurc sensing in Arabidopsis. It was shown that alternative H2A.Z histone containing nucleosomes acts, like regulators of ambient temperature transcriptome. In plan ts short 21~24 nucleotides long non coding RNAs (sRNA) have been recently recognised as important gene express ion regulators both transcriptionally and post-transcriptionally Furthermore sRNAs also have a major function during epigenetic regulation of chromatin structure. In this work we use deep-sequencing and computational methods to identify, profile and describe conserved and non-conserved sRNAs in Arabidopsis thaliana during different ambient tcmpcraturc conditions. Fúrthermore we also identify sRNA cleaved mRNAs through genomic~scale high-throughput sequencing. Understanding the molecular mechanisms of ambient tcmpcrarurc response of sRNAs will give us important knowledge that can be adapred to crop species and used in future breeding programs. This is particularly important, because the projecred increases in mean global tcmpcrarurc as well as predicted extremes of tcmpcraturc in [he next 100 years suggesüng significant threats to wild planrs and agricultural production.
NARIC - ABC, Gödöllő, Hungary
Ivett Baksa [email protected]
3
Danube Conference on Epigenetics
2014 . November 19-21
The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription
Tiago Baptista (1,2,3,4), Chen-Yi Wang (1,5) , jacques Bonner (1,2,3,4) , Cheng-Fu Kao (5), László Tora (1,2,3,4) and Didier Devys (1,2,3,4)
1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 IlIkirch, France; 2. UMR7104, Centre National de la Recherche Scientifique, 67404 IlIkirch, France; 3. U964, Institut National de la Sariré et de la Recherche Médicale, 67404 Illkirch, France; 4. Universiré de Strasbourg, 67404 Illkirch, Cedex, France; 5. Institute of Cellular and Organismic Biology, Academia Sinica,
Taipei 11529, Taiwan
The SAGA (Spt-Ada-GcnS acetyltransferase) coactivator complex contains distínct chromatin-modifying activities and is recruited by DNA-bound activators to regula te the expression of a subset of genes. Interestingly, recent srudies have reported little overlap berween genome-wide SAGA~binding profiles and changes in steadystate expression upon deplerion of subunits of the complex.
As indicarcrs of SAGA recruitment on chromatin, we monirored in S. cerevisiae the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which arc respectively deposited or removed by SAGA. Changes in these modifications after depletion of the corresponding enzyme demonstrared that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In egrcement with this, we show that SAGA plays a eritical role for RNA polymerase II recruitment at all expressed genes.
Moreover, through quantification of newly synthesized RNA, we demonstrared that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Finally, comparative dynamic transcriptome analysis (cDTA) revealed that all genes showed impaired transcription upon SAGA subunits deletion, which is compensated by an overall decrease on the decay rate.
Thus, our study discloses a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.
1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, France; 2. UMR7104, Centre National de la Recherche Scientifique, 67404 Illkirch, France; 3. U964, Institut National de la Sanré et de la Recherche Medicale, 67404 Illkirch, France; 4. Universiré de Strasbourg, 67404 IlIkirch, Cedex, France
Tiago Baptista
[email protected]
4
Danube Conference on Epigenetics
2014 . November 19-21
Gross Chromosomal Rearrangement (GCR) based on centro mere fission coincide with massive changes of gene expression profile of HUVECs.
University of Debrecen, Medical Faculry Department of Biochemistry and Molecular Biology
Genomic instability is a hallmark of cancer. Mutational changes or breaks of DNA can easily lead to tumor development, although [here is no general factor identified that ultimately induce cancer development. Analyses of genomic data of many different cancer cell lines show that many differenr genomic changes can apparently result in similar malignant transformation of the cells. However, a growing number of studies has shown that centromere instabiliry may have a fundamental contribution to this patholegical event, especially when the tumor becomes more invasive. Our study reveals that gross chromosomal rcarrangcmcnt based on centromere fission may result in dramatic changes in both the morphology and physiology of human cells derived from umbilical cord. During in vitro maintenance of HUVEC cells many cell lines have, apparently spontaneously, changed their morphology and growth pattem in a similar way. The cells became spindie shape, more com pact, without cell surface protrusions, they losr the contact inhibition of growth and they seems to be immortal as they arc able to proliferate over 200 population doubling. Furthermore, the cells have some level of tumorigenic porenrial as they are able to form colony on sof t agar. FACS measurement of 30 different CD proteins clearly separate them from HUVECs. Karyotype analysis (classical, giemsa stained) revealed that the chromosomes of the different cell lines have similar structural abnormalities, apparently most of the C group chromosomes arc broken at the centromeric region, while telomere FISH showed that the broken chromosomes have telomere cap at the broken ends too. Nevertheless, the viability test demonstrares that these cells are as viable as HUVECs and cornet assay have not derecred DNA damage. In vitro, they arc able to spontaneously differentiare inte contractile myotubes upon persisrenr confluency of the primárv cells, and also osteegenie differentiation of these cells shows the presence of alizarin stained calcium deposirs, however we were not able to differentiare them toward adipogenic dircetion. The Affymetrix micro array results revealed massive changes in gene expression profile of the transformed cells compared to HUVECs. More than 13,000 gene expression have significantly changed and the location of the altered genes differ from random distribution. The investigation of epigenetic markers by merhylation sensitive restrietion of the DNA found less methylated DNA in the case of transforrned cells.
AlI together our results suggest that Gross Chromosomal Rearrangement based on centromere fission accompanied with epigenetic changes of the DNA can push the cells to a state of regained growth porenrial which share s many characteristic with tumoric phenotype. These cells can provide a unique opporruniry to study this specific chromosomal aberration and cancer pathology
Debreceni Egyetem
Beáta Barta-Tóth [email protected]
5
Danube Conference on Epigenetics
2014 . November 19-21
Determination of ERa regulated genes by the mcta-analysis of transcription factor binding sites in MCF-7 cell line derived from various experimental methods
Dóra Bojcsuk l , Attila Horváthl,2, Edina ErdŐsi, László Steiner2, Endre Barta 1, 2 and Bálint L. Bálintl
Estrogen receptor alpha (ERa) is a member of the nuclear receptor super family and is known as a key hormoneregulated transcription factor in three-quarters of the breast cancer cases. The activation of ERa by 17~-estradiol results binding of the receptor [Q transcription regulatory regions of the DNA and influences the expression of specific genes.
One of the greatest opportunities in research is the ability to mánage large datasets and draw biologically relevant conclusions from them. Currently, there arc many ways to investigate functional genomics features of sequencing data; S0, to elucidare the effect of ERa transcription factor binding in MCF-7 cell line we re-analyzed ali available ChIP-seq, RNA-seq, GRO-seq and DNaseI hypersensitivity as weil as H3K4me2 experiments with bioinformatical merhods.
\Ve matched the consensus ChIP peaks to GRO-seq, DNaseI hypersensitivity and H3K4me2 signals and to level of RNA-seg expression - that were influenced by esrradiol treatment -, and each binding site was given a score based on the cumulative change of the differenr features. Next, we decided to invcsrigatc further those genes that were previously annorated to the binding sires with the highest score. After defining the possible genes that are influenced by cstrogcn treatment we identified SNPs that may affect the binding events.
In the literarure, a part of our identified genes were published in association with breast cancer, but some genes were not published; so, later on we plan to derermine the realationship berween genes and breast cancer and to válidate the effect of these SNPs in these binding sites under laboratory conditions.
1 University of Debrecen, Medical and Health Science Center, Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatic Core Facility,
2MTA-DE "Lendulet" Immunogenomics Research Group, University of Debrecen, Debrecen, Hungary Dóra Bojcsuk
[email protected]
6
Danube Conference on Epigenetics
2014 . November 19-21
Heterochromatin and Alzheimer disease: loss of the physiological role of Tau protein regulating pericentromeric heterochromatin.
Mansuroglu Z#, Mokrani-Benhelli H#, Marcaro V#, Delattre L*, Sul tan A*, Violer M*, Soues S#, Lefebvre B*, Buée L*, Galas M-C*, Bonnefoy E*.
#Université Paris Descartes, Centre Interdisciplinaite Chimie Biologie-Paris, 45 rue des Saints-Peres, 75006 Paris, France.
*Université de LilIe 2, Inserm UMR 837, rue Michel Polonovski, 59045 LilIe, France.
Alzheimer disease (AD) is a dévastating neurodegenerative disease leading to massive neuronal dysfunction and cell death. Post-mortem AD brains are histopathologically characterízed by extracellular beta-amyloid (A~) pépride plaque s and intracellular Tau neurofibrillary tangles. AD treatment based on amyloid eascade hypothesis has proved to be totally ineffecrive. Su ch AD therapeutic failure derives from incornplere understanding of the complex mechanisms of AD that need to be forrher investigated. Although the microtubule-associated Tau protein is mostly considered as a cytosolic protein, a nuclear localization of Tau has been described in neuronal and non-neuronal cells. Here, we describe results indicative of a physiological role for Tau protein regulating pericentromeric heterechromatin (PCH) structures in primary cultured neurons. Electron microscopy, chromatin immunoprecipitation and confocal immunofluoerescence analysis of wild type (\X1T) and Tau deficienr (KaTau) primary neuronal cultures, demonstrate that Tau interacts with pericentromeric DNA sequences and plays an essential role ensuring the subnuclear, clustered distribution of H3K9me3 and HPla, which are hallmarks of pericentromeric structures. Comparative analysis of \VT and Kf.I'Fau neuronal cultures indicated an almost total loss of H3K9me3 and HPla clusters in KO'Tau respect to WT neurons. A very similar situation can be observed in AD respcet to control brains. Such loss of H3K9me3 and HPla clusters was not the result of a loss of either the HPla protein or the H3K9me3 epigenetic mark but was correlated with an abnormal rate of sena/anusens non-coding pericentromeric RNAs observed in K'O'Fau compared to WT neurons. The effect of the loss of PCH structures in K'O'Fau neurons upon gene expression regulation was analyzed with respect to the expression of the PCH-regulated interferon-beta gene that has been shown to be abnormally up regulated among AD patien ts. We hypothesize that loss of the physiological function of Tau protein regulating neuronal PCH structures can induce deletenous effects in AD brains.
Université Paris Descartes, Centre Interdisciplináire Chimie-Biologie-Paris, 45 rue des Saints-Peres, 75006 Paris, France.
BONNEFOY
eliette. [email protected]
7
Danube Conference on Epigenetics
2014 . November 19-21
Acetylations of Ftz-Fl and histone H4KS are required for the fine-runing of ecdysone biosynthesis during Drosophila metamorphosis
Barbara N. Borsos 1, Tibor Pankotai 1,2,*, Dávid Kovács 1, Christina Popescu 1, Zoltán Páhi 1, Imre M. Boros 1,2
1 Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary 2 Institute of Biochemistry, Biological Research Center, Szeged, Hungary
The molting during Drosophila development is tightly regula ted by the ecdysone hormone. Several stcps of the ecdysone biosynthesis have been already identified but the regulation of the entire process has not been clarifled yct. \Ve have previously reporred that dATAC histone aceryltransferase complex is necessaty for the steroid hormone biosynthesis process. To reveal possible mechanisms controlled by dATAC we made assumptions that either dATAC may influence directly the transcription of Halloween genes involved in steroid hormones biosynthesis or it may exert an indirect effect on it by acetylating the Ftz-Fl transcription factor which regulates the transcription of steroid converting genes.
Here we show that the lack of dATAC complex resulrs in increased mRNA level and decreásed protein level of Ftz-F1. In this context, decreásed mRNA and increased protein levels of Ftz-Fl were detected upon treatment of Drosophila S2 cells with histone deacetylase inhibitor trichostatin A. We showed that Ftz-Fl, the transcriptional activator of Halloween genes, is acetylated in S2 cells. In addition, we found that ecdysone biosynthetic Halloween genes are transcribed in S2 cells and their expression can be influenced by deacetylase inhibirors. Furthermore, we could detect H4KS acetylation at the regulatorv regions of disembodied and shade Halloween genes, while H3K9 acetylation is absent on these genes.
Based on our findings we conclude that the dATAC HAT complex plays a dual regulatory role in Drosophila steroid hormone biosynthesis through the acetylation of Ftz-Fl protein and the regulation of H4KS acerylation at the promoters of Halloween gen es.
This research was supported by TÁMOP 4.2.4. A/2·11·1·2012·0001 and HURO/1101/173/2.2.1.
Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary
Barbara N. Borsos [email protected]
8
Danube Conference on Epigenetics
2014 . November 19-21
Thermal separation of ere recombinase expression from enzyme activity leads to high frequency removal of both the selection marker and the recombinase gene from transgenic barley in auto-excision strategy
Csaba Éva (present address): Department of Applied Genomics, Centre for Agricultural Research, Hungarian Academy of Sciences, H-2462 Martonvásár Hungary
Klára Mészáros: Cereal Breeding Department, Centre for Agricultural Research, Hungarian Academy of Sciences, H-2462 Martonvásár Hungary
Csaba Éva, László Tamás: Department of Plant Physiology and Plant Molecular Biology, Eötvös Loránd University, H-1117 Pázmány Péter sétány I/C Budapest Hungary
Selection marker genes (SMGs) are essential for the genetic modificarion of most plant species. Their presence in the genome is however unnecessary after the transformation process and even generates high environmental and consumer concerns. The SMG can be remeved from the plant genome if it was co-introduced wi th a sire-specific recombinase gene in an autó-excision strategy. Our proof of concept was that high level Cre recombinase express ion and thus high recombination frequenev could be obtained by using a cold-inducible promoter for [he induction of ere gene. It was assumed form the fact [hat this enzyme has barelv any activity at low tcmpcraturc and therefore by applying cold induction, it would not delere its own gene while it is needed. The hypothesis was tested by producing transgenic barley that contained the wcs120 cold inducible promoter-dríven ere recombinase gene and the SMG bar, both flanked by the recombinase recognition site lox. The results supported our hypothesis as both ere and the SMG could be removed from 96% of the planrs treared with the most effective treatment containing induction of ere gene at 40C for 3 days and subsequent enzyme activation at 240C. The changes caused by the excision were fully inherited in the progeny. Mostly low copy transgenic plants were treared at the earliesr developmental phase, which may prevented genetic mosaicism and thereby also contributed to our success.
Department of Applied Genomics, Centre for Agricultural Research, Hungarian Academy of Sciences
Csaba Éva [email protected]
9
Danube Conference on Epigenetics
2014 . November 19-21
Identification of STAT6 as a transcriptional repressor during alternative macrophage activation
Zsolt Czimmerer l *, Bence Dánielt *, Attila Horvath 1 *, Gergely Nagyi, Laszlo NagyI,2
1. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Egyetem rér I. Debrecen, Hungary H-4010
2. MTA-DE "Lendület" Immunogenomics Research Group, University of Debrecen, Egyetem tér l. Debrecen, Hungary
* These authors contributed equally to this work.
Macrophages are participating in the mainrenanec of tissue homeostasis, protection against pathogen infections and tissue repair. Macrophage phenotypes and functions are influenced by their cytokine milieu. The two end poinrs of macrophage polárizarion are INFyjLPS-mediated classical (Ml) and IL-4/IL~13-induced alternative (M2) macrophage activation. Ml-type macrophage activation results in enhanced bacrericidal capacity and proinflammatory properties. In contrast, M2-type macrophages have anti-inflammatory properties, and therefore participate in tissue regeneratien and protection against parasitic infections. IL-4 activates both IL4Ra-]AKSTAT6 and PI3K pathways resulting in alternative macrophage activation-specific gene expression changes. Although, the mechanism of IL-4/STAT6-dependent transcriptional activation is well understood, [he IL-4- regulated transcriptional repression is not characterízed. We combined genome-wide ChIP-seC) and RNA-seC) analysis for better understanding of the IL-4-mediated negative regulation of gene expression in murine macrophages. We characretized the STAT6 cistrome and H4 acetylaton in IL-4 stimulated mouse macrophages and we found extensive overlap between S'I'ATű-bound and H4 acerylated genomic regions. We observed that one cluster of IL-4-induced STAT6 peaks was associated with decreásed H4 acetylation. Next, we corripared the STAT6-bound genomic regions to ILA-rebTUltated genes and we could show extensive association berween decteased H4 acerylation at STAT6-bound region and IL-4-mediated repression. In addition, we identified several immunologically televant IL-4-repressed genes in association with decreásed H4 acetylation at STAT6-bound regions. Taken together, these results suggest that IL-4 may medulate immunological properties of macrophages via STAT6-mediated direct transcriptional repression. Currently we are in the process of identification of the mechanism of IL-4jSTAT6-mediated direct transcriptional repression in macrophages using genomic and biochemical approach es.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Egyetem [ér l. Debrecen, Hungary H-4010
Zsolt Czimmerer [email protected]
10
Danube Conference on Epigenetics
2014 . November 19-21
A role for Rif1 in silencing of X-linked gene clusters in mammals
Lucia Daxingerl,2, Harald Oey2, Luke Isbel2 and Emma Whitelaw2
lLeiden University Medical Centre, Leiden, The Netherlands; 2La Trobe Institute of Molecular Sciences, Bundoora, VIC, Australia
\Ve have cartied out a chemical mutagenesis screen to identify genes that are required for establishment and maintenance of epigenetic state in the mouse. Mutant lines identified from this screen are called Mommes (Modifiers of murine merasrable epialleles). The human homologues of ma ny of these have been found to be associated with human disease.
One mutant line, MommeD18 was identified to earry a mutation in Rap interacting factor 1 (Rifl). RiflMommeD18 is a suppressor of variegation and express ion of a GFP transgene is significantly higher in mutant corripared to wild type mice. Rifl has been shown to be involved in DNA damage response, replication timing and epigenetic silencing of subtelemeres in mammals. Here, we perforrned RNAseq on E9.5 wild type and homozygous embryos to study the role of Rifl in transcriptional repression. \Ve find up-regulation of X-linked ampliconic regions in mutant embryos. These gene clusters arc expressed predominantly in spermatogonic cells and in the brain. AdditionaHy, we find that Rifl is required to repress a limited number of germ-line specific genes. We obtained similar results using hRlFl knockdown cell lines. Our findings suggest a role for Rifl in the silencing of independendy acquired genes on the human and mou se X chromosomes in somatic cells. While the function of these gene clusters is largely unknown, members of these gene families arc of ten expressed in human tumors and are known as cancer testis antigens.
Leiden University Medical Centre
Lucia Daxinger [email protected]
11
Danube Conference on Epigenetics
2014 . November 19-21
Mapping the Estrogen Receptor alpha binding sites derived from ER positive cell lines in the light of the 1000 Genomes Project
Edina ErdŐsi, Dóra Bojcsukl, Lilla Ozgyinl, Attila Horváthl, Endre Barta l , and Bálint L. Bálinti
1 University of Debrecen, Medical Faculty, Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatic Core Facility
Estrogen Receptor alpha (ERa) is a hormone-regulared transcription factor expressed in most of endocrinerelated cancers (sueh as breast and endomerrial caneer) in women. ERa is an important target for endocrine therapy of breast cancer, although resistance to treatment occurs in a significant number of the patien ts. Differences in response to the treatment can be derived from genetic variations between individuals. The aim of our study was to imrestigate the effects of SNP-s in the ERa binding sires (ERBSs).
ERa ChIP-SeC) data were colleeted from public database. We selecred four ER positive cell lines derived from breast (NfCF-7 and T47D) and endometrial cancers (ECC-l and Ishikawa). Raw data were re-analyzed by our bioinformatic pipeline to derermine the common and individual ERBSs of each cell line. The resulting peak sets were compared with the dbSNP database, then the common ERBSs were annotated, giving 29 intronic geneassociated ones. These genes encode protems including DNA-binding ones, regulatorv enzymes and some which have role in cell adhesion. Some of these were selcered to be validated by experimentál methods. \Ve investigated the presence of ERBS by chromatin immunoprecipitarion followed by ~PCR (ChIP-~PCR) in the control and estradiol (E2) treared MCF-7 cells and in parallel we examined the gene expression changes upon the treatment. Results of the ChIP experiments showed ER enrichment at the selecred ERBSs in E2 treared MCF-7 cells. Genes hosting the ERBSs showed significant changes in expression upon ligand treatment. Characterizaeion of the newly characterized ER target genes may help us to understand the effects of SNPs on ER binding events.
University of Debrecen, Medical Faculty, Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatic Core Facility
Edina Erdős [email protected]
12
Danube Conference on Epigenetics
2014 . November 19-21
In situ functional analysis of a maintenance element in the bxd cis-regulatory region of Drosophila
Dávid Farkas, Gabriella Kozma, Izabella Bajusz, Ferenc ]ankovics, Henrik Gyurkovics and László Sipos (Institute of Genetics, Biological Research Center, Szeged, Hungary)
In the post-genomic era, one of the main challenges facing biology is answering the question of how different cell types and cell lineages, deriving from the zygote, utilize the very same genome differently during development. Part of the answer must be sought at the level of higher order chromatin structure.
The alteration of the chromatin structure is a substantial process of epigenetic regulation of gene expression. In Drosophila, the bithorax complex (BX-C) is an exquisitely convenient model system to study epigenetic regulation. In Drosophila, rwo classes of genes, the Polycomb group and the trithorax group, are required to act in concert to maintain the expression of numerous key regula tor genes by regulating the chromatin structure. The POLYCOMB group proteins are epigenetic silencers required for the repression of the target gene by condensating its chromatin structure, whereas the TRITHORAX group protems arc known to be epigenetic activators with the function to keep the target gene in an open chromatin conformation. The regulation of chromatin conformation depends on special DNA sequences called Polycomb Response Elemencs (PREs) containing binding sites for the POLYCOMB group proteins and the Trithorax Response Elemencs (TREs) containing binding sires for the TRITHORAX group proteius.
In the bxd/pbx cis-tegulatory region, the PRE (bxd PRE) is the neigbbor of the TRE (bxd TRE) establishing a so call ed Mainrenance Element (NlE), which is responsible for maintaining the active or the inactive chromatin conformation from the late embryonic stagcs to the adulc stagc. The in situ function of the bxd PRE is well known, contrarily, the in situ function of the bxd TRE element is not known yet.
Our aim is to reveal the true function of the bxd TRE in its original context, to shed light on irs role as an activator in the regulation of the distant Ubx promoter and in the regulation of the local chromatin state.
Institute of Geuerics, Biological Research Center, Szeged, Hungary
Dávid Farkas [email protected]
13
Danube Conference on Epigenetics
2014 . November 19-21
CLASS I HISTONE DEACETYLASES IN THE REGULATION OF ADIPOSE TISSUE FUNCTIONALITY
Alessandra Ferrari, Erika Fiorino, Serena Barilla. Nico Mitro, Donatella Caruso, Uliano Guerrini, Emma De Fabiani, Maurizio Cresrani
Dipartimento di Scienze Farmacologiche e Biomolecolari Universita degli Studi di Milano
Histone deacetylases (HDACs) are epigenetic regulators involved in metabolic homeosrasis. Our laboratory demonstrared that class I selective HDAC inhibition improves obese and diabetic phenotype in genetic model of type 2 diabetes (db/db mouse). In the light of these results, we performed fúrther srudies to characterize the effect of class I HDAC inhibirion in a model of obesity induced by a high fat diet (HFD, 60% calories from far). C57BL6/J mice fed HFD for !7 weeks and then rreated with class I selecrive HDAC inhibitor MS275 showed 10% reduction of body weight, improved glucose clearance, and better resistance to cold exposure, as a result of iruproved thermogenic capacity of brown adipose tissue (BAT) confirrned by higher expression of typreal BAT markers (Di02, ElovI3). In white adipose tissue (\VAT) MS275 reduced adipocyte size and increased the express ion of markers of fat functionality (Glut4, Pparg, Fabp4). lipid catabolism (Cpt! b, Lead) and mirochondrial biogenesis (Tfam, CytC). Viscera! WAT of MS275 treared mice showed increased oxidativé capacity, as shown by higher expression (70%) of Ucpl, suggesting "browning" of this far depor. The next stcp was to investigate mechanism underlies MS275 effects in WAT. In both subcutaneous and viscera! \VAT there are different cell populations, including preadipocytes and mature adipocytes. In vitro experiments in C3HI0Tl/2 preadipocytes demoostrared that cells differentiated in the presence of MS275 showed, at the end of differenuatíon, increased expression of Pparg and of its main targcts (Fabp4, Glut4, Acrp30); conversely no effects were observed in in vitro fuHy differenriared adipocytes treated with MS275, demonstrating that class I HDACs are important players in early stagcs of adipocyte differentiarion. Our in vitro and in vivo results suggest that selectíve inhibitien of class I HDACs improves obese and eliabetic state, by ameliorating adipose tissue functionaliry, mostly by "reprogramming" preadipocyre fate.
Universiti degli Studi di Milano
Alessandra Ferrari alessandra.ferrari [email protected]
14
Danube Conference on Epigenetics
2014 . November 19-21
Monitoring the endocannabinoid system during chondrogenesis
Authors: Zsófia Főldvári l , Csilla Somogyi 1 , Csaba Mattal, Tamás ]uhászl, Róza Zákányi
Affiliation: 1 University of Debrecen, Faculty of Medicine, Department of Anatomy, Histology and Embriology
Osteoarthritis, a disease leading to pain and finally loss of function, influences the elderly people's quality of life. The currently available surgical techniques aiming to cure osteoarthritis are not satisfactory, tberefore there is a niche for new, cell-based regenerative methods. According to the latest results in the field of cartilage research, CB 1 and CB2 rcccptors arc expressed in mature and arthritic chondrocytes and cannabinoid products can attenuate the degradation of proteoglycan matrix with the inhibition of cytokine-indneed NO production.
Ouring the in vitro part of our work high density chondrogenic cell cultures (HOC) were esrablished from limb buds of 11,5 day-old mouse embryos. The mRNA expression of the members of Endocannabinoid System (ECS) was monirored by semiquantitative RT-PCR and the [wo cannabinoid receprors were derecred by western blot and immunocytochemistry, To evaluate the effect of CB1 agenist (anandamide: AEA) and inverse agonist (AM251) on chondrogenesis chondrogenic markers and matrix constituents were monitored by RT-PCR technique and dimethyl-methylene blue (DMlYIB) staining, respectively. Ex vivo, we derecred the mRNA expression of the ECS members in cartilage samples of different developmental stagcs and observed the spatiotemporal distribution of the CB1 receptor in mou se embryonic limb bud s and in the limbs of young mice. Our results show the presence of each examined receptor and enzyme of the ECS at mRNA level in mou se HOC. CB1 protein is expressed in HDCs, limb buds of a I Scday-old mouse and in the articulat cartilage of young animals. CB 1 receptor is present during in vivo and in vitro chondrogenesis and the applied pharmacons in a concentration dependenr way influence chondrogenesis. This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1- 201 2-0001 'National Excellence Program'.
University of Debrecen, Faculty of Medicine, Department of Anatomy, Histology and Embriology
Zsofia Foldvari [email protected]
15
Danube Conference on Epigenetics
2014 . November 19-21
Role of epigenetic regulatory mechanisms in sport physiology
Zsuzsanna Gaál (University of Debrecen, Department of Physiology) Attila Mokánszki (University of Debrecen, Department of Physiology) János Fodor (University of Debrecen, Department of Physiology) Ildikó Balatoni (University of Debrecen, Depattment of Physiology) László Csernoch (University of Debrecen, Department of Physiology)
The term epigenetics includes all mechanisms that influence gene expression without any changes in the sequence of the DNA. Based on the data of preliminary results, emerging amou nt of evidence supports the importance of DNA methylation, histone modification and non-coding RNAs, especially microRNAs (miRNAs) in determining individnal skills in sport.
Main aims of our research include elucidating distinct epigenetic patterns according to different kinds of sport activity, and describing key epigenetic regulatory mechanisms that regulate skeleral muscle differentiation as well. A certain group of microRNAs (e. g. miR-l, miR-133ajb and miR-206) is of ten mentioned in the literature as .myomiks". Besides regulating differentiarion some of them also have a role in skeletal muscle hypertrophy mainly by regulating the IGF-I / Akt signaling pathway. A few connections between ageing and altered miRNA express ion levels arc also known.
In consequence of physical exercise, characteristic changes in histone code have been observed. These alterations are mediated by several mechanisms including decreásed level of miR-33, HDAC-phosphorylation and subsequent nuclear export of HDAC4 and HDACS. Ariother example of connections berween different types of epigenetic regulatorv mechanisms is that miR-206 represses hypertrophy of myogenic cells but not muscle fibers via inhibiton of HDAC4.
MiR-33 has also an impact on DNA-methylation pattern: physical exercise inhibits miR-33-mediated inhibition of AMPK and CaMK, rherefore the activaton of the PPARy-coactivator PGC-Ia increases following exercise partly because of a dose-dependenr hypomethylation of its promoter region.
Experiments seeking for connections between forrher epigenetic changes, achieved results in different kinds of competitive sporrs, ageing, differentiation and hypertrophy of skeleral muscle may contribute to personalize sport activity which has a significance in public health too.
University of Debrecen, Department of Physiology
Dr. Gaál Zsuzsanna [email protected]
16
Danube Conference on Epigenetics
2014 . November 19-21
Epigenetics imp act on the early aging phase of white adipose tissue
Grera M.P. Giordano Attianese, Aurélien Naldi,
Barbara Toffoli,
Carine \Vinkler,
Michaél Baruchet,
Béatrice Desvergne
and Fedérica Gilardi
Ali authors are from Center for Integrative Genomics, University of Lausanne
Epigenetic mechanisms are key regulators of the transcriptional changes occurring during the senescence process of several mammalian tissues. Due to its very dynamic function and changes throughout life, white adipose tissue (\VAT) is a very interesting model of both aging and consequent age-related disorders. However, rbere is still a gap in knowledge of the physiological role of epigenetics during WAT aging. The aim of this study is to investigate how chromatin remodeling events contribute to the progression of WAT aging, particularly during its very precocious phase, when the interference of the age-relared systemic body decline is negligible. To this goal we colleered WAT from 3 and 12 month s old mice, as representative of young~ and middle-adulthood, respectively. Our results showed that 12 month s old mice arc characterízed by mild hypertrophy, accompanied by downregulation of genes involved in DNA repair and angiogenesis, but no inflammation, thus representing a good model of \VAT early aging. A new method for chromatin immunoprecipitation from WAT was employed to evaluate genome~wide the landscape of a series of histone marks and RNA Pol II by using Chflz.seq analysis. In parallel, RNA-seq provided an unbiased evaluation of gene expression in the two representative groups. The combination of ChIP-seq and RNA-seq data obrained in the same experimental conditions allowed assessing the actual impact of epigenetic changes on mRNA levels. Our results will improve the knowledge on the molecular mechanisms underlying early aging and will contribute to the identification of new potential therapeutic targcts for fat related age-associated metabolic diseases.
Center for Integrative Genomics University of Lausanne Genopode Building
Federica Gilardi [email protected]
17
Danube Conference on Epigenetics
2014 . November 19-21
The DNA methylation cofactor UHRFl is required for gene expression reprogramming in cardiac hypertrophy
Carolina M. Grecol, Pierluigi Carullo 1,2, Paolo Kunderfranco 1,2, Francesca Rusconi 1 ,2, Lorena Zentilin3, Elisa di Pasqualel,2, Mauro Giacca3, Robette Papaitl,2, Gianluigi Condorellil,2,4
Heart failure, a leading cause of mortality worldwide, is accompanied by cardiac hypertrophy, a process associated with significant transcriptional changes characretized by the re- expression of fetal genes. Epigenetic mechanisms (DNA methylation and histone rnodificarions) play a key role in defining the gene expression profile without modifying the actual DNA sequence. Although DNA methylation is a keyelement of gene expression regulation, its role in this pathology remains unclear. \Ve rcport that the DNA methylation cofacror ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) is a stress-induced cardiac gene controlled by serum response factor (SRF), a key transcription factor for the hypertrophy response. We found that UHRFl mediated centractile dysfunction and promoted the adaptíve bioenergetic response of hypertrophic cardiomyocytes by binding w the promoters of genes encoding SERCA2 - a key ion pump for contractility - and genes involved in multiple metabolic pathways. UHRFl repressed the transcription of these genes by recruiting DNA methyltransferase (DNMT)3b and histone methyltransferase (HMT)G9a leading to de novo methylarion of DNA and dimethylation of histone H3 at lysine 9 (H3K9me2), respectively, Indeed, cardiomyocyte-specific silencing of Uhrfl and pharmacological inhibition of both DNA and histone H3K9 merhylation improved the cardiac function of mice subjected w pressure overload. Taken together, these findings identify UHRFl as a eritical mediator of the gene re-programming process of cardiac hypertrophy and indicare that inhibition of this epigenetic nerwork could be a promising therapeutic strategy for heart failure.
1 Humanitas Clinical and Research Hospital
2Institute of Genetic and Biomedical Research (IRGB)
3International Centre for Genetic Engineering and Biotechnology (ICGEB) Carolina Greco
[email protected]
18
Danube Conference on Epigenetics
2014 . November 19-21
Studying DNA-binding features of chromatin architectural factors via bacterial growth
Malre Prell, Institute for Biochemistry and Molecular Biology, Medical School, RWTH Aacben University, 30 Pauwelsstrasse, Aachen, Germany 52074.
Hiltrud Königs, Institute for Pathology, Medical School, RWTH Aachen University, 30 Pauwelsstrasse, Aachen, Germany 52074.
Beruta Hermanns-Sachweh, Institute for Pathology, Medical School, RWTH Aachen University, 30 Pauwelsstrasse, Aachen, Germany 52074.
Ferdinand Kappes, Institute for Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, 30 Pauwelsstrasse, Aachen, Germany 52074.
The DEK oncogene is a unique non-histone chromosomal factor with no known enzymatic activiry. DEK participates in the regulation of the repressing epigenetic histone mark H3K9Me3. On the other hand, a large body of in vitro work revealed that DEK exhibits DNA and chromatin binding functions (e.g. DNA supercoiling, DNA bending, and sequence-independent binding to non-B-form DNA structures) that are inherent to the family of chromatin architectural factors. However, the biological relevance of DEK's DNA binding activities in the cellular nucleic acid metabolism has remained elusive. Our recent results now show that DEK binds to the entry and exit site of the nucleosome in vitro and that knocking down DEK expression in cells results in a marked reduction in the nucleosomal repeat length.
In order to detail the role of DEK's DNA-binding activities in shaping chromatin structure in vivo, we sought to create "DNA-binding-dead" mutants. Our initial rational mutant design founded on information deduced from the NMR structure of DEK, in silico modeling, and homology comparisons, however, yielded no such desired mutants. Therefore, we employed a random mutagenesis approach, which we combined with arather unusual read-out system: bacrerial growth. This was possible as multiple lines of evidence showed that DEK expression in bacteria leads to a severe compaction of the bacrerial nucleoid, thus highlighting DNA binding activities as the source of growth attenuation. Based on rbese findings we lined up a novel screening procedure that indeed yielded "Ioss-of-function" DEK mutants. lnitial testing of these mutants in newly created DEK knockout cell lines showed that DNA binding activities of DEK arc vital for overall proper chromatin structure and forrher suggcsts that DEK is a vital chromatin architectural protein. Moreover, combining random mutagenesis with bacrerial growth may be a help fuI novel tool for studying DNA binding features of other chromatin-associated proreins.
Institute for Biochemistry and Molecular Biology, Medical School, R\X1TH Aachen University, 30 Pauwelsstrasse, Aachen, Germany 52074.
HaihongGuo
haihongguo [email protected]
19
Danube Conference on Epigenetics
2014 . November 19-21
The proto-oncoprotein FBI-l interacts with MBD3 to recruit the Mi-2/NuRDHDAC complex and BCoR and to silence p21WAF /CDKN1A by DNA methylation
Won-Il Choil, Bu-Nam Jeonl, Jae-Hyeon Yoonl, Dong-In Kohl, Myung-Hwa Kiml, Mi-Young Yul, Kyung-Mi Lee1, Youngsoo Kim2, Kyunggon Kim2, Suj in Susanne Hur3, Choong-Eun Lee4, Kyung-Sup Kim l , and ManWook Hurl*
lDepartment of Biochemistry and Molecular Biology, BK21 Project for Medical Science, Severance Biomedical Research Institute, Yonsei University School of Medicine, 50 Yonsei-Ro, SeoDaeMoon-Gu, Seoul, 120-752, Korea, 2Department of Biomedical Sciences &
Biomedical Engineering, Seoul National University College of Medicine, 103 Daehangno, Seoul 110-799, Korea, 3Sookmyung Girls' High School, 91 Dogok-Dong, Gangnarn-Gu, Seoul, 135-505, Korea, 4Department of Biological Science, Sungkyunkwan University, Suwon 440-746, Korea
The tumour suppressor gene CDKNIA (encoding p2IWaf/Cipl) is thought to be epigenecically repressed in caneer cells. FBI-l (ZBTB7A) is a proto-oncogenic transcription factor repressing the
ARF and p2IWAF/CDKNIA genes of the p53 pathway. FBI-I interacts directly with MBD3 (Methyl CpG binding domain protein 3) in the nucleus. \Ve demonstrared that FBI-l binds both
non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-l, where it enhances transcriptional repression by FBI-l. FBI-l also interacts with the corepressors NCoR, SMRT, and BCoR to repress trauscription. :MBD3 regula tes a molecular interaction between the corepressor and FBI-l. MBD3 decreases the interaction between FBI-l and NCoRjSMRT but increases the interaction berween FBI-I and BCoR. Because MBD3 is a subunit of the Mi-2/NuRD-HDAC complex, FBI-I recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HPI. MBD3 and BCoR play significant role in the recruitment of the Mi-2/NuRD-HDAC complex and the NuRD complex associared proteins, DNMTs and HP. By recruiting
DNMTs and HPI, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKNIA by DNA methylation.
Department of Biochemistry and Molecular Biology, BK21 Project for Medical Science, Severance Biomedical Research Institute, Yonsei University School of Medicine, 50 Yonsei-Ro, SeoDaeMoon-Gu, Seoul, 120-752, Korea
Man-Wook Hur
[email protected]
20
Danube Conference on Epigenetics
2014 . November 19-21
Involvment of endothelial progenitor cells in neovascularization is regulated by histone acetylation
1 Florin Iordache, lAndrei Constantineseu. lEugen Andrei, 2Carmen Curutiu, 1 Horia Maniu
l Institute of Cellular Biology and Pathology "Nicolae Simionescu"of Romanian Academy, Department of Fetal and Adult Stem Cell Therapy, Bucharest, Romania
2University of Bucharest, Faculty of Biology, Departament of Microbiology-Immunology, Bucharest. Romania
Neovascularization is a complex process involving a series of interconnected steps leading to the forrnation of new blood vessels when vascular lesions occur in adult organs. The major steps involved arc the chemotaxy, proliferation, migration, adherence and differentiation of endothelial progenitor celis (Epe) to the target situs, and their organization in vascular networks. Recent data regarding the epigenetic changes showed that histone acetylation regulates pluripotency of stem cells and their differentiation capacity by controlling the expression of genes and transcription facrcrs that are required for differentiation. Our aim was to investigate the role of histone acetylation in neovascularization and EPC differentiation. To test this hypothesi s we have used qRT~PCR and flow cytomctry assays for characterization of EPC and mesurements of telomerase activity, western blot analysis of PCNA, wound-healing assay and impedance mesurements to invcsrigarc proliferation, cell motility, adhesion and chemotaxy of EPe. Neovascularization potential of EPC cultured on collagen based scaffolds was assessed using Matrigel assay and scanning electron microscopy. The results showed that histone acetylation downregulate the processes of proliferation, adherence, migration and differentiation of EPC inhibiting neovascularization. Microscopy analysis revealed that in acetylated state EPC lose their abiliry to form vascular tube-like structures on synthetic collagen scaffolds and on matrigel basement membrane matrix, suggesting that neovascularization potential of endothelial cells is inhibited by acetylation. The new findings of our data indicare that histone acetylation medulate the poceses involved in EPe differentiation and regulates neovascularization, therefore investigating these patterns of acerylation can improve the future development of stem cells applications in tissue engineering and regenerative medicine.
Institute of Cellular Biology and Pathology "Nicolae Simionescu"of Romaniari Academy, Department of Fetal and Adult Stem Cell Therapy, Bucharest, Romania
Florin Iordache [email protected]
21
Danube Conference on Epigenetics
2014 . November 19-21
SEPT9 and SFRPl DNA hypermethylation in colorectal cancer
Alexandra Kalmarl,3, Reinhold Wasserkort2, Sandor Spisakl,3, Gabor Valczl,3, Barnabas Wichmannl,3, Kinga Toth l , Katalin Leiszter l , Barbara K. Bartak l , Theo deVos4, Bela Molnarl,3, Zsolt Tulassay1,3
Background Septin 9 and secreted frizzled-relared protein 1 play role in colorectal cancer. Out aims were to analyze DNA methylation patterns in DNA methylation-regulated genes in healthy and diseased colonic epithelial and stromal cells.
Merhods Colonic epirhelial and stromal cells were colleered using laser capture microdissection (LCM). Direcr bisulfire sequencing was used to analyze the methylation status of SEPT9 and SFRPl genes in notmal (n=3), adenomatous (n=3) and colorectal cancer (0=3) samples. SEPT9 and SFRP1 protein expression was assessed using immunohistochemistry on healthy (n=IO), adenomatous (n=14) and eRe (n=13) samples. Stromal myofibroblasts were detecred by aS:MA immunohistochemistry, the immunopositive cells were LCM se para ted and SFRPI DNA methylation was assessed by high «solution melting analysis (HRM).
Results The regions analysed in SEPT9 were unmethylated in notmal tissues except for a methylation boundary derecred downstream of the latgest CpG island within SEPT9. In adenoma and tumor samples, the epithelial cells displayed markedly incteased methylation levels (>80%,p<IO-4), but only within one of the epG islands investigated. In stromal cells increased methylation (up to SO%,p<10~4) was only seen in tumor patiems and in histologicaIly notmal tissue close to tumor, but not in adenoma. Analyzed region of SFRP1 showed remarkable increase in the adenoma and tumor epithelial cells. SEPT9 and SFRP1 protein levels showed significant (p<O,OS) decreasement in adenoma and tumor tissue samples. aSMA immunopositive myofibroblasrs were identified as the main source of stromal SFRP1 protein, that was found to be downregulated by DNA hypermethylation only in carcinoma, but not in adenoma.
Conclusion Hypermethylation of SEPT9, SFRP1 could be detecred in adenoma and cancer samples corripared to healthy controls. DNA methylation alterations originated in epithelral cells, stromal cells appear to acquire hypermethylation only subsequently via field effect.
1. 2nd Dept of Internal Medicine, Semmelweis University, Budapest, Hungary
2. Epigenomics AG, Berlin, Germany
3.Molecular Medicine Research Unit, Hungarian Academy of Science, Budapest, Hungary Alexandra Kalmar
[email protected]
22
Danube Conference on Epigenetics
2014 . November 19-21
DNA hypermethylation and upregulated miRNA21 expression lead s to decreased mRNA expression of C0L1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in leftsided colorectal adenoma and cancer
Alexandra Kalmárl,2; Bálint Péterfial,2, Péter Hollosi3,4, Zsófia B. NagyI, Barbara K. Bartak l , Barnabás \Vichmann2; Árpád V Patai l , Pál Michellerl, Ilona Kovalszky3, Béla Molnárl, 2, Zsolt Tulassayl,2
Background: Epigenetic mechanisms can contribute to colorectal cancer formatien. Out aims were to identify DNA methylation markers and miRNAs playing role in eRe development on the basis of gene expression alterations along the adenoma-carcinoma formation.
Methods: Whote genome expression profiling was performed by using HGU133 Plus 2.0 microarrays on healthy colonic (49), adenoma (49) and CRC (49) samples and on laser microdissected (LCM) epithelial and stroma! cells from healthy (6) and eRe (6) samples. Methylation status of genes wi th gradually altering expression were analyzed on macrodissected (10) and LCM (5) healthy colonic, adenomatous biopsy (10) and LCM (5), macrodissected (10) and LCM (5) left-sided colorectal cancer samples using pyrosequencing, In silico miRNA predictien for the selceted genes with miR\VALK algorithm, miRNA expression was analyzed on CRCs (3), adenomas (3) and normal tissue adjacent to tumor (NAT)(3) samples using Exiqon microRNA Ready-re-Use PCR Human Panel 1+ II. PTGDR and SFRPl immunohistochemistry experiments were performed.
Results: A set of 18 trauscripts showed decreasing expression (p::SO,Ol) in biopsy samples along CRC formation. COLJA2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylarion both in adenomas and tumor samples corripared to NAT, while BCL2, PRIMAI and PTGDR showed hypermethylarion only in CRC group. miR-21 was significantly (p<O,Ol) upregulated in adenoma and tumor samples compared to healthy controls that can influence the expression of genes without remarkable DNA methylation alteration. PTGDR and SFRPl protein levels decreased along adenoma-carcinoma sequence.
Conclusion: Gene expression-based screening was found to be a suirable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selecred markers or miR21 upregulation might result in reduced expression and may contribute to colorectal can cer formatien.
1- 2nd Department of Internal Medicine, Semmelweis University, Hungary
2- Molecular Medicine Research Group, Hungarian Academy of Sciences, Hungary
3- 1 st Department of Pathology and Experimentál Cancer Research, Semmelweis University, Hungary Kalmár Alexandra
[email protected]
23
Danube Conference on Epigenetics
2014 . November 19-21
Identification of multiple segment-specific enhancer elements in the bxd/pbx cisregulatory region of Drosophila
Péter Kaltenecker
Biological Research Centre, Hungarian Academy of Sciences
László Sipos
Biological Research Centre, Hungarian Academy of Sciences
The homeotic Ulrrabithorax (ubx) gene dererrnines the identity of parasegment (PS) 5 and 6 in the Drosophila embryo. In PSS the abx/bx, in PS6 the bxd/pbx cis-regulatorv region controls the expression of Ubx. These two parasebTffients correspond to the region spanning from the posterior half of the sec ond thoracic segment [Q the anterior part of the first abdominal segment (Al) in adult fly. Disruption of the bxd function results in the transfotmations of the posterior half of the haltera and the anterior part of Al inte the corresponding parts of PSS. Such transformation of the haltera into wing suggcsts the disfuncrion of the pbx enhancer in bxd mutants. However, the Al segment-specific enhancer has not been identified yct. Our aim is to identify this enhancer locared presumably in the distal part of the bxd domain, as suggesred by our preliminaty results.
To generate deletions in the distal part of the bxd region we induced FRT-mediated recombinations berween differenr P-element insertions locared in trans. Some of these deletions carry an intact P-element wi th a marker gene, which were mobilized to obrain clean delerions. Derivative lines were analyzed phenotypically and by PCR; some of the deletions were sequenced.
Our results confirm that the Al segment-specific adult enhancer is locared in a -12 kb region at the distal part of the bxd domain. U sing overlapping deletions, we distributed this fragment inro three sub-regions. Our observations suggest that all of these sub-regions contain elements, act as enhancers with differenr intensiry, Consequently, there arc multiple enhancer elements responsible for the formarion of the adult Al segment.
Biological Research Centre, Hungarian Academy of Sciences
Péter Kaltenecker kaltet888@b'ffiail.com
24
Danube Conference on Epigenetics
2014 . November 19-21
Intercellular trafficking of the nuclear oncoprotein DEK
Anjan K. Saba, Dcparnncm of Internal i\lcdicinc, Division of lnfcctious Discascs, University of i\lichigan, 1150 \'(( Medical Center Dr., Ann Aroor,!'IH 48109-5640 Amruta i\lundadc, Department of lmcrnall\-ledicinc, Dh·ision of lnfcctious Discascs, University of l\'lichigan, 1150 W. Medical Center Dr., Ann Arbor, /\11 48109-5640 Ania Dcut~mann, Department of Biology, Unh'crsit)' of Konstanz, Univcrsitatsstraűc 10, Konstanz, Gcrmany
David Rosmarin. Whitchcad Institute, J\HT, ') Cambridge Center, Cambridge, Massachusetts 02142
J\hurcrn Legendre, Department of Internal ]\'Icdicinc, Division of lnfccrious Discascs, Unh'crsit)' of i\lichigan, 1150 W. Medical Center Dr., Ann Arbor, Ml 48109-5640 Nicolas Chatain, Institute for Biochemistry and J\'lolccular Biolog)', J\lcdical School, R\VJ'I-] Aachcn Unh'ersit)', 30 Pauwclssrrassc, Aaehcn, Gcrmany 52074
Zcina AI-Obaidi, Department of Internal Medicine, Division of lnfccrious Discascs, Unh'ersity of Michigan, 1150 W Medical Center Dr., Ann Arbor, Ml 48109-5640 Barbara S. Adams, Department of Pcdiarncs, Division of Rheumatolog)', University of Michigan, 1500 E. Medical Center Dr., Ann Arbor, MI 48109-5718
Hiddc Plocgh, Whitehead Institute, MIT, 9 Cambridge Center, Cambridge, Massachusetts 02142
Elisa Ferrando-J\Ia)', Department of Biology, Unh'ersit)' of Konstanz, Univcrsitatssrraűc 10, Konstanz, Germany
Nirit Mor-Vaknin, Department of Internal Medicine, Division of lnfccrious Discascs, University of Michigan, 1150 W Medical Center Dr., Ann Arbor, MI 48109-5640 David 1'11. Markovitz, Department of Internal Medicine, Dh'ision of lnfccrious Discascs, Unh'ersit)' of Michigan, 1150 W. Medical Cemer Dr., Ann Arbor, 1'111 48109-5640
DEK is a conserved non-histone protein and the only representative of its own protein class. Multiple intra- and extracellular functions have been identified that intimately tie this abundanr constituent of metazoan chromatin to autoimmune disorders and tumorigenesis. Whereas the roles of DEK in tumor biology arc most likely due to irs intracellular chromatin-modulating functions (e.g. control of hererechromatin integrity), functions in the pathogenesis of auto-immune disorders are more artributable to the extracellular activities of DEK (e.g. secretion of DEK from macrophages and role as a chemotactic factor in the extracellular milieu).
Here we now show that exogenous DEK can penerratc cells, transloeatc to the nucleus, and there carry out its bona fide endogenous nuclear functions. DEK internalizaeion is a heparan-sulfare-dependent process, and cellular uptake of DEK inte DEK knock-down cells cotrects the phenotypic aberrations characteri stic of these cells: global heterechromatin deplerion and DNA repair deficits. Additionally, adjacent cells can take up DEK secreted from synovial macrophages as seen in transwell culture systcms. These findings unify the extracellular and intracellular activities of DEK and suggest that this paracrine-like loop, which shuttles a protein vital for heterechromatin integrity in and out of cells, might represent an unexpecred new layer of epigenetic regulation.
Institute for Biochemistry and Molecular Biology, Medical School, R\X1TH Aachen University, 30 Pauwelsstrasse, Aachen, Germany 52074
Ferdinand Kappes [email protected]
25
Danube Conference on Epigenetics
2014 . November 19-21
Dynamics of transcription factor binding across liver development
Panagiota Karagiannil, Panagiotis Moulosl, Bianea M. Schmitt2, Dominique Schmidt2, Duncan T. Odom2 and lannis Talianidisl
l Biomedical Sciences Research Center Alexander F1eming, 16672 Vari, Greece
2University of Cambridge, Cancer Research UK Cambridge Institute, Robinson \Vay, Cambridge, CB2 ORE, UK
Liver development is characterízed by a functional switch from a major hemopoietic site in early embryonic stagcs to the main metabolic organ in posciaral life. This shift is accompanied by dramatic changes in the hepatic transcriptional programs. In the current study, we monitored gene expression across development in murine liver, using global transcriptome analysis by RNA sequencing in seven developmental stagcs. Consisrenr with the functional switch and in egrcement with other studies, we observe a large number of differentially expressed genes among developmental stagcs. To mechanistically address the regulation of these transcriptional program s, we performed genome-wide occupancy analysis of two major he pati c transcription factors, CjEBPa and HNF4a, by performing ChIP sequencing of these facrors across differenr developmental stagcs. Our analysis indi ca tes a very large number of promoters occupied by these rwo facrors. Interestingly, this occupancy is highly dynamic, characretized by gain and loss of binding sires during development. Furthermore, comparative analysis of binding with expression data reveals that CjEBPa and HNF4a occupancy does not always correlate with expression of their transcriptional targcrs. We propose that these two factors, in addition to functioning as typical transcription factors, recruiting [he transcriptional machinery, also have a bookmarking role. This bookmarking function allows the factor to bind specific loci and keep rhem in an open chromatin conformation, allowing for their transcriptional activarion at a later stagc.
BSRC ALEXANDER FLEMING
FLEMING STREET 34, 16672 VARI, GREECE
Panagiota Karagianni [email protected]
26
Danube Conference on Epigenetics
2014 . November 19-21
Selective upregulation of the expression of plasma membrane Ca2+ATPase isoforms upo n differentiation and 1,25-(OH)2-D3-vitamin treatment of colon can cer cells
1 Polett Ribiczey, Hungarian Academy of Sciences, Membrane Biology Research Group, Budapest, Hungary 2 Béla Papp, INSERM, UMR U978, Bobigny. France
3 László Homolya, Hungarian Academy of Sciences, Membrane Biology Research Group, Budapest, Hungary 4 Ágnes Enyedi, National Blood Center, Department of Molecular Cell Biology, Budapest, Hungary
5 Tünde Kovács, Semmelweis University, Department of Medical Biochemistry, Budapest, Hungary
Signal transduction pathways regulated pardy by cellular Ca2+ signaling control several cell functions including cell growth and differentiation. Plasma membrane Ca2+ATPases (PMCAs) are essential in the adjustmerit of the resting cytoplasmic Ca2+ concentration and in the regulation of global and dynamic Ca2+ signals.
Our previcus studies have shown that PMCA4b expression is upregulated during short chairi fatty acid (SCFA)~ induced differentiation of gastric and colon carcinoma cell lines, as weil as, following treatment with specific histone deacetylase (HDAC) inhibirors such as trichostatin A. Here, we show upregulation of PMCA4b expression upon post-confluency-induced differentiarion of both enterocyte- and goblet cell-type colon cancer ceils, while we could not detect marked changes in PMCAlb expression during this process. We also studied the effect of 1,25~(OH)2~D3 (Vit~D3) on the expression of the PMCAs and of selcered differenriarion markers in cntcrocytc-rypc Caco-Z colon cancer cells. In post-confluent culture s these cells are known to differentiate towards a normal enterocyte-Iike intestinal epithelial phenotype. Unlike in the differentiation induced changes in PMCA expression, Vit-D3 selectively stimulated PMCAlb expression, while having no effect on PMCA4b expression in preconfluent and early post-confluent Caco-Z cells. Vit-D3 also did not affect the expression of other differentiation markers. Similar data were obrained using DLD~l colon carcinoma cells. The PMCA~specific immunofluorescent signal, locared basolaterally, was significantly higher in Vir-Dő-rreáred cells, underlining the Vit-D3-induced upregulation of PMCA expression in differentiated Caco-Z cells. \Ve suggest that while PMCAlb has a housekeeping function in colon cancer cells, PMCA4b par ticipates in the reorganization of the Ca2+ signaling machinery during cell differentiation. The subceilular localizatien of PMCAlb and the selective Vit-D3- dependent upregulation of its expression indicare that this iso form may have a crucial role in Vit-Dő-stimulared intestinal Ca2+ absorption.
Semmelweis University
Department of Medical Biochemistry 1094 Budapest. TŰzoltó utca 37-47. Tünde Kovács, PhD [email protected]
27
Danube Conference on Epigenetics
2014 . November 19-21
DNA methylation analyses of the alternative promoter regions of the glucocorticoid receptor gene in infant stress reactivity
Emese Krukl, Krisztina Lakatos2, Lívia Kende Őzéné2, Boglárka Bekecs2, Zsófia Erzsébet Horváth 1, Ildikó Tóth2,Judit Gervai2, Zsófia Nemodal
1 Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
2 Institute of Cognitive Neuroscience and Psychology, Research Centre for Natural Sciences, Hurigarian Academy of Sciences, Budapest, Hungary
Inter-individual differences in stress reactiviry have been linked to genetic and epigenetic variations in the hypothalamic~pituitary-adrenal (HPA) system, especially in the glucocorticoid receptor (NR3Cl) gene. The finetuning of the HPA function during development has been in the center of stress-relared studies, giving strong support for biological mechanisms of gene~environment interaction. Following model-building animaI studies of maternal deprivation, findings from human srudies also indicate the importance of DNA methylation changes of the NR3Cl gene 1 F promoter region in response to childhood maltreatment and suboptimal parental care both in hippocampus brain region and peripheral tissues, such as leukocytes. In addition, neonatal leukocyte DNA from cord blood samples of mothers with depression or anxiery during pregnancy showed increased methylation of certain CpG-sites of the NR3Cl gene 1 F promoter. \Ve hypothesized that normal variation in maternal behaviour, especially anomalous maternal behaviour can have an influence on children's stress regulation, possibly by modifying the expression of key regulatory elements, such as the NR3Cl gene. Since saliva samples have been successfully used in adults to assess stress reactivity by measuring cortisollevels and to evaluate DNA methylation differences of the NR3Cl gene lF promoter, we used this non-invasive sampling method in 100 orie-year-old children in a mildly stress-evoking situation. Atypical matemal behaviour was assessed by the AMBIANCE coding scheme focusing on affective communication errors. Because the NR3Cl gene has multiple tissue-specific alternative promotcrs, and leukocytes and epithelial cells express only lB and lC, whereas the majority of the promoters (lB-G) are expressed in hippocampus brain region, we aimed to analyze the different alternative promoters in infant saliva samples. DNA methylation levels were measured by pyrosequencing of the lB, lC, and 1 F promoter regions.
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Emese Kruk [email protected]
28
Danube Conference on Epigenetics
2014 . November 19-21
DYNAMIC NATURE OF THE METHYLATION LANDSCAPE OF THE HEART
Paolo Kunderfranco
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Carolina Greco
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Roberre Papait
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Condorelli Gianluigi
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Cardiac hypertrophy is associared with significant transcriptional changes characretized by the re- expression of fetal genes. Epigenetic mechanisms play a key role in defining the gene expression profile without modifying the actual DNA sequence. Recently, DNA methylation has been found to play a role in gene transcription regulation during heart failure, indi ca ting that DNA methylation in the heart is a dynamic process. Therefore, we analyzed the genome-wide transcription and methylation/hydroxymethylation status of cardiomyocytes isola ted from embryonic, neonatal, adult and hypertrophic (1 week pressure-overloaded) mou se heart with RNA, MeDIP and hMeDIP (methylated/hydroxymethylated DNA immunoprecipitation) high-throughput sequencings. We found that genomic regions enriched in 5-hmC were profoundly dynamic: in particular, adulr cardiomyocytes were significantly enriched in 5-hm C at gene bodies, and hypertrophy was associared with a shifr of this mark to intergenie regions and repetitivé elements, recapitulating the landscape found in embryonic cardiomyocytes. We then assessed if there was a relationship between 5-hmC and gene transcription, as previously described for neurons. We found that the presence of 5-hmC at promoter regions was associated with low levels of transcription whereas the presence at gene bodies with high levels of transcription. Integrative analysis of previously generated ChIP-seq data revealed a genome-wide strong co-localization of 5-hmC with active histone marks. GO analysis of genes harboring 5-hmC uncovered distinct gene sets for biological processes, molecular function and KEGG signature in embryonic, neonatal, adulc and hypertrophic cardiomyocytes. Finally, differential analysis during the hypertrophic response revealed astrong enrichment of 5-hmC on a set of repetitive elements, which were also characterízed by loss of H3K9me3, and concained predicted binding site for cardiac specific TFs. Our study offers the first comprehensive analysis of 5-hmC in cardiomyocytes, and provides evidence that this epigenetic mark plays an important role in the heart under normal and stressed conditions.
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Paolo Kunderfranco [email protected]
29
Danube Conference on Epigenetics
2014 . November 19-21
SerpinBl0 (SPB10, Bomapin) participation in UVB stress response in keratinocytes
Zsuzsanna Újfalueli 1, Barbara N. Borsos 1, Imre Miklós Boros 1,2
1 Department of Biochemistry and Molecular Biology, University of Szeged, Faculty of Science and Informatics, Hungary
2 Institute of Biochemistry, Biological Research Center, Szeged, Hungary
Bomapin (SPBIO) is a serine protease inhibitor that promotcs cell proliferation under normaI circumstances in hematopoietic and myeloid leukaemia cells, while in the absence of growth facrcrs it can indu ce ápoprosis.
Based on these finding it seemed interesting wbecher in cancer evolving cells this protein has a gain of function effect. We have previously shown that SPB 10 mRNA level is incteased upon UVB treatment in keratinocyte Hker E6SFM cell line. To test the cell Line specificity and to learn abour the possible biological function of SPBiO induction upon UVB treatment we used immertalised keratinocyte (Hker E6SFM, HaCat) and melanoma (A375) cell lines respectively, As a first step we analysed the kinetics of induction by derermining the mRNA level in a time-depenclent manner (2h, 8h, 24h). Interestingly we found that SPBiO mRNA level increased 2 and especially 8 hours after UVB irradiation (80 mJ/cm2) while 24h after the treatment the mRNA decreásed in all examined cell lines. Accordingly Bomapin seems to be involved in the UVB induced stress response. We also investigated the mRNA level of SPBiO upon lower level (20 mJ/cm2) UVB irradiation in primer keratinocyte cell line and found that the gene expression was also increased. The elevared mRNA level resulted in higher SPBiO protein level in a time dependent manner (2h, 8h, 24h) in Hker cells, but nuclear localizanon was not influenced by UVB treatment. Our preliminary data suggest that the SBPiO protein could be a player in the UVB induced stress response. As a forrher step we plan to test whecher other stress signals play a role effect SPBiO mRNA level as well. Additionally we plan to investigate the function of SPBiO and its possible role in DNA damage signallieg pathway in keratinocytes.
This work was supported by grants from TÁMOP-4.2.2-08/1-2008-0001, and TÁMOP-4.2.2.A-ll/l/KONV- 2012-0035.
Department of Biochemistry and Molecular Biology, University of Szeged, Faculry of Science and Informatics, Hungary
Hajnalka Majoros [email protected]
30
Danube Conference on Epigenetics
2014 . November 19-21
Promoter analysis of high molecular weight glutenin genes of wheat indicates separate regulatory mechanisms
Szabolcs Makai, Csaba Éva. László TAMÁS, Angélajuhász
An analysis of the promoter regions of wheat HM\V-GS genes and their differenr alleles arc presented and correlated to their respective expression data measured in silico. Expressions of transcription factors involved in the regulation of Glu-Al, -B'l and -Dl genes were computationally analysed. Promoter regions of HMW-GS genes are highly conserved, ver their motif compositions, polymorphisms and motif occurrences arc specific to genes ra ther than to alleles. Promoter differences are reflected in the corresponding expression dynamics. The most distincr promoter s belong to Glu-l Bx genes that are always highly expressed and miss a few binding sites present in ali other HMW-GS genes. Interesting finding that HM\V-GS promoters can be divided into 1-200 bp long cis regulatory modul es. Results show that at leasr two distinct mechanisms arc involved in HMW-GS transcription regulation that controls the two paralogs of HMW-GS genes. The in silico expression analysis of involved transcription facrcrs show that trauscript level of rbese facrors vary by genotypes and this may largely account for the variance express ion of the same alleles ac ross genotypes. In order to study the function of the identifled promoter modules, a biolistic transient expression system was adapred for leaves of two species, bread wheat (cv. Chinese Spring) and Brachypodium dstachyon. Bombardment with a construct containing a constitutive prornoter-driven uidA reporter gene resulted in numerous blue spots after hisrochemical staining. The studv of HM\V promotcrs containing truncated modules is under way.
Agricultural Research Institute of Hungarian Academy of Science
Szabolcs Makai [email protected]
31
Danube Conference on Epigenetics
2014 . November 19-21
PKC-mediated phosphorylation of BCL11B Serine 2 residue negatively regula te s its interaction with the NuRD complexes during human CD4+ T cell activation.
Marion DUBUISSEZI, Ingrid LOISONI, Han VORNG2, Olivier ROHR3, Anne TSICOPOULOS2 and Dominique LEPRlNCEl
(I) Institut de Biologie de Lille, CNRS UMR8161. (2) CIIL INSERM UlOI9, Institut PASTEUR de Lille, Université Lille Nord de France, Lille, France
(3) Institut de Parasitologie et de Pathologie Tropicale, EA7292, Universiré de Strasbourg, 67000 Strasbourg, France.
The transcription factor BCLI1B, also named CTIP2, is a major protein iruplicated in various aspects of development, function and survival of T cells. BCLI1B is regulated by post-translational modiflcations (PTMs) such as SUMOylation and phosphorylation in murine thymocytes. These PTMs medulate the transcriptional activity of BCLI1B switching it from a transcriptional repressor in inactivated thymocytes to a transcriptional activator in activated thymocytes. Indeed, BCLllB is phosphorylated on several Serine residues by MAPK and then SUMOylated to recruit the coactivator P300 to activare Id2 transcription.
Here, we showed that BCLllB is able to interact via its conserved N-terminal MSRRKQ motif with endogenous MTAl and MTA3 proreins to recruit various NuRO complexes. Furthermore, PKC-induced phosphorylation of BCLllB Serine 2 regulates NuRD recruitment by dampening the interaction with MTA proreins. A newly obcained phosphospecific antibody, BCLllB pSer2, derecred an increased phosphorylation of BCLllB in vivo upon anti-C03jC028 activarion of human C04+ T cells. In addition, through RT-gPCR and ChIP analyses, we showed that BCLllB represses transcription of the IL2 and Id2 genes with the NuRD complexes in nonactiváted C04+ T cells. FoHowing activation of C04+ T cells, BCLllB activates transcription of IL2 and Id2 by recruiting P300.
In conclusion, our results add a new stcp in the inrerconnecred PTMs switching BCLllB from irs repressor function to its activátor function. Firsdy, BCLllB is phosphorylated on Serine 2 by PKC. Then, BCLllB is SUMOylated to recruit P300 and to activate IL2 and Id2 transcription. Finally, two mechanisms allow stopping IL2 and Id2 aerivation : transcriptional repression of BCLllB express ion and dégradarion of BCLllB proreins by [he proteasomc.
These results provide the first evidence that phosphorylation of BCLllB Serine 2 is essential for derepression of IL2 and Id2, two key BCL1IB target-gcries during CD4+ T~cell activarion.
Institut de Biologie de Lille, CNRS UMR8161, Universiré Lille Nord de France, Lille, France
Oubuissez Marion [email protected]
32
Danube Conference on Epigenetics
2014 . November 19-21
Differentially methylated fetal cfDNA marker identification from maternal plasma in Down-syndrome pregnancies
Maros-Szabó Zsl, Batta ZI, Jámbrik KI, Domokos BI, Galamb Á2 l Larkbio Kft, Debrecen, Hungary
22nd Department of Obstetrics and Gynaecology, Semmelweis University, Budapest, Hungary
DNA methylation pattem differs significantly between individuals especially between the mother and the ferus. \Vith the identification of chromosome specific methylation markers that arc uniquely found in the fe tus but not in the mother, numerical chromosome aberrations are derecrable.
Our aim was to detect trisomy 21 from maternal plasma investigating the differential methylation patterns of the mother and the foerus. MedIP-Seq was performed on pooled cfDNA extracred from the plasma of healthy nonpregnant women, from healthy umbilical cord blood plasma and from DNA of healthy umbilical cord enelothel cells. Methylation patterns were examined and regions that are uniquely methylated in the ferus were selecred as potential markers. A set of markers together with control regions (identicaIly methylated in mcther and ferus) were than tested with CJPCR in plasma of healthy pregnant women and in plasma from women carrying ferus with Down-syndrome. Variations in copy number values revealed chromosome trisomy.
A number of 125 chromosome 21-specific regions were identified where fetal methylation was overrepresented. Altogether 11 marker primer and 4 control primer pairs (chr 20) were designed and tested in CJPCR following MedIP. Out of these, 7 pairs worked as desired and a combination of 3 primer pairs yielded the most sensitive and specific results.
Our investigation proved that fetal numerical chromosome aberrations are derectable from maternal plasma using differentially methylated dDNA regions. \Vith MedIP and subsequent qPCR we were able to distinguish healthy and affecred plasma on a relatively low number of samples (9 affected and matched control samples, respectively). The application of the merhod in clinical setting needs to be further verified.
Larkbio kfr
Maros-Szabó Zs [email protected]
33
Danube Conference on Epigenetics
2014 . November 19-21
SYSTEMATIC, ROBUST, REVERSIBLE DNA METHYLATION PRECEDE SPORADIC, RANDOM MUTATIONS IN COLORECTAL ADENOMADYSPLASIA-CANCER DEVELOPMENT
Bela Molnarl, 2, Balint Peterfia 1, 2, Alexandra Kalmarl, 2, Barnabas Wichmann 1, 2, Arpad V. Patai 1, Zsolt Tulassayl,2
INTRODUCTION: Colorectal adenoma-dysplasia-cancer (AD-eRe) is characterised by sequential mutations of certain genes, methylation of selected genes occurs in high frequency in early can cer stagcs. \Ve aimed to evalate the role of epigenetic and genetic alterations in AD-eRe development.
METHODS: DNA was isolated from biopsies (20 normals; 33 adenomas; 17 CRCs). A multiplex PCR panel was designed to amplify mutation hot spots of 12 genes (e.g. APC, BRAF, EGFR), these were sequenced by GS Junior instrument. Methylation analysis of 94 genes was performed on Human Colon Caneer EpiTect Merhyl II Signature peR Array. Whole genome expression analysis was performed with HGU133plus2.0 microarrays from 49 normal, 49 adenoma and 49 CRC speeimens and on 5-Aza rreated HT29 cells. Immunohistochemistry was performed using tissue microarrays.
RESULTS: Mutations were found in 76% of adenomas and 78% of [he CRCs. Average number of mutations was 1; 1,8; 1,9 and 2,3 in low grade AD, high grade AD, carcinomas and serrared AD, respectively, The APC suppressor gene was mutated in adenomas more frequently than in carcinomas (36% vs. 24%). The most frequently mutated genes were APC, TP53 and KRAS wi th 36%, 18% and 26% frequencies in adenomas and 24%, 47% and 45% frequencies in carcinomas. DNA methylation of a gene set (e.g. SFRP1, MAL, SST) was found in 100 % of the investigated colorectal adenoma and cancer specimens. Ariother set of genes (DKKl, SLI3, TMEFF2) was hypermethylated in adenomas and cancers in >75% of the cases. In adenomas 56 genes, in dysplasias 40 in cancer 37 genes were methylated. Decreásed mRNA and protein expression could be resrored by demethylation treatment.
CONCLUSION: Methylation in early premalignant stagcs were followed by increasing number of soma tic mutations along AD-CRe. Demethylation treatment reversed the systematic methylation alterations. Epigenetic alterations precede somatic mutations of AD-CRC and show higher significance in CRC development than genetic.
1 - 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary
2 - Molecular Medicine Research Unit, Hurigarian Academy of Sciences, Budapest, Hungary
Béla Molnár [email protected]
34
Danube Conference on Epigenetics
2014 . November 19-21
A novel method to predict regulatory regions based on histone mark landscapes
Gergely Nagy l Bence Dániel l László Nagy 1,2 Endre Barta 1,2
1. Department of Biochemistry and Molecular Biology, University of Debrecen, Faculry of Medicine, Debrecen, Hungary
2. MTA-DE "Lendület" Immunogenomics Research Group, University of Debrecen, Faculry of Medicine, Debrecen, Hungary
Macrophages as phagocytes and professional antigen presenting cells play eritical roles in both innace and adaprive immunity. Main transcription facrcrs ac ting during their differentiation and function are known, but the behavior and co-operation of these facrors still remained unexplored. We introduce a new approach [Q map nucleosomefree regions using exclusively active enhancer and core promoter marking histone modification ChIP-seq data. We could detect approximately 56,000 potential active enhancers/promoters showing different lengths and histone modification shapes. Beside the highly enriched PU.1 and C/EBP sites, we could also detect binding sites for RUNX and AP-I, as weil as for the MiT (MITF-TFE) family and MEF2 proteins. The PU.I and C/EBP transcription factots are known for trans forming cells inte macrophages. The other transcription facrors found in this study can play a role in macrophages as weil, since it is known that the MiT family proreins are responsible for phagocytic activity and the MEF2 protems specify monocytic differentiation over the granulocyte direction. Our results imply that this method can provide novel information abour transcription factor organization at enhancers and core promoters as weil as abour the histone modifications surrounding regulatory regions in any immune or other cell types.
Department of Biochemistry and Molecular Biology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary
Gergely N agy [email protected]
35
Danube Conference on Epigenetics
2014 . November 19-21
Characteristic and verified microRNA expression patterns in colorectal adenomacarcinoma sequence
Zsófia Brigitta Nagyi, Barnabás Wichmann2, Alexandra Kalmárl, Barbara Kinga Barták l , Nha Lel, Bálint Péterfia2, István Füril, Zsolt TulassayZ, Béla Molnár2
miRNA expression alterations can be observed in colorectal cancer (eRe), however dysregulation of miRNA might be present in various stagcs, such as adenoma. High-throughput screening platforms became available recently:miRNA microarrays, RT-gPCR panels with hundreds of miRNA specific oligos.
Our aim was to identify the miRNA expression alterations berween normal colenie tissue (N), tubular adenoma (ADT), tubulovillous adenoma (ADTV) and eRe samples. Another purpose was to derermine the level of miRNA by different methods and in several types of samples such as fresh frozen, FFPET.
Six t Y fresh frozen biopsy samples (N=20 N. ADT=II, ADTV=9,CRC=20) were collected; and, total RNA were isola ted. A miRNA microarray experiment was perforrned. Then, a series of pools of RNA from the same groups of samples was made to validace the data of microarray by RT~qPCR. Then, RT~qPCR data of FFPET tissues (N=3, ADTV=3, CRC=3) were compared to the microarray and PCR results from fresh-frozen samples. miRNA-mRNA interactions were predicted based on algorithms and validated by biopsy mRNA microarray data. miRNA-126 was selected to be visualized by in situ hybridization.
Out of the 1733, the derectable miRNAs, was a small percentage (N=442, AD=460, CRC=441). 12 miRNA were upregulated and 11 miRNA were downregulated only in neoplastic lesions (AD+CRC) compared to N. 11 miRNA showed altered expression berween ADT and ADTV Expression levels of 9 miRNA were found [Q be changed berween ADT, TV and CRC groups based on microarray data. MiRNA expression data could be confirrned by RT-PCR in both FFPE and FF samples. miRNA-126 arrayexpression results could be confirrned by in situ hybridization. MAP3Kl were identified as targcts of miR-196a in MAPK signaling pathway.
A smaIl number miRNA showed alteration during neoplastic development, but their impact is remarkable and systematic elemenrof the upstream mRNA pathways. The identified miRNA expression changes arc reproducible in FFPE tissues as well.
1 2nd Department of Internal Medicine, Semmelweis University, Budapest 2Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest
Nagy Zsófia Brigitta [email protected]
36
Danube Conference on Epigenetics
2014 . November 19-21
GLUT10 facilitates dehydroascorbic acid uptake in the endoplasmic reticulum: lessons from arteria] tortuosity syndrome
Csilla E. Némcth l , Paola Marcolongo2, Alessandra Gamberucci2, Rosella Fukcri2, Angiolo Benedetti.Z, Nicoletra Zoppi3, Marco Ritclli3, Nicola Chiarelli3, Marina Colombi3, Andy \\Iillacrt4, Paul J. Coucke4, Péter LŐwS, Pál Gróf6, Szilvia Nagyl, Tamás Mészáros l , Éva Margittai7, Gábor l
1 Department of Medical Chemistry, Molecular Biology and Parhobiochcmistry, Semmelweis University, Budapest, Hungary. 20cpartment of Molccular and Developmental Medicine, University of Siena, Siena, ltaly.
3Division of Biology and Genetics, Department of Molecular and Translational Medicine, Medical Faculty, University of Brescia, Brcscia, ltaly.
4Center for Medical Genetics, Ghent University, Ghenr, Belgium.
50cpartment of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary. 6Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
7lnstitute of Human Physiology and Clinical Expcrirucntal Research, Semmelweis University, Budapest, Hungary.
Ascorbare (AA) is a cofactor for iron/2-oxoglutarate dependent dioxygenases, which are locared in the nucleoplasm and in the endoplasmic reticulum (ER) lumen and have been shown to participate in the epigenetic regulation of gene expression via histone and DNA demethylation. Thus, transport of AA or its oxidized form dehydroascorbare (D HA) inte these compartments is required for epigenetic regulation. Although DHA transport in the ER membrane has been demonstrated, the molecular mechanism has not been elucidared.
Recerit findings verified that mutations in the gene SLC2A 10 encoding glucose transporter 10 (GLUTI0) are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder. However, neither the subcellular localizatien nor the transported molecule of GLUTI0 has been identified. \Ve report here that GLUTI0 is aDHA transporter in the ER and nuclear envelope (NE). Transport measurements in cells whose plasma membrane was selectively permeabilized showed that D HA transport and accumulation was markedly reduced in fibroblasrs from ATS patients and in GLUTI0 silenced immortalized human fibroblasrs. Reexpression of GLUTI0 in patients' fibroblasrs restcred DHA transport activiry, Measurement of DHA uptake in subcellular fractions of fibroblasrs showed that ER transport was reduced in patien ts. GLUTI0 protein produced by in vitro translation and incorporated inte liposomes efficiently transported DHA. Long-term incubation in the presence of AA res ul ted in a twofold higher eteady-state intracellular AA concentration in control fibroblasrs. Transmission electron microscopy with immunogold staining revealed that AA taken up was mainly loealized to nucleoplasm.
Our data demonstrace that GLUTIO facilitares DHA entry into the ER lumen and probably to the nucleoplasm; the missing function of AA as a cofacror for dioxygenases in these compartments can be a decisive factor of ATS pathomechanism. Further studies on DNA/histone demethylation are in progress.
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
Csilla Németh [email protected]
37
Danube Conference on Epigenetics
2014 . November 19-21
Interrogating heritable changes of chromatin-associated molecular phenotypes in human lymphoblastoid trios
Lilla Ozgyin l. Attila Horvath 2. Dora Bujcsuk 1. Endre Barta 2.
Balint Laszlo Balint 1.
Proper regulation and fine-runing of gene expression at chromatin level plays a key role in health and disease. Alrerarions in transcription factor regulatory networks and associated molecular phenotypes such as histone modifications and chromatin state at cis-regulatorv elements could guide our attention to genomic sites that may contribute to complex diseases. Geneme- Wide Association Studies have idenrified numerous multifactorial disease-associared Single Nucleotide Polimorphisms (SNPs) and Copy Number Variations (CNVs) using casecontrol design that fall into intergenie regions. These are likely to concern regulatory regions (rSNPs) and some of them are themselves referred to as expression Quantitative Trait Loci (eQTLs) indicating their role in gene express ion regulation. DNA methylation, apartly transgenerationally inheritable epigenetic modification can also be causative to gene express ion changes by abolishing TF binding to their purative binding sires (fFBSs).
In our srudies, we aim to identify and characterize heritable genetic variation- or DNA methylation-associated changes in transcription factor binding, the correlation of these changes with histone modifications and gene expressional outcome. At first, we reanalysed ENCODE ChIP-seq data from CEU trios to identify CTCF and PAXS binding sites in B-lymphoblastoid cell lines and then we used ANOVA and DiffBind analyses to identify significant TFBS occupancy ditferences among individuals. We next scanned these selected CTCF or PAXS binding regions for motif-disrupting variations using 1000 Genomes Project's personalized genotypes and also investigated methylation events that might underlie the loss or gain of TF binding ability. Based on these in silico results, we validated some sires of differential CTCF binding using ChIP-gPCR and we plan to válidate merhylation data by sire-specific High Resolutin Melting analysis (HRNl).
1.University of Debrecen, Faculty of Medicine, Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatics Core Faciliry
Lilla Ozgyin [email protected]
38
Danube Conference on Epigenetics
2014 . November 19-21
Genetic and epigenetic risk factors in Tourette Syndrome
Luca Pagliarolil,2, Andrea Vereczkeil, Tamas Aranyi2 and Csaba Barta l
Introduction
The aim of this work is the identification of candida te genes involved in the pathogenesis of Tourette Syndrome (TS) and possibly also iruplicated in obsessive compulsive disorder (OCO) and arreurion deficit hyperactivity disorder (ADHD); the selection of miRNAs regula ting the expression of those genes; the study of genetic variants in the candiclate genes involved in this regulation, thus identifying possible targcts in the pathomechanism of TS.
Methods
Starting from the list of alI candidarc genes for TS we cross-referenced berween the set of genes that were also iruplicated in OCD and ADHD. Then, we searched miRNAs targeting rhese candidace genes (n=30) seeking for a correlation between data form differenr darabases.
Results
\Ve were able to track down a list of candidace miRNAs. First, we compared and contrasted the target miRNAs from the differenr darabases to identify a short-list for each gene. Second, we looked for a connection berween miRNAs targeting different genes within our list. Third, we identified some miRNAs (eg. mir-200, mir-374, mir- 429) in our top ranking gene set as well as some others with the capacity to regulate the highest number of genes in our total gene set (such as mir-520, mir-302). Finally, we searched for genetic variation in regulatory sequences of these gen es.
Discussion
The lack of information and the dissonance between the available darabases required certain adjustments and a personal elaboration of the data in order to esrablish a credible list of genes and miRNAs for subsequent functional assays. This project impjemented a combined and systematic approach to identify plausible candida te gene-miRNA pairs and genetic variants in 3'- UTR regulatory regions for forrher functional studies in order to shed some light on the pathophysiology of TS.
1 Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
luca pagliaroli [email protected]
39
Danube Conference on Epigenetics
2014 . November 19-21
Interaction of linker histone H1 with the nucleosome
Corinne Crucifix(I),Jan Bednar(2), Imtiaz Lone(3). Dimitar Angelov(3), Stefan Dimitrov(2). Patrick Schultz(l) l-Institute of Generies and Molecular and Cellular Biology (IGBMC), IlIkirch, France
2~]oseph Fourier University, Grenoble, France
3-Université de Lyon, Lyon, France
The genetic information is packed inte chromatin and the level of compaction is modulared by a number of protein facrcrs and epigenetic modifications that influence the accessibiliry of the DNA. A major player in the regulation of chromatin compaction is the linker histone H 1 family, which binds to the nucleosome core particle (Nep) only in the presence of linker DNA, which connects to successive nucleosomes. The Nep is composed of 147 bp of DNA wrapped around ahistone octamer formed by two copies of each core histones H2A, H2B, H3 and H4. Nucleosomes arrays form loose and exrended chromatin fibers which can be further compácted inte 30 nm fibers. Histone Hl is a tripartire protein composed of an evolutionary conserved globular core domain and two variable and highly charged C- and N-terminal unstructured flanking regions. DNA footprinting analysis showed that Hl protccts nucleosomal DNA close to the dyad axis and also binds to the linker DNA. Lowresolution electron microscopy showed [hat Hl modifies the angle berween the entry and the exit linker DNA, which are brought into close contact. This slight movement is believed to be the central mechanisms driving chromatin compaction. To analyze in more details the mode of interaction of the linker histone with DNA highresolution structural information is needed and several laboratories in the world try to co-crystallize Hl with NCP. Co-crysrals were obrained in several labs but the density of the bound Hl remains elusive. Here we present high-resolution cryo electron microscopy data showing [he location of histone Hl wirhin the nucleosome and highlighting irs dynamic rcarrangcmcnt, a possible cause for its phantom behavior in X-ray crystallography.
Institute of Generies and Molecular and Cellular Biology (IGBMC), IlIkirch, France
Gabor Papai [email protected]
40
Danube Conference on Epigenetics
2014 . November 19-21
GENETIC AND EPIGENETIC ANALYSIS OF COLORECTAL SERRATED POLYPS REVEALS A COMMON DNA METHYLATION SIGNATURE IN SESSILE SERRTAED ADENOMAS AND HYPERPLASTIC POLYPS
Árpád V. Patai l , Barbara Kinga Bartákl, Bálint Péterfial,2, Tamás Micsik3, Alexandra Kalmárl,2, Réka Horváthl, Árpád Patai4, Zsolt Tulassayl,2, Béla Molnárl,2
Introducrion: Serrared polyps include neoplastic sessile serrared adenomas (SSA), traditional serrated adenomas (TSA) and non-neoplastic hyperplastic polyps (HP). SSA and TSA arc hypothetized to be precursor lesions of an alternative pathway of colorectal cancer, abundant in genes with aberrant promoter hypermetylation.
Aims and methods: Out primary aim was to study 12 selecred gene mutations (APC, BRAF, CTNNBl, EGFR, FBXW7, KRAS, MSH6, NRAS, PIK3CA, SMAD2, SMAD4, TP53) and promoter methylation status of 96 genes in serrared polyps. Pathological records were searched for "serrated" and "hyperplastic polyps" in the archive of Ist Department of Pathology, Semmelweis University, Budapest (between 2011 and 2013). Hyperplasric polyps under 5 mm in size and locared in the sigma and rectum were excluded. DNA was isolated and 10 samples (4 SSA, 1 TSA and 5 HP) were chosen for forther analysis. Amplicons for gene mutations were sequenced by a GS Junior Instrument (Roche). DNA methylation percentages of 96 genes were deremuned using Methyl-Profiler PCR array system (Qiagen). 5 samples from normal colonic mucosa were used as control.
Results: Analysis of DNA methylation revealed 9 methylated genes (BAGE, CCNAI, H19, MAGEAI, MGXI, PTGIS, RUNX3, SPARC, UGT1AI) in both normal and serrared samples. 12 genes (BNCI, DKK2, GALR2, OPCML, PCDHIO, PDUM4, SFRPI, SFRP2, SUT3, TACl, VIM, WIFI) were hypermethylated in 7 serrated polyps and 1 gene (SST) were hypermethylated in 9 serrared polyps, but in none of the norma! samples. TSA exhibited only 2 hypermethylated genes (CALCA, SST) versus normal mucosa. There was no significant molecular difference observed between SSA and HP. 2 SSAs exhibired BRAF mutation. Conclusions: Our combined mutation and DNA methlyation analysis revealed a common DNA merhylation signature present in SSAs and HPs, but not in TSA. Further investigation is needed to better characterize the molecular background of this newly recognized colorectallesions.
1- 2nd Department of Internal Medicine, Semmelweis University, Budapest
2- Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest
3- l st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest Patai V. Árpád
[email protected]
41
Danube Conference on Epigenetics
2014 . November 19-21
Assessing the role of newly identified regulatory and effector molecules in models of skeletal muscle regeneration and atrophy
Andreas Parsales 1, Tamas Varga 1, Konstantina Lyroni 2, Gergely Nagy 1, Attila Pap l, Petros Tzerpos 3, Attila Horvárh 1, Bence Daniel I, Szilard Políska 1, Balint L. Balint 1, Christos Tsatsanis 2, Balazs Oezso 5, Benediere Chazaud 6, Charalambos G. Spilianakis 3,4, Lászlo Nagy 1,7,8.
Affiliations:
(1) Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4032, Hungary. (2) Department of Clinical Chemisrry, School of Medicine, University of Crcre, Herakliori 71003, Crere, Greece.
(3) Institute of Mclecular Biology and Biotechnology, Foundation for Research and Technology-Hellas.
(4) Department of Biology, University of Crcre, Heraklion, Greece.
(5) Department of Pathology, Faculrv of Medicine, University of Debrecen, Nagyerdei Kn. 98, Debrecen 4032, Hungary. (6) INSERJ\·I, U1016, Institut Cochin, 75014 Paris, France.
(7) Sanford-Burnham Medical Research Institute, Orlando, FL 32827, USA.
(8) i'vITA-DE "Lendület" Immunogenomics Research Group, University of Debrecen, Debrecen, Hungary
Macrophages are necessaty for skeleral muscle regeneration after injury and protcet against muscle atrophy while promoting muscle recovery. Hindlimb unloading and reloading arc characterízed by a major loss of muscle force and are associared with c1assic leukocyte infiltration during recevery from muscle atrophy. Muscle recruits inflammatory monocytes/macrophages that switch roward an anti-inflammatory profile upon phagocytosis of debris. In vitro, pro-inflammatory macrophages stimulate myoblasr proliferarion, wbereas anti-inflammatory macrophages stimulate their differentiation. Thus, macrophages are involved in both phases of skeletal muscle regeneration: first, inflammation and c1eansing of necrosis, and then myogenic differentiation and tissue repair.
By studying macrophage gene regulation in inflammation and tissue regeneration and by expression profiling inflammatory tissue macrophages we identified novel regulatory and effecror molecules. The contribution of these molecules is being evaluated using full body and macrophage-specific knock-outs and the dynamics of skeletal muscle regeneration were derermined using histology and morphometry.
Cistromic and epigenetic changes might also reveal the méchanism behind regulated expression changes. Therefore in parallel we are also looking at altered macrophage-specific gene expression changes in muscle clerived macrophages using genomic approaches. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seC)) offers high resolution, genome-wide analysis of DNA-protein interactions. Nevertheless, current standard merhods require abundanr starting material in the range of 10-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisitiori of biologically relevant epigenetic data. Here by utilizing a ChIP-seCJ protocol optimízed for low cell numbers (down to 20,000 cells/IP), we assessed the performance of the ChIP-seCJ technique on a series of decreasing cell numbers. The optimized method presented here considerably reduces the input requirements for performing ChIP-seCJ. It exrends the applicability of the technique to isolated primary cells and rare cell populations (e.g. hindlimb unloading/reloading induced macrophages, bio bank human samples etc.), and in many cases it is expected to Iessen the need for cell culture and any related epigenetic changes associared with it. In addition, this study highlights a challenge characteristic to ChIP-seCJ from low cell numbers: as cell input numbers decrease, levels of unmapped sequence reads and PCRgenerated duplicare reads increase. We discuss a number of solutions to overcome the effects of reducing cell number that may help to further improve ChIP performance.
University of Debrecen
Andreas Patsales [email protected]
42
Danube Conference on Epigenetics
2014 . November 19-21
Effect of methylation on the nanomechanics of dsDNA
Csaba L Pongor
Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
Pasquale Bianco
Laboratory of Physiology, BIO cj O Department of Physics, University of Florence, Florence ,Italy
Miklós Kellermayer
Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
In its physiological environment DNA is constantly exposed to mechanical stress. The nanomechanical properties of DNA influence not only its response to stress but also its interaction with proreins. Despite its crucial role in epigenetics, little is known about how methylation affects the nanomechanical properties DNA. To investigate the impact of methylation on DNA nanomechanics, here we marupulated single molecules of chemically or enzymatically methylated DNA and compared their properties with those of the non-methylated. As model molecule we used a 3312-base-pair long sequence of lambda-phage DNA that met the criteria of a CpG island. Chemically methylated DNA was prepared with PCR contairung 5-methyl-CTP in the reaction mixture. For enzymatic methylation M.SssI methyltransferase was used. Single DNA molecules were mechanically manipulated with a force-measuring optical tweezers instrument in repeated stretch-relaxation cycles. The equilibrium shape of surface-adsorbed DNA molecules was measured by using atomic force microscopy (AFM). We found that the molecular con tour length, bending rigidity and intrinsic stiffness were significantly greater in methylated DNA compared with the unmethylated form, indicating that methylation leads to both structural and nanomechanical alterations. Furthermore, the cooperative overstretch transition was significantly longer in the methylated form of the molecule, suggesting that the dynamics of intramolecular rcarrangcmcnts are also affected. AFM measurements of DNA molecules adsorbed to mica surface substantiated the significant reduction of molecular con tour length in methylated DNA. By contrast, the apparent bending rigidity of the surface-adsorbed methylated DNA was increased, which is most likely caused by interactions between DNA and the mica surface. In sum, merhylation leads to an axial compaction of dsDNA structure combined with increased bending flexibilityand elasricity in the low-force, entropic-enthalpic regime. Conceivably, modulation of DNA structure and nanomechanics caused by methylation leads to a complex control of structural accessibility and association kinetics of DNA-binding proreins.
Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
Csaba István Pongor [email protected]
43
Danube Conference on Epigenetics
2014 . November 19-21
Proteomic interrogation reveals wide-spread functions of the chromatin associated DEK oncogene
Christian Preisinger
IZKF Aachen
Faculty of Medicine R\X1TH Aachen University Aachen
Germany
Alexander von Kriegsheim Sysrems Biology Ireland Conway Institute University College Dublin Ireland
Christinne Becker
DEK is an abundant and unique non-histone chromosomal factor, which plays multiple roles in DNA-dependent as weil as in DNA-independent cellular processes. Additionally, DEK has been identified as a bona fide oncogene with mounting data showing that high DEK level correspond with poor progriosis in a number of hard-ro-trear tumors. On a molecular level, DEK has vital functions in the maintenance of the repressive epigenetic mark H3K9Me3, which in rurn provides a rational of how DEK might function in rumorigenesis, e.g. aberranr silencing of tumor suppressor genes upon DEK overexpression. However, the preci se molecular functions of DEK, and in particulat its role in tumorigenesis, are only poorly investigated. In order to gain deeper insight inro its multifacered biology, we sought to deci ph er interaction partners and post transcriptional modifications (PTMs) of this unique nuclear factor. For this purpose a Localisatien and Affinity Purification (LAP)-tag has been eloned either N- or C-terminally to DEK and was inducibly expressed in the HEK 293 Flp-In T-REx cell system. Subsequenr immunoprecipitation regimens using a GFP-trap were used to purify DEK-interacting facrcrs which were subjecred to Le-MS/MS analyses using a Q Exactive. In total ~400 specific interactions facrors were identified wi th roles in various cellular processes, e.g. ribosome biogenesis, mRNA processing, transcriptional regulation, nucleolar biology, and chromatin architeemre and remedeling.
Taken together, we used an unbiased proteomic approach to identify DEK-specific interaction partners and PTMs, confirmed known as weil as novel interaction partncrs, and validated some newly found inreracrions partners for their biological relevance in DEK function. This study will certainly help in better understanding the pro-tumorigenic function of this protein, and will evenrually lead to novel srrarcgics in tumor therapy that target this biochemically distinct nuclear factor.
Institute for Biochemistry and Molecular Biology Medical School
RWTH Aachen University Malte Prell [email protected]
44
Danube Conference on Epigenetics
2014 . November 19-21
Prognostic significance of gene expression and DNA methylation analysis of selected DNA repair genes in bladder cancer using artificial neural approach
A. Wojtczyk ~ Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland p. Schlichtholz - Institute of Oceanology, Polish Academy of Sciences, Sopor, Poland
M. Presler - Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
A. Mirowska, - Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland J. Michajlowski - Department of Urology, Medical University of Gdansk, Gdansk, Poland
M. Matuszewski - Department of Urology, Medical University of Gdansk, Gdansk, Poland
B. Schlichtholz - Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
Epigenetic inactivation of DNA repair genes in caneer has been reported for several DNA repair pathways including BER, NER, and MMR. It remains to be derermined which genes have the highest prognostic significance.
The aim of the study was to examine the expression and DNA methylation status of the selecred genes mainly from DNA repair systcms in the transitional cell carcinoma (fCC) of urinary bladder, and investigate its prognostic relevance using artificial neural approach. This study evaluared gene expression status for four selecred DNA repair genes (MBD4, TDG. MLHI, MLH3) and promoter methylarion status for MGMT, MLHI, and proapóproric DAP-kinase. In addition, DNMT1 and HOXA5 mRNA expression was derermined.
A total of 60 patiems with TCC were evaluated. Real-time PCR was used to examine the gene expression in tumor and adjacent non-cancer tissue samples. The frequenev of abetrarit methylation was assessed with merhylation specific PCR (MS P) or COBRA method.
The relative level of MBD4, MlH1 and MlH3 mRNA was decreásed in 40%, 42% and 41% of tumor samples, respectively while the remaining samples showed lower or no difference in expression. No significant changes were observed in case of TDG mRNA level. The increased level of DNMT1 mRNA was observed in 38% of tumor samples and HOXA5 mRNA in 46%. The overall methylation frequenev in tumor samples was 19% for MLH1, 34% for MGMT, and 45% for DAPK. Moreover, a set of three genes capable of aceuratelv predicting the outcome of bladder cancer patients was selcered using an artificial neural network bioinformatics approach with a median prediction accuracy of 90%. This srudy showed that our selected genes and artificial neural nerwork can be successfully applied to the prediction of outcome in bladder cancer with a reasonably high performance.
Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
Schlichtholz [email protected]
45
Danube Conference on Epigenetics
2014 . November 19-21
Factors Affecting the Severity of Sickle Cell Anaemia
Marthew F. Shannon (1), Swee Lay Thein (2) and RebeccaJ. Oakey (1). (1 )Department of Medical & Molecular Generies
(2)Depattment of Molecular Haematology
King's College London. London UK.
Extreme variation in disease severity is observed between Sickle Cell Anaemia (SCA) patients. In mild case s patients live largely unaffecred live s, wbereas in severe cases patients can experience multiple strokés and organ failure during childhood. Some genetic facrors have been associated with this variation, these known facrors do not account for all of the variation observed, and we hypothesise that there are additional genetic modifiers involved. Additionally, discordancy has been observed berween monozygotic twins, leading us to hypothesise that epigenetic variation also plays a role in mediating SCA phenorvpe severiry. This project aims to identify novel genetic and epigenetic modifiers of SCA. \Ve will also investigate the epigenetic impact of treatrnent with hydroxyurea, a cytotoxic agent that has been shown to increase foeral haemoglobin levels in SCA pati en ts. Hydroxyurea is the most commonly prescribed therapy for SCA, Its mechanism of action is not fully understood, and we hypothesise [hat it indirectly affects DNA methylation. Using whole exome sequencing of severe and mild SCA patients we will identify novel modifier genes and variants, which will be validated in a collection of over 400 DNA samples from SCA patients with a broad range of phenotypic severity. Top candidates will be investigated by modelling their function through the use of CRISPR genomic edi ting. Using an in vitro culturing technique, nucleared erythroid progenitors arc expanded from peripheral blood, facilitating investigation of gene expression and epigenetic marks in [he SCA affected cell [)'pe. This technique will be used to iriterrogate differences in gene express ion berween monozygotic twins discordant for SCA phenotypic severiry, Erythroid progenitors sampled from SCA patients prior to treatment with hydroxyurea, and after 6 months, will be assayed for gene expression and DNA methylation patterns, and changes as a result of treatment will be studied forther.
King's College London
Matthew Shannon [email protected]
46
Danube Conference on Epigenetics
2014 . November 19-21
Epigenetic regulation of retinoic acid dependent embryonic stem cell differentiation
Zoltan Simandil, Attila Horvath l , Ixchelt Cuaranta-Monroy l , Erik Czipal, László Imrel, Gábor Szaból, Endre Barta l , Sascha Sauer2, Bálint L.Bálintl and Laszlo NagyI,2
1 University of Debrecen, 2Max Planck Institute for Molecular Genetics, 3Sanford-Burnham Medical Research Institute
Retinoids are morphogens and have been iruplicated in cell fare commitment of embryonic stem cells (ESCs) to neurons. Their effects are media ted by RAR and RXR nuclear receptors. However, early transcriptional and epigenetic events resulting in cell-type specific gene activation or repression is less understcod.
Comprehensive genome-wide studies were carried out to derermine how RAR:RXR targcts arc defined in early stem cell differentiarion. We provide data how co-activator P300 and co-repressor SMRT and HDAC3 are involved in chromatin remodeling. Moreover we show two novel co-factors, Protein aRginine Methyl Transferase (PRMT) 1 and 8, which also play key roles in determining retinoid regulared gene expression and cellular specification in a multistage neuronal differentiation of murine ESCs. PRNlTl acts as a selective modulator, providing the cells with a mechanism to reduce the porcncy of retinoid signals on regulatory "horspors". PRNlT8 is a retinoid receptor target gene itself and acts as a cell type specific transcriptional co-activator of retinoid signaling at later stagcs of differentiation. Lack of either of them leads to reduced nuclear arginine methylation, dysregulated neuronal gene expression and altered neuronal activity.
Overall, we show how weIl esrablished and novel co-regulators control key transcriptional events of stem cell differentiation.
Funding: Scienrific Research Fund (OTKA K100196 and OTKA F68254), TÁMOP422_2012_0023 VÉDELEM, Hungarian Brain Research Program _ Grant No. KTIA_13_NAP-A-Ij9.
Department of Biochemistry and Molecular Biology, University of Debrecen
Zoltan Simandi [email protected]
47
Danube Conference on Epigenetics
2014 . November 19-21
RUNX3 gene methylation and protein expression downregulation is associated with glioma progression
Skinute D, Vaitkiené P, Sreponaitis G., Simanavióius E., Mikuóiűnas M., Kazlauskas A.
Introducrion. Gliomas are the most common and aggressive among primary malignant brain tumors with significant heterogeneity in histology, molecular profile and patient outcome. However, molecular targcts that could provide reliable diagnostic and prognostic information on this type of cancer are currently unknown. Recent studies show that certain phenotypes of gliomas such as malignancy, resistance to therapy and relapses are associated with the epigenetic alterations of tumor-specific genes. Runt-related transcription factor 3 (RUNX3) is a canelidate tumor suppressor in a variery of tumors, its inactivation due to methylation is related to carcinogenesis. RUNX3 was shown to harbor frequerit tumor-specific promoter methylation in glioblasromas. Aim. The aim of the study was to identify RUNX3 promoter methylation and protein express ion in gliomas, and to estimate association between methylation/ expression alteration, malignancy grade and patient clinical characteristics.
Methods. The methylation status and protein expression levels of RUNX3 were measured by methylation-specific PCR and Western blot in differenr malignancy grade glioma tissues.
Results. It was shown that RUNX3 was frequently methylated and downregulated in glioblasromas. RUNX3 gene methylation was derecred in 77.3% of glioblasromas (n=49) as corripared to 0-11.4% in low grade gliomas (n=87). Decreased protein expression was observed in 48.6% of glioblastomas (n=26), as corripared to 11.4-22.9% of low grade gliomas (n=61). RUNX3 gene methylation did not correlated with protein levels in gliomas. Survival analysis showed that gene methylation and protein expression downregulation were associated with shorter patient survival.
Conclusions. The results demonstrarc that RUNX3 gene methylation and protein expression downregulation are glioma malignancy dependent and contribute to tumor progression.
Laboratory of Neurooncology and Genetícs, Neuroscience Institute, Lithuanian University of Health Sciences, Eiveniu str. 4, Kaunas, LT 50009, Lithuania
Daina Skinute [email protected]
48
Danube Conference on Epigenetics
2014 . November 19-21
DNA hypomethylation in tumors leads to the activation of oncogenic microRNAs
Axelle Loriot, Aurélie Van Tongelen, Jordi Blanco, Charles De Smet*
Group of Genetics and Epigenetics, de Duve Institute, Universiré carholique de Louvain, Brussels, Belgium
DNA methylation patterns of ten become altered in cancer cells. Alterariens include hypermethylation of selecred prcmotcrs, leading to silencing of eritical genes such as tumor suppressor genes, and hypomethylation of numerous other DNA sequences. We have shown that genome hypomethylation in tumors results in the activation of a group of germline-specific genes, which use printarily DNA methylation for repression in soma tic tissues. These genes, which were originally discovered because their activation in tumors leads to the expression of tumor-specific antigens, were named cancer-germline (CG) genes. Recently, we identified a novel CG transcript (CT-GABRA3) displaying DNA hypomethylation-dependent activarion in various tumors, including melanoma (65%) and Jung carcinoma (40%). Importandy, CT-GABRA3 harbors a microRNA (miR-lOS), which was recently identified as a promoter of cancer metastasis by irs abiliry to weaken vascular endorhelial barriers following exosomal secretion. CT-GABRA3 also carries a microRNA (miR-767), which we found to target TET1, a tumor suppressor gene exerting chromatin regulatory functions. Together our studies reveal that DNA hypomethylation contributes to tumor progression via activation of CG-type micoRNAs with oncogenic potennal. Moreover, we anticipate that, by targeting TET1, one of these microRNA can lead to further epigenetic dysregulation of tumor genomes.
Group of Genetics and Epigenetics, de Duve Institute, Universiré carholique de Louvain, Brussels, Belgium
Charles De Smer [email protected]
49
Danube Conference on Epigenetics
2014 . November 19-21
Daniel Srockholm 1,2,3, Tamas Aranyi 1,2,4,Thibaut Wiart 2,Anne Galy 1,2, Andras Paldi 1,2,3
1/UMR951 2/Genethon 3/EPHE 4/UEVE
lbis, rue de l'Intetnationale, 9100 Evry, France
The Illumina 450k Merhylation Assay measures the average methylation level of 450000 selecred CpGs that represent 99% of human genes and CpG islands of the genome. Traditional methods of analysis are based on the idenrificarions of methylation changes that are higher than a fixed threshold in order to distinguish signal from the experimentál noise. Insread of this approach, we used 3 parametérs to assess the difference between the control and experimental replicates. The first parameter is based on our "double average technique'' (DAT). DAT aliows the identification of very smali, but coordinared differences between rwo series of samples without a fix threshold. The second parameter quantifies the level of clustering of the identified CpGs according to their genomic localization. The third parameter describes the exrent of DNA methylation (bera-value) changes. These parametérs are used to classify different treatments. Consenred Methylated CpGs (CM-CpGs) present in ali members of the same class arc then identified. This pipeline can lead to the genome wide identification of a significant number of highly conserved CpG methylation changes despite a small extent of methylation differences.
Genethon
Daniel Srockholm [email protected]
50
Danube Conference on Epigenetics
2014 . November 19-21
The effect of histone modifications on nucleosome stabilityand the benefit of pannicking
László Imre1, Zoltán Simándi2, Attila Horváth2, László Halász l , György FenyŐfalvi1, Péter Nánásil, András Szántó l , Hiroshi Kimura3, László Nagy2, Lóránt Székvölgyil and Gábor Szabó l
l Department of Biophysics and Cell Biology, University of Debrecen,2Department of Biochemistry and Molecular Biology, University of Debrecen, 3Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
The effect of various posttranslational histone tail modifications (P'I'Ms) on nucleosome stabiliry was compared by exposing agarose embedded nuclei to treatments with salt or intercalater dyes, determining the remaining fracrion of hisrones using PTM specific antibodies and laser scanning cytomctry Sreep elution profiles could be measured in nuclei of all phases of the ceil cyde by both salt and intercalator treatment in the case of H3K4me3 and H3K27ac marks, while the nudeosomes carrying a number of differenr other marks were relatively resistant, similarly to bulk histone-Of-P Destabilization of the H3K4me3 marked TSS proximal nudeosomes was uniform along the genome, as revealed by chip sequencing, when doxorubicin was used as the intercalater. Nicking treatments of the nuclei did not affect the stability of nucleosomes carrying H3K4me3 or H3K27ac, while those of the second group were ail destabilized. To interpret these results we suggest that the H3K4me3 and H3K27ac active marks specify dynamic nudeosomes accomodating already relaxed DNA sequences, while most other nucleosomes hold the DNA in constrained superhelices. In accordance with this hypothesis, endogeneous nicks were mapped by chip sequencing in the vicinity of active promotcrs of human peripheral blood lymphocytes as weil as mouse embryonic stem ceils. In nuclear halos, two topologically isolated chromatin domains were demonstrared in ail phases of the cell-cyde, superhelical loops and the nuclear lamina enclosed compartment harboring nicks. Importantly, the latter domain accomodates the sites of in vivo nu deo side analogue incotporation upon trauscription as well as replication. These observarions lend support for a model where the role of transient nicks in transcriprional regulation and higher-order chromatin organization are integrated. Support: OTKA 72762,101337, Fulbright fellowship (G.Sz), TÁMOP 4.2.2.A-II/I/KONV-2012-0023, TÁMOP 4.2.4. A/2-I 1-1-2012-0001
Dept. Biophysics and Cell Biology, University of Debrecen
Gabar Szabo [email protected]
51
Danube Conference on Epigenetics
2014 . November 19-21
A novel cancer-germline transcript carrying pro-metastatic miR-10S and TETtargeting miR-767 induced by DNA hypomethylation in tumors
Axelle Loriot; Group of Genetics and Epigenetics; de Duve Institute; Universiré Carholique de Louvain; Brussels, Belgium
]ordi Blanco; Group of Genetics and Epigenetics; de Duve Institute; Université Carholique de Louvain; Brussels, Belgium
Current affiliation: Physiology Unit; School of Medicine; Universitat Revira i Virgili; Reus, Spain
Simon Klaessens; Group of Generies and Epigenetics; de Duve Institute; Universiré Carholique de Louvain; Brussels, Belgium
Julie Cannuyer; Group of Genetics and Epigenetics; de Duve Institute; Universiré Carholique de Louvain; Brussels, Belgium
Nicolas van Baren: Ludwig- Institute for Caneer Research Ltd: Centre du Cancer des Cliniaues: Universitaíres Genome hypomethylation is a common epigenetic alteration in human tumors, where it of ten lead s to aberranr activation of a group of germline~specific genes, commonly referred to as "cancer-ger mline'' genes. The cellular functions and tumor promoting potential of these genes remain, however, largely uncertain. Here, we report identification of a novel cancer-germline transcript (CT-GABRA3) dispJaying DNA hypomethylation-dependent activation in various tumors, including melanoma and Jung carcinoma. Importantly, CT-GABRA3 harbors a microRNA (miR-lOS), which has recently been idenrified as a promoter of cancer metastasis by its abiliry to weaken vascular endochelial barriers following exosomal secretion. CT-GABRA3 also carries a microRNA (miR- 767) with predicted target sires in TETI and TET3, [wo members of the ten-eleven-translocation family of tumor suppressor genes, which are invoJved in the conversion of S-methylcytosines to S-hydroxymethylcytosines (ShmC) in DNA. Decreased TET activity is a hallmark of cancer; here, we provide evidence that aberranr activation of miR-767 contributes to this phenomenon. We demonstrace that miR-767 represses TETI/3 mRNA and protein expression and regula tes genomic ShmC levels. Additionally, we show that high CT-GABRA3 transcription correlates with reduced TETI mRNA levels in vivo in Jung tumors. Together, our study identified a cancer-germline gene that produces microRNAs with oncogenic potential. Moreover, our data indicare that DNA hypomethylation in tumors can contribute to reduced ShmC levels via aerivation of a TET~targeting microRNA.
Group of Genetics and Epigenetics; de Duve Institute; Universiré Carholique de Louvain; Brussels, Belgium
Aurélie Van Tongelen [email protected]
52
Danube Conference on Epigenetics
2014 . November 19-21
METHYLATED SEPTIN 9 DETECTION IN TISSUE AND PLASMA OF COLORECTAL NEOPLASIA AND THE RELATIONSHIP TO THE AMOUNT OF CIRCULATING CELL-FREE DNA
Kinga Tóth" 1, Reinhold \Vasserkort2, Ferenc Siposl, Alexandra Kalmárl, 3, Barnabás \Vichmannl, Katalin Leiszter l , Gábor Valczl, MátkJuhászl, Pál Miheller l , Árpád V. Patail, Zsolt Tulassayl , 3, Béla Molnár!, 3
INTRODUCTION:Methylated Seprin 9 was evaluated as aspecific biomarker for colorectal cancer in plasma samples. However, it hasn't been investigated how methylated DNA derecred in plasma relares to the occurrence of methylated DNA in colon tissue.
AIMS, METHODS:Aim of this study was to quantitatively compare levels of methylated SEPT9 in matched plasma and tissue samples of healthy, adenoma and eRe case s; and to derermine the amount of circulating free DNA (dDNA) and the expression of Septin-9 protein in tissue. Plasma and matching biopsy samples were colleeted from 24 patients with no evidence of disease (NED), 26 adenomas and 34 CRe. RT-PCR assay was used [Q derermine the amount of DNA in samples and the portion of DNA rnerhylared at a locus of SEPT9 after bisulfire conversion. Septin-9 protein expression was determined using immunohistochemistry
RESULTS:ln tissue samples, percent of methylated reference (PMR) values of SEPT9 above a selcered threshold of 1% were detecred in 4.2% of NED, 100% of adenoma and 97.1% of CRe. PMR differences were found significant (p<O.OOl) between NED vs. adenoma and NED vs. eRe. In plasma samples SEPT9 PMR values, using 0.01% cut-off level were derecred in 8.3% of NED, 30.8% of adenoma and 88.2% of CRC cases. Significant PMR differences were observed in comparisons berween NED vs. CRC (p<O.OI) and adenoma vs. eRe (p<O.Ol). Significant differences (p<O.Ol) in the amou nt of cfDNA were found berween NED and eRe and amodest correlation was observed between mSEPT9 concentration and cfDNA in plasma of cancer patients (R2=0.48).
Protein expression of Septin-9 in tissues derermined by IHC was inversely correlated to SEPT9 methylation levels.
CONCLUSION:Methylated SEPT9 was derecred in healthy tissues only at low levels, but significantly elevated in adenoma and CRC tissues. In plasma samples, elevared mSEPT9 values were derecred in CRC, but not in adenomas. Tissue levels of mSEPT9 alone aren't sufficient [Q predicr mSEPT9 levels in plasma.
l2nd Department of Internal Medicine. SEMMELWEIS UNIVERSITY, Budapest, Hungary,
2Extracorporeal Immune Modulation Unit, Fraunhofer Institute of Cell Therapy and Immunology, Rostock, Germany
Tóth Kinga
[email protected]
53
Danube Conference on Epigenetics
2014 . November 19-21
UVB induces a major genome-wide rearrangement of RNA polymerase II at transcribed human genes
Ákos Gyenisl, David Umlauf 1, Zsuzsanna Ujfaludi 3, Imre Boros 3, Tao Ye 2, László Tora 1
1 Cellular signaling and nuclear dynamics program, 2 Microarrays and deep sequencing platform, Institut de Génétigue et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104 CNRS, UdS, INSERM U964, BP 10142, F-67404 ILLKIRCH Cedex, CU de Strasbourg, France.
3 University of Szeged, Faculty of Sciences and Informatics, Department of Biochemistry and Molecular Biology. Középfasor 52, H6726, Szeged, Hungary
Transcription of DNA is continuously disrurbed by damaged DNA triggered by various genotoxic effects from endogenous and also environmental sources. Transcription coupled repair (fCR) has been described to take part in the rapid restoration of blocked transcription by elimination of DNA lesions from the transcribed strand of active gen es. However, the mechanism of TCR in individual target genes has been well srudied, the precise global mechanism by which the action of RNA polymerase II (pol II) transcription is regulated foUowing UVB irradiation during the DNA repair processes is still not well understcod.
ln order to srudy the effect of UV on Pol II transcription we neared MCF7 human celis with the non-lethal dose of UVB and we accessed the DNA~bound Pol II distribution. We found that about 90% of the promoters of expressed genes showed reduced Pol II occupancy 2-4 hours foUowing UVB irradiation, which was resrored to "norma}" or higher levels 5-6 hours after the treatment. Interestingly, we found a smaller set of the activé genes, where the enrichment of Pol II was not decreásed after UVB irradiation at the promoter regions, but increased throughout the entire transcription unit. \Ve also observed that promotcrs, wbere Pol II clearance occurred, the behaviour of TFIIH but not of TBP highly resembled that of Pol II suggesting that at these genes TFIIH might be sequestered for DNA repair upon UVB treatment.
ln conclusion, our study uncovers a global negative regulation of Pol II transcription initiation on the large majority of transcribed genes foUowing non-lethal UVB irradiation, with the exception of a small subset of genes (including regulators of repair, cell growth and survival), wbere Pol II escapes this negative regulation.
University of Szeged, Faculty of Sciences and Informatics, Department of Biochemistry and Molecular Biology, Középfasor 52, H6726, Szeged, Hungary
Zsuzsanna Új faludi [email protected]
54
Danube Conference on Epigenetics
2014 . November 19-21
Reliable ChIP-seq results with the Diagenode iDeal ChIP-seq kit for Transcription Factors and MicroPLEX library Preparation kit
Anne-Clémence Veillard, Jan Hendrickx, Irina Panteleeva, Heléne Pendeville, Miklos Lacz.ik, Céline Saba tel and Dominique Poncelet
DIAGENODE SA, LIEGE SCIENCE PARK / rue du Bois Sainr-Jean 3 / 4102 Seraing / Belgium
Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become the gold standard for whole-genome mapping of protein-DNA interactions. However, although ChIP-seC) is a powerful tool, the ChIP-seq method requires optimized protocels ensuring high recevery and increased signal-to-noise rario. This is even more important for the study of Iowabundant proreins or transcription facrors. The Diagenode iDea} ChIP-seq kit for Transcription Factors has been developped with fully optimized rcagcnts to enable sucessful ChIP on non histone proteins. Moreover, this kit has been thoroughly validated by Diagenode for ChIP-seC) on an Illumina sequencer. Actually, due to the small amounts of DNA recovered after ChIP with transcription facrcrs Diagenode has developed a library preparatien protocol for limited quantities of DNA. The MicroPlex Library Preparatien Kit requires only picogram amounts of ChIP'd DNA inputs for librárv preparation compatible with the Illumina® platfotms. In this postcr, we demonstrace the successful use of the Diagenode iDeal ChIP-seC) kit for Trauscription facrcrs in combination with the :MicroPLEX Library Preparation kir for Chlfc.seq.
Diagenode SA, LIEGE SCIENCE PARK / rue du Bois Saint-Jean 3 / 4102 Seraing / Belgium
Anne-Clémence Veillard [email protected]
55
Danube Conference on Epigenetics
2014 . November 19-21
Genome-wide epigenetic and transcriptional effects of ERI<J/2 activation in hepatocytes
Borbála VetŐl, Caroline Bacquet l , Attila Horváth2, Szabolcs Sipeki 3, Endre Barta2, Dávid ]ónás2, László Budayl, Bálint L. Bálint2, László Nagy 2, András Váradi 1 , Tamás Arányil
l Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences Budapest, Hungary
2Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen Debrecen, Hungary
3Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University Budapest, Hungary
Our previous results have shown that the expression of ABCC6 gene is downregulared by activation of the ERK 1/2 signaling pathway. Here we repott that ERKI/2 is capable of directly phosphorylating HNF4a (hepatocyte nuclear factor 4 alpha), a major regula tor of metabolic genes and ABCC6 in hepatocytes. Our ChIP-qPCR results in human HepG2 cells demonstrace that the decrease of HNF4a binding on regulatorv regions of the hepatic genes ABCC6, APOAI, BLVRA, BLVRB, HPD and PKLR happens upon rapid (30 mins) and 24 hours' activation of ERKI/l. \Ve also performed genome-wide analysis (ChIP-Seq) and observed that the number of genome-wide HNF4a binding sires is decreásed similarly to the binding in the target genes. In order to cor relate epigenetic modifications with transcription factor binding, we investigated the genome-wide H3K27ac profile in control and ERKI/2-activated cells. H3K27ac (acerylared lysine 27) is associared with active enhancers. The H3K27ac peaks were diminished genome-wide and at the tissue-specific genes, as well. These data suggest that HNF4a binding occurs in H3K27ac regions and the disappearance of the TF also lead s to the loss of H3K27acmarked open chromatin structure.
Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences Budapest, Hungary
VetŐ Borbála [email protected]
56
Danube Conference on Epigenetics
2014 . November 19-21
Functional characterization and gene expression profiling of Drosophila melanogaster short dADA2b isoform-containing dSAGA complexes
Nóra Zsindelyl, Tibor Pankotai 1 , Edith E. Vamos2, Orbán Komonyil, László Bodail and Imre M. Boros l , 2
1 Department of Biochemistry and Molecular Biology, University of Szeged, Kőzépfasor 52, H-ó72ó Szeged, HUNGARY
Zlnstitute of Biochemistry, Biological Research Center, Temesvári krt. 62, H-6726 Szeged, HUNGARY
Histone acetyltransferase (HAT) complexes play a role in chromatin structure modifications which might lead to changes in gene expression. The GeNS protein is the catalytic component of several multiprotein HAT complexes which modify chromatin structure by acetylating specific lysine residues at the N-terminal tails of histone H3 and H4. ADA2 proreins together with ADA3, SGF9 and GCNS form the acetyltransferase module of GeN 5-containing HAT complexes, which play roles in modulating HAT activiry and specificity of the complexes. In Drosophila OUf group has described two ADA2 proreins (dADA2a and dADA2b) in two GCNS-conraining HAT complexes, ATAC and dSAGA which have differenr histone specificiries. The dADA2b-containing dSAGA complex is involved in the acerylation of nucleosomal H3 at lysine (K)9 and K14. Furthermore, analysis of [he dAda2b gene revealed that by alternative splicing it gives rise to two mRNAs (dAda2bS and dAda2bL).
\Ve examined wberher [he two dSAGA specific dADA2b subunits could contribute to the functional complexity of dSAGA in transcription regulation. Here we prcscnt findings showing that during Drosophila development the two dADA2b isoforms, which differ in their C-terminal domains, arc expressed at various levels. Genetic complementation experiments indicare that dADA2bS alone can support development but cannot fully complement dAda2b mutations. In the presence of dADA2bS, the SAGA-specific H3 acetylation level is partially resrored in dAda2b mu tan ts. Comparison of whole transcriptome profil es of dAda2b null and dAda2bS transgene-carrier dAda2b nulllarvae indicates partialoverlap between affecred genes. mRNA levels corresponding to selected genes which are either up- or down-regulated in dAda2b mutants are altered by dADA2bS expression to differenr extents, ranging from cornplete restoration to no restoration at alI. The short isoform of dADA2b seems to be more capable of restoring los t dSAGA functions that cause mRNA level up-regulation than those that lead [Q decreásed mRNA levels.
These data support the hypothesis that different isoforms of dADA2b contribute to the functional variations of dSAGA multiprotein HAT complexes.
Department of Biochemistry and Molecular Biology, University of Szeged, Középfasor 52, H-ó72ó Szeged, HUNGARY
Nóra Zsindely [email protected]
57
Danube Conference on Epigenetics
2014 . November 19-21
Sequentia! Partitioning of Histone Methylation and Demethylation Activities Determines the Robustness of Natura! Transdifferentiation
Steven Zuryn,l Arnaud Ahier,l Manuela Portoso,2 Esther Redhouse \Vhite,l Marie-Cbarlotre Morin,l Raphaél Margueron.Z Sophie Jarriaultl
l Department of Development and Stem Cells, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104/INSERl\! U964, Universiré de Srrasbourg, 67404 Illkirch CU Srrasbourg, France. Zlnstitut Curie, INSERl\! U934, CNRS UMR3215, 26, Rue d'U1m, 75015 Paris, France.
Postmitotic somatic cellular identity is generally a stable feature of multicellular organisms. However, there are naturally occurring instances whereby cells can rransdifferentiare inro other cell types with disrincr functions. Documenred examples are rare, but in certain cases, extrémely precise and efficient reprogramming outcomes are observed, representing as vet unexploited avenues in which to probe the molecular mechanisms that ensure robust cell conversion. Using unbiased and then targeted genetic screens, we report that a conserved H3K27me3/me2 demethylase, ]MJD-3.1, and the H3K4 methyltransferase Set1 complex act to ensure invariant transdifferentiation of post-mitotic Caenorhabdiris elegans hindgut cells inte motor neurons. At single-cell resolution, we find that perfect conversion, particularly under stressful cellular conditions, requires [he precise organization of each histone modifying activiry into sequential, discrere phases of conversion. This functional and dynamic partitioning is achieved through a combination of active nuclear degradation of ]M]D-3.1 and separable modular interactions between each histone modifier and transcription factors that have conserved roles in cell pluripotency and terminal fare selection. Our results draw parállels berween epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.
Department of Development and Stem Cells, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104/INSERM U964, Universiré de Srrasbourg, 67404 Illkirch CU Srrasbourg, France.
Steven Zuryn [email protected]
58
