Detailed programme soon!
Confirmed Speakers
Alexander van Oudenaarden Hubrecht University, The Netherlands
Mario Nicodemi Universita' di Napoli "Federico II", Italy
Suzana Hadjur Cancer Institute, University College London, UK
Wouter de Laat Hubrecht University, The Netherlands
Amos Tanay Weizmann Institute, Israel
Leonie Ringrose Humbold University, Germany
Andrew Pospisilik Max Plank Institute for Immunobiology and Epigenetics, Germany
Iannis Talianidis Institute of Molecular Biology & Genetics, BSRC Al. Fleming, Greece
Ueli Schibler University of Geneva, Switzerland
Ferenc Muller University of Birmingham, UK
Fischer Tamas University of Heidelberg, Germany
Frye Michaela Wellcome Trust – MRC, Cambridge Stem Cell Institute, UK
Eric Miska Gurdon Institute University of Cambridge, UK
Erika Watson University of Cambridge, UK
Colot Vincent IBENS, France
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Detailed Agenda with Abstracts of the 2014 Conference
Wednesday 19. November
Location: HOTEL GELLERT, First Floor,TEA Room
16.00 Registration
18.00 Opening Lecture: Paolo Sassone-Corsi:
Common Threads: Epigenetics, Metabolism and the Clock
University of California, Irvine, introduced by László Tora
19.15 Welcome Dinner in the Gellert Hotel, First Floor: Duna and Gellért Rooms
Thursday, 20. November
Location: Research Center for Natural Sciences
8.30 Registration
9.00-10.30 Session 1,
Chair: Iannis Talianidis
Maxime Dahan, Laboratoire Physico-Chimie Curie Institut Curie, CNRS UMR168:
Probing the target search of DNA-binding proteins in mammalian cells, one molecule at a time
For many cellular functions, DNA-binding proteins (DBPs) need to find specific target sites in the genome. Facilitated diffusion (FD), namely the combination of one-dimensional motion along non-specific DNA and three-dimensional exploration, is the dominant model for the target search (TS) of DBPs. Yet, this model has hardly been tested in vivo, particularly in the complex environment of a mammalian nucleus, and it is still controversial whether it accelerates association to specific DNA binding sites. To address that question, we have implemented a TS assay using human cells with a unique target locus for an inducible exogenous searcher, the tetracycline repressor (TetR). Using single-molecule tracking and in situ biochemical measurements of association kinetics, we directly characterize the mobility of TetR, its transient interaction with non-cognate DNA and the kinetics of binding to the specific locus. Overall, we find that the searcher follows a FD strategy but that the search kinetics is not limited by the diffusive motion but by the low association efficiency to non-specific DNA sites. We will also discuss how these measurements can be extended to endogeneous DNA-binding proteins.
Ido Amit, Weizmann Institute:
Shaping the blood: Lessons from Chromatin and single cell RNA dynamics
Thirty years of dedicated research have enabled the categorization of functionally similar hematopoietic cells into a lineage tree by means of a combination of a small number of cell surface markers. Nevertheless, our understanding of the hematopoietic cell lineage remains coarse and biased, limited by preselecting of specific markers. A wealth of new studies highlight the crux of using cell populations for genome-wide measurements as even state-of-the-art classification approaches are limited and retain a heterogeneous population masking gene expression heterogeneity, uncharacterized cells and functions.
In order to understand in a comprehensive and unbiased approach the immune cell lineage and activity in vivo, we have developed an automated massively parallel RNA single cell sequencing approach (MARS-Seq) for measuring the genome wide expression of tens of thousands of single cells in their native context. Importantly, MARS-Seq enables transcriptome measurement of thousands of cells per experiment.
I will demonstrate how using this combined experimental and computational approach; we characterize the cell fate and activity of thousands of immune cells from mouse spleen in various physiological contexts, focusing of the dendritic cell (DC) lineage. We show that using our single cell approach we can build a high resolution ab initio immune identity map without the use of any predefined cell markers. Focusing on subpopulations of DCs, I will demonstrate that these DC populations include highly heterogeneous mixtures of transcriptional states that are only coarsely approximated using surface marker sorting and how these cells respond differently to stimulation. I will discuss how, our approach is greatly expanding the knowledge of transcription regulation heterogeneity and immune functional diversity beyond the limited numbers of cells/marker currently used to describe populations of immune cells. Finally, I will combine these results with unpublished work on chromatin regulatory maps of DC to connect between chromatin dynamics and gene expression heterogeneity.
Rickard Sandberg, Karolinska Institutet:
Single-cell RNA-seq reveals principles of allelic expression in mammalian cells
Analyses of gene expression across large numbers of single cells can reveal cell-to-cell variability. To this end, my lab has been developing single-cell RNA-seq methods (e.g. Smart-seq2) that combine full-length transcript coverage with high sensitivity and accuracy. We have performed global analyses of gene expression in hundreds of individual cells from mouse preimplantation embryos of mixed genetic background (CAST/EiJ x C57BL/6J) as well as in somatic cells. Using strain-specific SNPs we investigate allelic expression in these cells during preimplantation development to characterize X-chromosome imprinting and allelic expression patterns of autosomal genes. Although expression from both paternal alleles is generally observed in analyses of diploid cell populations, no study has addressed allelic expression patterns genome-wide in single cells. Importantly, we demonstrate that allelic expression is independent for genes across all expression levels making this a genomic principle. Additionally, our study revealed that allelic transcription fluctuations generated widespread random monoallelic expression in single cells, and we discuss its functional consequences. Finally, we explored to what extent random monoallelic expression was clonality propagated through cell division through single-cell transcriptome analyses of clonally related somatic cells.
10.30-11.00 Coffee Break
11.00-13.00 Session 2
Chair: Wendy Bickmore
Brian McStay, Centre for Chromosome Biology, Galway
From NORs to nucleoli and genomic stability of rDNA arrays in human cells
Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Nucleoli, sites of ribosome biogenesis and key regulators of cellular growth are the most prominent of these. They disappear during mitosis, reforming around prominent poorly characterized chromosomal features, nucleolar organizer regions (NORs) on human acrocentric chromosome short-arms. By examining the effects of depleting UBF, a nucleolar specific HMG box protein that binds extensively over rDNA chromatin, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Through construction of synthetic NORs and synthetic nucleoli we prove that nucleolar biogenesis is a staged process where UBF-dependent mitotic bookmarking precedes function dependent nucleolar assembly.
Isolation of repetitive, highly transcribed rDNA within the nucleolar interior prompted us to investigate the response of nucleoli to DNA double strand breaks (DSBs) within these essential genes. Targeted introduction of DSBs into rDNA, but not closely linked sequences, results in ATM-dependent inhibition of rDNA transcription by RNA polymerase I, coupled with withdrawal of rDNA from the nucleolar interior to NOR anchoring points at the periphery. This localized response renders rDNA accessible to the repair machinery. Most significantly, repair is carried out by the homologous recombination pathway, independent of cell cycle stage. These results suggest that rDNA repair can be templated by repeats in cis and point to a role for chromosomal context in maintenance of their genomic stability.
Claire Rougeulle, Univ Paris Diderot
Non-coding RNAs and X-inactivation plasticity in mammals
X chromosome inactivation (XCI) in mammals is an essential process which is developmentally regulated and tightly linked to the cellular context and to the potency of the cell. XCI is also a well-known example of large scale gene regulation controlled by long non-coding RNAs (lncRNAs). Xist, which coats the X chromosome from which it is expressed and induces its silencing, was one of the first lncRNA to be discovered, and its function in XCI has been well documented, at least in the mouse. Since the initial discovery of Xist more than 20 years ago, several additional lncRNAs have been proposed to participate to the regulation of XCI, but their exact contribution remains for the most part elusive. In addition, we know now that X-inactivation strategies are tremendously variable among mammals, and the extent to which lncRNAs contribute to this variation deserve specific attention. Several lncRNAs involved in XCI have orthologs in various mammalian species, but their function has not been addressed in species other than mouse; others are poorly conserved and may be restricted to a particular group of mammals.
We have recently identified a novel lncRNA, XACT, which appears to be specific to the humans or to closely related primates. XACT displays the unique property of coating the active X chromosome in humans, and its expression is restricted to early embryonic stages, in which XCI displays important plasticity. We will discuss possible function for XACT in controlling X chromosome activity in humans, and the divergence in XCI strategies in mammals.
Pantelis Hatzis, Fleming Institute, Athens
The long non-coding RNA WNTRLINC1 regulates transcription, stemness and carcinogenesis in the intestinal epithelium
The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation and proliferation in the intestinal epithelium. Mutations in pathway components APC, AXIN or β-catenin lead to aberrant transcriptional activity of the TCF4/β-catenin complex, the endpoint of the Wnt pathway, and are the primary transforming factor in colorectal cancer. Our research on Wnt-mediated transcriptional regulation has identified long intergenic non-coding RNAs (lincRNAs) as novel targets of the Wnt pathway. One of these, termed WNTRLINC1, positively regulates factors required for intestinal stem cell maintenance. It is required for continued proliferation of colorectal carcinoma cells and is significantly upregulated in colorectal cancer patient samples. It exerts its impacts by modulating the chromatin accessibility of transcription regulatory factors with which it interacts. WNTRLINC1 represents thus a new player involved in normal and abnormal intestine physiology and a putative novel diagnostic and therapeutic target in colorectal cancer.
Wendy Bickmore, University of Edinburgh
Transcription and nuclear organisation: chicken and egg
There are some well-established relationships between aspects of the spatial organisation of the nucleus and gene expression. Sequences at the nuclear periphery are often associated with the repression of transcription, the most active regions of genomes are often found in the centre of the nucleus, loci repressed by certain epigenetic pathways are found in a compact chromatin state. But who comes first: does structure direct function (transcription) or vice versa?
I will describe experiments that try to break the links between chromatin/nuclear organisation - especially at the nuclear periphery - and the act of transcription. By specifically perturbing either nuclear/chromatin organisation or transcription I will discuss the extent to which we can begin to understand the relationships between structure and function in the nucleus.
13.00- 14.30 Buffet Lunch and Poster Session
14.30-16.00 Session 3
Chair: Laszlo Nagy
Puri Pier Lorenzo, Sanford-Burnham Institute for Medical Research, La Jolla, CA. USA and Fondazione Santa Lucia. Rome, Italy.
Epigenetic networks regulating skeletal muscle regeneration and fibro-adipogenic degeneration in health and diseases.
The ability of skeletal muscle to repair upon acute injury by regeneration of new myofibers relies on functional interactions between different cell types, including muscle satellite stem cell (MuSCs) , fibro-adipogenic progenitors (FAPs) and cells from the inflammatory infiltrate (i.e. macrophages, eosinophils). In chronic degenerative disorders, such as muscular dystrophies, the integrity of this network is progressively compromised, leading to a switch from compensatory regeneration to an alternative, pathogenic repair by deposition of fibrotic and fatty material. We have shown that this switch is largely dependent on the phenotype adopted by FAPs, and have identified an HDAC-regulated network, by which microRNAs control the composition and activity of the SWI/SNF chromatin remodeling complex to direct chromatin remodeling toward two alternative - promyogenic or fibro-adipogenic - phenotypes of FAPs. This network provides the rationale for the ability of HDAC inhibitors to promote regeneration and prevent fibrosis and fat deposition in dystrophic muscles.
Laszlo Nagy, University of Debrecen and Sanford Burnham Institute
Cistromic and long-range interactions of lineage- and signal specific transcription factors integrate macrophage specification and control lipid signaling
Cellular differentiation and subtype specification is principally governed by lineage specific and signal specific transcription factors. Importantly, the interaction of these transcription factors should be interpreted in the context of a distinct cell-type specific genomic architecture. However the contribution of genomic interactions and the identity of enhancer networks regulating induced genetic programs are still very poorly understood and difficult to map. Here we show that the combination and bioinformatic integration of steady state mRNA levels, genome-wide localization (ChIP-Seq) of transcription factors, architectural elements, histone marks and nascent RNA production (GRO-Seq) allows for the unraveling of signal specific enhancer networks and the regulated target genes. We used macrophages and the paradigm of lipid-activated nuclear receptor signaling to identify the enhancer network operated by the liganded Retinoid X Receptor in the context of M0 and M2 macrophage activation states. The M0 macrophage RXR cistrome has at least 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features 387 liganded-RXR bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin limited functional domains. These findings were molecularly validated using 3C and 3C-Seq and we show that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded-RXR controlled enhancers and provides the macrophage with a novel inducible program. M2 macrophage polarization redistributes RXR and leads to the establishment of additional transcriptional including nuclear receptor signaling pathways.
Dirk Schübeler, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland:
Setting and reading DNA methylation
Recent advances generated genomic DNA methylation maps during cellular differentiation at unprecedented resolution. Combined with functional assays this revealed that dynamics in DNA methylation coincide with changes in regulatory activity and that transcription factors play an important role in shaping methylation patterns. This tightly links DNA methylation with underlying DNA sequence features and suggests that a substantial fraction of methylation changes occur downstream of gene regulation.
In order to create functional genomic binding maps of trans-acting factors involved in reading and writing DNA methylation we use a controlled biotin tagging approach that enables to identify protein domain contribution to chromosomal localization, a system utilized this to map all MBD domain proteins and a set of isoforms and disease causing mutants in order to better understand the readout of DNA methylation by this family of proteins (Baubec et al., Cell 2013).
In order to gain insights into pattern generation we determined the genome-wide distribution and locus-specific activity of the enzymes responsible for de novo DNA methylation in mammals. DNMT3A and B share general binding preference for the CG dinucleotide yet we observe additional distinct binding preferences. Bound regions are preferentially remethylated when reintroducing individual enzymes into cells that lack DNA methylation suggesting that recruitment is the primary level of guiding activity. De novo methylation is excluded from active regulatory regions and is directed to linker regions between positioned nucleosomes.
In case of DNMT3B preferential binding in stem cells occurs at actively transcribed genes and importantly is required to maintain genic hypermethylation. Mutational analysis shows that the PWWP domain is necessary for this targeting suggesting that lysine 36 of histone H3 links histone and DNA methylation at active genes. This work suggests that locus specific targeting of de novo methyltransferases is a highly dynamic process required for maintaining integrity of the methylome.
16.00-16.30 Coffee Break
16.30-18.30 Session 4
Chair: Béla Molnár
Tamas Aranyi, Institute of Enzimology, RCNS
Infection of human cells with lentiviral vector leads to highly reproducible genome-wide DNA methylation changes
Lentiviral vectors (LV) are efficient tools for gene transfer. They are frequently used as vectors in vivo and in vitro in laboratory experiments and clinical trials. After infection LVs integrate the genome and interact with the chromatin. It is therefore of major importance to investigate their effect on the DNA methylation of human cells. In the present study we tested in primary human haematopoietic and Jurkat cells the effect of infection of both wild-type and integration deficient LVs. We used the Infinium Illumina450K BeadChip assay and developed the double average technique (DAT) to detect DNA methylation changes. We demonstrated that independently of the capacity of vectors to integrate soon after infection a great number of CpGs undergo DNA methylation increase. These methylation changes revealed to be highly reproducible in preferentially targeted genomic regions. Interestingly, these regions are found in CpG islands of non-expressed genes. The consequences of these epimutations will be discussed.
Balint L Balint, University of Debrecen
Epigenetic tools for mapping chromatin modifications in clinical samples
Understanding the person-to-person variability in diseases by using the unprecedented amount of available genomic data may be approached as never before. We propose to investigate the impact of individual genetic variations in breast cancers. We performed a meta-analysis of all the publicly available datasets from different breast cancer end endometrial cancer cell lines. In order to improve the reproducibility of ER alpha ChIP in samples with low cell numbers we developed a nanoparticle based spike-in control. With this tool we can not only controll the ChIP reaction but also to measure the binding capacity of an antibody and to compare different ChIP protocolls in a more accurate way than before.
Attila Németh, Biochemistry Center Regensburg
Molecular mapping of nucleolus-associated chromosomal domain dynamics during cellular senescence
Nucleolus-associated chromosomal domains (NADs) were identified genome-wide in human HeLa cervix carcinoma (Németh et al., PLoS Genet 6, e1000889.) and HT1080 fibrosarcoma cells (van Koningsbruggen et al., Mol Biol Cell 21, 3735-3748.) providing a snapshot of genome organization around the nucleolus. Yet, questions about the dynamics of nucleolus-associated chromatin remained to be answered. Since large rearrangements in the nuclear and nucleolar architecture occur during cellular aging we decided to map NAD reorganization in this fundamental biological process. Nucleoli of IMR90 human lung embryonic fibroblasts were isolated from a young, proliferating cell population, as well as from senescent cells. Nucleolus-associated DNA was analyzed on high-resolution microarrays by comparative genomic hybridization. Additionally, RNA was extracted from the same cell populations and subjected to global gene expression analysis to compare genome-wide transcriptome and NAD profiles, further to identify chromatin regulators of cellular aging. The results of genomics analyses are currently validated in single cell 3D-immuno-FISH experiments. In summary, our investigations provide novel insights into the spatial and transcriptional dynamics of the nucleolus-associated genome during cellular senescence.
Judit Balog, Leiden University Medical Center
The role of chromatin repressor SMCHD1 in the development of human disease
Epigenetic screens in different organisms identified known and new epigenetic modifiers of chromatin stability and plasticity. SMCHD1 (Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1) was first identified in a screen for epigenetic modifiers involved in repeat mediated epigenetic repression in mice harboring a multicopy GFP transgene (Ashe et al. 2008). SMCHD1 is a non-canonical member of the SMC family of proteins shown to be involved in epigenetic repression of the inactive X chromosome and a few autosomal loci. Little is known about repeat mediated epigenetic repression in human cells, but we identified SMCHD1 mutations in a subgroup of patients (2%) affected by a rare muscular dystrophy, Facioscapulohumeral Muscular Dystrophy (FSHD) (Lemmers et al. 2012.). A key element in the pathomechanism of FSHD is the germline transcription factor DUX4 which is ectopically expressed in muscle of FSHD individuals but not of healthy controls, leading to cell death. The DUX4 open reading frame resides in the D4Z4 macrosatellite repeat unit, the building blocks of repeat arrays that can consist of up to 100 units at human chromosomes 4q and 10q. Several lines of evidence suggest that DUX4 expression is associated with an epigenetic derepression of the D4Z4 array in somatic cells, either by contraction of the D4Z4 repeat array to a size of 1-10 units (FSHD1) or by mutations in SMCHD1 (FSHD2). The aim of our study was to determine the epigenetic requirements of DUX4 expression in human primary myoblasts and differentiated myotubes. We found that DUX4 expression dynamically changes during myogenic differentiation, being highly increased in myotubes. A series of control and FSHD myoblasts and myotubes were measured for different epigenetic marks at the DUX4 promoter region, including DNA methylation, repressive and permissive histone modifications and the presence of SMCHD1. This, in combination with knock down experiments of D4Z4 chromatin modifiers, identified SMCHD1 as key repressor of DUX4 activity in skeletal muscle.
Ferenc Muller, University of Birmingham
Functional validation of disease associated human enhancer candidates using the zebrafish transgenic embryo as a scalable vertebrate model
Genome wide analyses such as ENCODE and FANTOM predicted a surprisingly large number of non- coding elements with suggested cis-regulatory function in mammals. However, functional validations of the candidate regulatory elements are mostly lacking due to limitations of transgenesis technologies and capacities. We have investigated the utility of the transgenic zebrafish embryo as a scalable in vivo vertebrate model to study the functionality of candidate human enhancers predicted by a combination of chromatin signatures (e.g. H3k4me1, H3K27 Ac, H2AZ), TF binding events and enhancer specific bidirectional transcription. These analyses indicated that despite the evolutionary distance between human and fish, 60% of the candidate enhancers exhibiting sequence conservation lead to reporter expression which recapitulates the expression patterns of either zebrafish or human genes associated with the candidate enhancers. For example, we have demonstrated the activity of enhancer candidates associated with Type 2 diabetes and fasting glycemia in the pancreatic islet of zebrafish larvae. Currently we are developing assays in order to improve the sensitivity of transgenesis tools so as to detect subtle changes in enhancer function caused by sequence variation (SNPs) associated with dysregulation in disease. To improve the reliability of zebrafish enhancer function assays, a targeted integration system mediated by PhiC31 integrase was developed with which we have demonstrated successful elimination of position effect variation commonly found in conventional, transposon-based transgenesis.
Tibor Pankotai, University of Szeged
Mechanistic insights into the transcriptional arrest in the presence of Double Strand Breaks
Double-strand breaks (DSBs) occur frequently in the genome during genome replication or by DNA damaging agents. DNA lesions affect fundamental DNA-dependent nuclear processes such as replication and transcription. We have developed an experimental system where DSBs are induced at coding regions of RNA polymerase II transcribing genes. We have started to study the kinetics of RNA polymerase II transcription inhibition in the presence of DNA breaks. We observed that induction of the break led to transcription inhibition and the restoration of transcription closely followed the dynamics of the repair of breaks. We confirmed by chromatin-immunoprecipitation that the break induction led to displacement of RNA polymerase II affecting both the elongation and the initiation of transcription. Our results show that this is dependent on one of the major kinase in DNA damage repair called DNAPKcs. We also investigated the downstream steps of RNA polymerase II removal and we claimed that it was a multistep process involving additional kinases and ubiquitin ligases NEDD4 and CUL3. At the last step of break dependent transcriptional silencing the RNA polymerase II is targeted for proteasome dependent degradation. These data demonstrate that the DNA damage repair complexes and proteasomal system have a synergistic and active role in transcriptional silencing during the DSB repair by removing the RNA pol II from the transcribing region. We show here that DNA lesions occurring at transcribed regions cause a transient repression until the lesion is repaired. This is probably a cell defense mechanism to avoid production of truncated or mutated transcripts in essential genes whose alterations in their gene expression would endanger cell viability. Understudying the role of DNAPKcs, in preventing RNA pol II bypassing a DSB might be a key in avoiding the production of mutated transcripts that could lead to cancerous phenotypes.
18.35-18.45 Demonstration of world’s fastest qPCR system xxpress (40 PCR cycles in 10 minutes)
18.45- 20.00 Wine and Cheese and Posters
Friday 21. November
Location: Research Center for Natural Sciences
9.00-10.30 Session 5
Chair: Imre Boros
Laszlo Tora, IGBMC, Strassbourg
The SAGA coactivator complex acts on the whole transcribed genome and is required for all RNA polymerase II transcription
The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.
Frank Holstege: Deciphering regulatory circuitry by genome-wide analyses
Iannis Talianidis, Fleming Institute, Athens
Epigenetic mechanisms regulating liver development and function
Histone or DNA modifications are considered as major determinants of epigenetic information for chromatin-templated processes such as transcription. In the liver, similar to other organs, specific histone modification patterns correlate with gene activity and represent important means of regulation of epigenetic states characteristic to different developmental stages, metabolic states or diseases.
However, histone or DNA modifications are not the only epigenetic signals that are responsible for the establishment, maintenance and reversal of metastable transcriptional states during liver development and various metabolic conditions. Trans-epigenetic signals, which eventually accumulate into complex gene networks and gradually emerging “promoter-marking” signals, that determine the potential of gene activation are equally important in the establishment of self-propagating transcription states.
The contribution of the above epigenetic mechanisms in the regulation of liver development and function will be discussed.
10.30-11.00 Coffee Break
11.00-13.00 Session 6
Chair: Petra Hajkova
Erica Watson, University of Cambridge
The transgenerational epigenetic effects of abnormal folate metabolism
Epigenetic changes or ‘epimutations’ accrued in the genome throughout a lifetime in response to environmental stressors may contribute to an increased risk for disease. If an epimutation occurs within the germline, it might be inherited by subsequent generations resulting in developmental defects and disease in their progeny. To study the mechanism of transgenerational epigenetic inheritance, we use a mouse model with a mutation in a key gene involved in folate metabolism (Mtrrgt) that disrupts the folate cycle. Folate is a vitamin important for the one-carbon metabolism and therefore it is necessary for the methylation of cell components (e.g., DNA). Highly controlled genetic pedigrees were used to study the specific effects of the Mtrrgt genotype in either maternal grandparent and revealed developmental abnormalities and widespread epigenetic instability in their grandprogeny at midgestation. This occurred even when the mother and the grandprogeny were genetically wildtype for Mtrr. Some of the abnormalities (e.g., neural tube, heart and placental defects) persisted for up to five generations and after embryo transfer experiments suggesting that the effect was independent of the maternal environment. Together, these data suggest that folate deficiency in humans may lead to transgenerational epigenetic inheritance of disease.
Piroska E. Szabó, Van Andel Institute, Grand Rapids
Epigenetic remodeling between generations
Epigenetic marks are faithfully propagated during cell divisions in the soma. However, the epigenome is thoroughly remodeled between subsequent generations. This is achieved by global remodeling events that affect DNA methylation and chromatin composition during the soma-germline and germline-soma transitions. To understand what dictates the pattern of de novo DNA methylation in the male germ line during soma-germline transition, we mapped DNA methylation, chromatin, and transcription changes in purified fetal mouse germ cells. Our results suggest that the pattern of de novo DNA methylation in prospermatogonia is dictated by opposing actions of broad, low-level transcription and dynamic patterns of active chromatin. Global de novo methylation occurred without any apparent trigger from preexisting repressive chromatin marks but was preceded by broad, low-level transcription along the chromosomes. DNA methylation was excluded only at precisely aligned constitutive or emerging peaks of active chromatin, at most CpG islands and some intracisternal A particles (IAPs). Resetting the maternal-or paternal-specific marking at differentially methylated regions (DMRs) of imprinted genes coincides with global epigenetic changes in the germ lines. We found that DNA methylation emerged in fetal male germ cells by default at each paternally methylated DMR in the presence of transcription-through and in the absence of active chromatin. On the other hand, each maternally imprinted DMR was protected in prospermatogonia from default DNA methylation among highly methylated DNA by an H3K4me2 peak and transcription initiation at least in one strand. We showed earlier that the germline-soma transition in the zygote involves global DNA hydroxymethylation in the paternally inherited genome. Recent studies revealed that certain protected regions resisted this global DNA demethylation. These studies suggest that histone marks play important roles in defining the epigenetic status of soma and germline by protecting specific loci from waves of global DNA demethylation and de novo methylation events.
J. Andrew Pospisilik, MPI for Immunobiology & Epigenetics,
Paternal diet defines offspring chromatin state and intergenerational obesity
The global rise in obesity has revitalized a search to understand genetic, and in particular, epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced Inter-Generational Metabolic Reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as two days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, de-silencing chromatin state-defined transcriptional units in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3 dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system regulates obesity-susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.
Petra Hajkova, MRC Clinical Sciences Centre, London
Dynamics of DNA modifications during epigenetic reprogramming in vivo
The processes of epigenetic reprogramming are characterised by the erasure of epigenetic memory at both DNA methylation and chromatin modifications levels. In vivo, in the context of normal embryonic development, genome-wide erasure of DNA methylation occurs in the zygote following the fertilisation and in the developing embryonic germ line. Recently, a number of studies have linked the DNA demethylation processes with the activity of Tet family of dioxygenases converting 5mC to 5hmC and higher oxidative intermediates (5fC and 5caC). In order to gain mechanistic understanding of developmental reprogramming, we have followed the kinetics of various DNA modifications in both developmental systems. I will discuss our findings on 5mC and 5hmC enrichment and localisation in the context of genome-wide DNA demethylation and locus specific transcriptional regulation.
13.00- 14.30 Buffet Lunch and Poster Session
14.30-16.00 Session 7
Chair: Richard Bártfai
Nabieh Ayoub, Technion Israel Institute of Technology
Dysregulation of KDM4A-D Lysine Demethylases Promotes Chromosomal Instability
Lysine demethylases (KDM), a new and exciting front of biological research, are implicated in multiple cellular processes including transcription, DNA damage response, cell-cycle regulation, cellular differentiation, senescence and carcinogenesis. KDM4A-D members demethylate H3K9 and H3K36 methylation marks. Recent studies show that various types of human cancers exhibit amplification or deletion of KDM4A-D members and thus implicating their activity in promoting chromosomal instability and carcinogenesis. How misregulation of KDM4 members promotes chromosomal instability remains largely unknown. In this meeting, I will present three novel functions of KDM4 family that provide molecular insights into the mechanisms by which KDM4 misregulation leads to chromosomal instability. The first is related to a new role of KDM4 protein in regulating the fidelity of mitotic chromosomes segregation. The second highlights a novel role of KDM4D in the repair of double-strand breaks. The third shows that KDM4 overexpression triggers H3K36me3 demethylation and disrupts the integrity of DNA mismatch repair. Collectively, our findings advance the link between cancer-relevant networks of epigenetic regulation and genome stability.
Maria Fousteri, Fleming Institute, Athens
Deciphering the dynamics of transcription in response to genotoxic stress
DNA damage may impede transcription challenging gene expression and cellular growth. Defects in the way cells restore damage-arrested transcription have been causatively linked to genomic instability as well as cancer, inborn disorders and premature ageing. We study the genome-wide response to genotoxic insults that lead to transcriptional arrest in the 3D chromatin background of the mammalian nucleus in healthy and disease related situations. We have investigated the effect of damage induction on actively transcribed chromatin and gene expression profiles at early and late time points, when all DNA damages and encompassing chromatin sites are expected to be repaired and RNA synthesis is restored in normal but not disease-derived cells. We find a reduction of RNA Polymerase II transcription initiation and an impediment of transcription elongation after damage induction that leads to a 3’->5’ shift in the occupancy of the elongating RNAPII. This increased binding of RNAPII corresponds to retained nascent RNA and lower levels of processed mRNAs. Our approach has provided important insights into the regulatory mechanisms that safeguard gene expression and genome integrity under stressed conditions or specifically promote apoptosis when transcription is permanently stalled and the cellular repair capacity is impaired.
Vincent Géli, Cancer Research Center of Marseille
The many faces of Set1 and of its subunits
In Saccharomyces cerevisiae, all H3K4 methylation is carried out by a single Set1 Complex (Set1C) that is composed of the catalytic (Set1) and seven other subunits (Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1 and Shg1). We recently uncovered that the PHD-containing subunit Spp1, by interacting with H3K4me3 and Mer2, was able to promote the recruitment of potential meiotic DSB sites to the chromosomal axis allowing their subsequent cleavage by Spo11. Therefore, Spp1 emerged as a key regulator of the H3K4 trimethylation catalyzed by Set1C and of the formation of meiotic DSBs. These findings illustrate the remarkable multifunctionality of Spp1, which not only regulates the catalytic activity of the enzyme (Set1), but also interacts with the deposited mark, and mediates its biological effect (meiotic DSB formation) independently of the complex. We will describe new insights illustrating diverse biological functions associated with the Set1-Complex and its subunits.
Tobias A. Knoch, Erasmus Medical Center
The detailed three-dimensional organization and dynamics of the human and mouse genomes
The dynamic three-dimensional chromatin architecture of genomes and the obvious co-evolutionary connection to its function – the storage and expression of genetic information – is still, after ~170 years, a central question of current research. With a systems genomics approach using a novel selective high-throughput chromosomal interaction capture (T2C) technique together with quantitative polymer simulations and scaling analysis of genomic structures and the DNA sequence, we determined the architecture of genomes with unprecedented molecular resolution and dynamic range from single base pair entire chromosomes: for several genetic loci of different species, cell type, and functional states we find a chromatin quasi-fibre exists with 5±1 nucleosome per 11 nm, which folds into 40-100 kbp loops forming aggregates/rosettes which are connected by a ~50 kbp chromatin linker. Polymer simulations using Monte Carlo and Brownian dynamics approaches confirm T2C results and allow to predict and explain additional experimental findings. This agrees also with novel dynamics information from fluorescence correlation spectroscopy (FCS) analysis of chromatin relaxations in vivo by M. Wachsmuth. Beyond, we find a fine-structured multi-scaling behaveour of both the architecture and the DNA sequence which shows for the first time, that genome architecture and DNA sequence organiztion are directly linked – again in detail on the base pair level. Hence, we determined the three-dimensional organization and dynamics for the first time in a consistent system genomics manner from several angles which are all in agreement as well as additionally also with the heuristics of the research of the last 170 years.
Consequently, T2C allows to reach an optimal combination of resolution, interaction frequency range, multi-plexing, and an unseen signal-to-noise ratio at molecular resolution and hence at the level of the “genomic” uncertainty principle and statistical mechanics, this opens the door to architectural sequencing of genomes and thus a detailed understanding of the genome with fundamental new insights with perspectives for diagnosis and treatment.
16.00-16.30 Coffee Break
16.30-18.30 Session 8
Chair: Gábor Szabó
Robert Schneider, IGBMC Strassbourg
Novel Players in Chromatin
One of the major goals of post-genomic biological research is to understand the molecular basis and physiological role of covalent protein modifications. These post-translational modifications (PTMs) can regulate protein interactions and/or stability and thus trigger particular downstream responses.
The best-characterised substrates for multisite PTMs are currently the histone proteins. Two of each of the four core histones (H3, H4, H2A and H2B) form the nucleosomal core particle around which 147 bp of DNA are wrapped. It has been suggested that PTMs of histones constitute a so-called "histone code". Nonetheless the set of characterised histone modifications is far from complete and many modifications are awaiting identification.
PTMs of histones are also clinically very important. Histone modifying enzymes have been found to be rearranged, mutated or deleted in many different types of. In particular the reversible nature of PTMs has led to the emergence of the promising field of epigenetic therapy.
One of key questions in the field is if histone PTMs can be causative for processes like transcription or are just by-products, with limited functional relevance. We recently demonstrated a causative function for lysine acetylation on the lateral surface of the histone octamer. We found that acetylations within the core of the nucleosome at positions that are in contact with the DNA are sufficient to stimulate transcription by modulating histone-DNA binding. Our model is that nucleosome function and signaling to the epigenome is directly regulated by specific lateral surface modifications.
Furthermore, we identified a novel mammalian histone modification and a new histone modifying enzyme that acts as guardian of transposable elements with a key role in maintaining genome integrity.
Imre Boros, University of Szeged
ADA contributions to HAT specificity
ADA2 proteins are components of various GCN5-containing histone acetyltransferase (HAT) complexes, which affect chromatin functions by histone modifications. In Drosophila dADA2a and dADA2b are present in histone H4- and H3-specific dATAC and dSAGA complexes, respectively. Both ADA2a and ADA2b are essential for the HAT catalytic activity of the corresponding complex, and in both complexes a GCN5-ADA2-ADA3 triad is the HAT module. We studied the contribution of different ADA forms to complex specific functions. We found that the functional characteristics of hybrid ADA2 containing HAT complexes depended on the C-terminal region of ADA2 subunit they contained. Our results demonstrate that the ADA2 C-terminal regions play important role in the specific incorporation of ADA2 into SAGA or ATAC type complexes, what than determines H3 or H4 specific histone targeting. ADA2b further contribution to complex variety is that it is expressed in two isoforms at varying levels in different stages of fly development. In the absence of dADA2bL, the dSAGA specific histone H3K9 and H3K14 acetylation is reduced, indicative of the formation of partially active HAT complexes. In concert with that, expression of dADA2bS rescues only partially dAda2b null mutants. Thus, different isoforms of dADA2b contribute to the functional versatility of GCN5 HAT complexes.
Ana Pombo, Institute for Medical Systems Biology, Berlin
Mechanisms of Polycomb repression in mouse embryonic stem cells
Polycomb repressor complexes are important chromatin modifiers with major roles in pluripotency and cancer. Polycomb-mediated gene silencing of developmental regulator genes in embryonic stem cells is accompanied by bivalent chromatin marks, and the presence of an unusual form of RNA polymerase II (RNAPII) at promoters and through coding regions. Polycomb-repressed RNAPII is phosphorylated on Ser5 but not Ser7 or Ser2 residues of the CTD, the later normally associated productive transcription.
To investigate the mechanisms of Polycomb repression in ES cells and the transcriptional activity of the associated RNAPII-S5p complexes, we have combined Chromatin Immunoprecipitation (ChIP) using S5p antibodies with RNA genome-wide sequencing, and investigate the effects of Polycomb knockouts on the transcriptional activity of Polycomb-repressed RNAPII complexes. We show that Polycomb silencing depends on mechanisms of RNA surveillance that actively degrade nascent RNAs produced at Polycomb repressed genes.
Lóránt Székvölgyi, University of Debrecen
Histone mutations driving human disease: a biophysical perspective
Substitution mutation of lysine 27 to methionine in the human H3F3A gene (coding for the histone variant H3.3) has been recently identified as a driver mutation for the brain tumor Pediatric Glioblastome Multiforme. The dominant negative disease phenotype caused the mutant histone is not understood at the molecular level. Here we apply various in vitro and in vivo model systems (E.coli, S. cerevisiae, human cell lines) and state of the art genetic and biophysical approaches to reveal how the histone H3K27M point mutation affect the nanostructure of nucleosome core particles (NCPs) and influence the integrity of chromosomes. We tested the viability and stress tolerance of H3K27M-expressing S. cerevisiae strains under different metabolic (YPD, YPGal, YPG) and stress conditions (H2O2, campthotecin, ethoposide, hidroxyurea, MMS, UV), and we find that the K27M mutation does not cause any growth defect in this model organism. We performed bulk- and single-molecule FRET measurements on in vitro reconstituted nucleosomes and observed a more opened conformation of the H3.3K27M nucleosomes, however the overall stability of the mutant NCPs has been similar to wild-type. Importantly, by FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) we reveal a significantly higher mobile fraction and faster repopulation rate of the H3.3K27M histones. The latter suggests that the disease caused by the H3.3K27M histone is related to the altered mobility and diffusion kinetics of mutant nucleosome core particles.
This work was supported by the European Union (FP7/MCA-CIG) and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1-2012-0001 ‘National Excellence Program’, also by the Hungarian Scientific Research Fund (OTKA-PD) and CRP-ICGEB international research grant.
20.00 - Closing Gala Dinner at the Hungarian Academy of Sciences (HAS)
Bus will leave from the RCNS (MTA-TTK) at 19.30 and from Gellert Hotel at 19.40.
The bus is organized only one way since the HAS is in the very hearth of the city with excellent pubs on the Pest side and nice walks on the Danube bank. More info about the walks and public transport in the MAPS Section.
Please sign up for bus transfer at the registration desk during the Thursday!
Location: Akademia Klub
Magyar Tudományos Akadémia
1051 Budapest, Széchenyi István tér 9.
Location: HOTEL GELLERT, First Floor,TEA Room
16.00 Registration
18.00 Opening Lecture: Paolo Sassone-Corsi:
Common Threads: Epigenetics, Metabolism and the Clock
University of California, Irvine, introduced by László Tora
19.15 Welcome Dinner in the Gellert Hotel, First Floor: Duna and Gellért Rooms
Thursday, 20. November
Location: Research Center for Natural Sciences
8.30 Registration
9.00-10.30 Session 1,
Chair: Iannis Talianidis
Maxime Dahan, Laboratoire Physico-Chimie Curie Institut Curie, CNRS UMR168:
Probing the target search of DNA-binding proteins in mammalian cells, one molecule at a time
For many cellular functions, DNA-binding proteins (DBPs) need to find specific target sites in the genome. Facilitated diffusion (FD), namely the combination of one-dimensional motion along non-specific DNA and three-dimensional exploration, is the dominant model for the target search (TS) of DBPs. Yet, this model has hardly been tested in vivo, particularly in the complex environment of a mammalian nucleus, and it is still controversial whether it accelerates association to specific DNA binding sites. To address that question, we have implemented a TS assay using human cells with a unique target locus for an inducible exogenous searcher, the tetracycline repressor (TetR). Using single-molecule tracking and in situ biochemical measurements of association kinetics, we directly characterize the mobility of TetR, its transient interaction with non-cognate DNA and the kinetics of binding to the specific locus. Overall, we find that the searcher follows a FD strategy but that the search kinetics is not limited by the diffusive motion but by the low association efficiency to non-specific DNA sites. We will also discuss how these measurements can be extended to endogeneous DNA-binding proteins.
Ido Amit, Weizmann Institute:
Shaping the blood: Lessons from Chromatin and single cell RNA dynamics
Thirty years of dedicated research have enabled the categorization of functionally similar hematopoietic cells into a lineage tree by means of a combination of a small number of cell surface markers. Nevertheless, our understanding of the hematopoietic cell lineage remains coarse and biased, limited by preselecting of specific markers. A wealth of new studies highlight the crux of using cell populations for genome-wide measurements as even state-of-the-art classification approaches are limited and retain a heterogeneous population masking gene expression heterogeneity, uncharacterized cells and functions.
In order to understand in a comprehensive and unbiased approach the immune cell lineage and activity in vivo, we have developed an automated massively parallel RNA single cell sequencing approach (MARS-Seq) for measuring the genome wide expression of tens of thousands of single cells in their native context. Importantly, MARS-Seq enables transcriptome measurement of thousands of cells per experiment.
I will demonstrate how using this combined experimental and computational approach; we characterize the cell fate and activity of thousands of immune cells from mouse spleen in various physiological contexts, focusing of the dendritic cell (DC) lineage. We show that using our single cell approach we can build a high resolution ab initio immune identity map without the use of any predefined cell markers. Focusing on subpopulations of DCs, I will demonstrate that these DC populations include highly heterogeneous mixtures of transcriptional states that are only coarsely approximated using surface marker sorting and how these cells respond differently to stimulation. I will discuss how, our approach is greatly expanding the knowledge of transcription regulation heterogeneity and immune functional diversity beyond the limited numbers of cells/marker currently used to describe populations of immune cells. Finally, I will combine these results with unpublished work on chromatin regulatory maps of DC to connect between chromatin dynamics and gene expression heterogeneity.
Rickard Sandberg, Karolinska Institutet:
Single-cell RNA-seq reveals principles of allelic expression in mammalian cells
Analyses of gene expression across large numbers of single cells can reveal cell-to-cell variability. To this end, my lab has been developing single-cell RNA-seq methods (e.g. Smart-seq2) that combine full-length transcript coverage with high sensitivity and accuracy. We have performed global analyses of gene expression in hundreds of individual cells from mouse preimplantation embryos of mixed genetic background (CAST/EiJ x C57BL/6J) as well as in somatic cells. Using strain-specific SNPs we investigate allelic expression in these cells during preimplantation development to characterize X-chromosome imprinting and allelic expression patterns of autosomal genes. Although expression from both paternal alleles is generally observed in analyses of diploid cell populations, no study has addressed allelic expression patterns genome-wide in single cells. Importantly, we demonstrate that allelic expression is independent for genes across all expression levels making this a genomic principle. Additionally, our study revealed that allelic transcription fluctuations generated widespread random monoallelic expression in single cells, and we discuss its functional consequences. Finally, we explored to what extent random monoallelic expression was clonality propagated through cell division through single-cell transcriptome analyses of clonally related somatic cells.
10.30-11.00 Coffee Break
11.00-13.00 Session 2
Chair: Wendy Bickmore
Brian McStay, Centre for Chromosome Biology, Galway
From NORs to nucleoli and genomic stability of rDNA arrays in human cells
Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Nucleoli, sites of ribosome biogenesis and key regulators of cellular growth are the most prominent of these. They disappear during mitosis, reforming around prominent poorly characterized chromosomal features, nucleolar organizer regions (NORs) on human acrocentric chromosome short-arms. By examining the effects of depleting UBF, a nucleolar specific HMG box protein that binds extensively over rDNA chromatin, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Through construction of synthetic NORs and synthetic nucleoli we prove that nucleolar biogenesis is a staged process where UBF-dependent mitotic bookmarking precedes function dependent nucleolar assembly.
Isolation of repetitive, highly transcribed rDNA within the nucleolar interior prompted us to investigate the response of nucleoli to DNA double strand breaks (DSBs) within these essential genes. Targeted introduction of DSBs into rDNA, but not closely linked sequences, results in ATM-dependent inhibition of rDNA transcription by RNA polymerase I, coupled with withdrawal of rDNA from the nucleolar interior to NOR anchoring points at the periphery. This localized response renders rDNA accessible to the repair machinery. Most significantly, repair is carried out by the homologous recombination pathway, independent of cell cycle stage. These results suggest that rDNA repair can be templated by repeats in cis and point to a role for chromosomal context in maintenance of their genomic stability.
Claire Rougeulle, Univ Paris Diderot
Non-coding RNAs and X-inactivation plasticity in mammals
X chromosome inactivation (XCI) in mammals is an essential process which is developmentally regulated and tightly linked to the cellular context and to the potency of the cell. XCI is also a well-known example of large scale gene regulation controlled by long non-coding RNAs (lncRNAs). Xist, which coats the X chromosome from which it is expressed and induces its silencing, was one of the first lncRNA to be discovered, and its function in XCI has been well documented, at least in the mouse. Since the initial discovery of Xist more than 20 years ago, several additional lncRNAs have been proposed to participate to the regulation of XCI, but their exact contribution remains for the most part elusive. In addition, we know now that X-inactivation strategies are tremendously variable among mammals, and the extent to which lncRNAs contribute to this variation deserve specific attention. Several lncRNAs involved in XCI have orthologs in various mammalian species, but their function has not been addressed in species other than mouse; others are poorly conserved and may be restricted to a particular group of mammals.
We have recently identified a novel lncRNA, XACT, which appears to be specific to the humans or to closely related primates. XACT displays the unique property of coating the active X chromosome in humans, and its expression is restricted to early embryonic stages, in which XCI displays important plasticity. We will discuss possible function for XACT in controlling X chromosome activity in humans, and the divergence in XCI strategies in mammals.
Pantelis Hatzis, Fleming Institute, Athens
The long non-coding RNA WNTRLINC1 regulates transcription, stemness and carcinogenesis in the intestinal epithelium
The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation and proliferation in the intestinal epithelium. Mutations in pathway components APC, AXIN or β-catenin lead to aberrant transcriptional activity of the TCF4/β-catenin complex, the endpoint of the Wnt pathway, and are the primary transforming factor in colorectal cancer. Our research on Wnt-mediated transcriptional regulation has identified long intergenic non-coding RNAs (lincRNAs) as novel targets of the Wnt pathway. One of these, termed WNTRLINC1, positively regulates factors required for intestinal stem cell maintenance. It is required for continued proliferation of colorectal carcinoma cells and is significantly upregulated in colorectal cancer patient samples. It exerts its impacts by modulating the chromatin accessibility of transcription regulatory factors with which it interacts. WNTRLINC1 represents thus a new player involved in normal and abnormal intestine physiology and a putative novel diagnostic and therapeutic target in colorectal cancer.
Wendy Bickmore, University of Edinburgh
Transcription and nuclear organisation: chicken and egg
There are some well-established relationships between aspects of the spatial organisation of the nucleus and gene expression. Sequences at the nuclear periphery are often associated with the repression of transcription, the most active regions of genomes are often found in the centre of the nucleus, loci repressed by certain epigenetic pathways are found in a compact chromatin state. But who comes first: does structure direct function (transcription) or vice versa?
I will describe experiments that try to break the links between chromatin/nuclear organisation - especially at the nuclear periphery - and the act of transcription. By specifically perturbing either nuclear/chromatin organisation or transcription I will discuss the extent to which we can begin to understand the relationships between structure and function in the nucleus.
13.00- 14.30 Buffet Lunch and Poster Session
14.30-16.00 Session 3
Chair: Laszlo Nagy
Puri Pier Lorenzo, Sanford-Burnham Institute for Medical Research, La Jolla, CA. USA and Fondazione Santa Lucia. Rome, Italy.
Epigenetic networks regulating skeletal muscle regeneration and fibro-adipogenic degeneration in health and diseases.
The ability of skeletal muscle to repair upon acute injury by regeneration of new myofibers relies on functional interactions between different cell types, including muscle satellite stem cell (MuSCs) , fibro-adipogenic progenitors (FAPs) and cells from the inflammatory infiltrate (i.e. macrophages, eosinophils). In chronic degenerative disorders, such as muscular dystrophies, the integrity of this network is progressively compromised, leading to a switch from compensatory regeneration to an alternative, pathogenic repair by deposition of fibrotic and fatty material. We have shown that this switch is largely dependent on the phenotype adopted by FAPs, and have identified an HDAC-regulated network, by which microRNAs control the composition and activity of the SWI/SNF chromatin remodeling complex to direct chromatin remodeling toward two alternative - promyogenic or fibro-adipogenic - phenotypes of FAPs. This network provides the rationale for the ability of HDAC inhibitors to promote regeneration and prevent fibrosis and fat deposition in dystrophic muscles.
Laszlo Nagy, University of Debrecen and Sanford Burnham Institute
Cistromic and long-range interactions of lineage- and signal specific transcription factors integrate macrophage specification and control lipid signaling
Cellular differentiation and subtype specification is principally governed by lineage specific and signal specific transcription factors. Importantly, the interaction of these transcription factors should be interpreted in the context of a distinct cell-type specific genomic architecture. However the contribution of genomic interactions and the identity of enhancer networks regulating induced genetic programs are still very poorly understood and difficult to map. Here we show that the combination and bioinformatic integration of steady state mRNA levels, genome-wide localization (ChIP-Seq) of transcription factors, architectural elements, histone marks and nascent RNA production (GRO-Seq) allows for the unraveling of signal specific enhancer networks and the regulated target genes. We used macrophages and the paradigm of lipid-activated nuclear receptor signaling to identify the enhancer network operated by the liganded Retinoid X Receptor in the context of M0 and M2 macrophage activation states. The M0 macrophage RXR cistrome has at least 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features 387 liganded-RXR bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin limited functional domains. These findings were molecularly validated using 3C and 3C-Seq and we show that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded-RXR controlled enhancers and provides the macrophage with a novel inducible program. M2 macrophage polarization redistributes RXR and leads to the establishment of additional transcriptional including nuclear receptor signaling pathways.
Dirk Schübeler, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland:
Setting and reading DNA methylation
Recent advances generated genomic DNA methylation maps during cellular differentiation at unprecedented resolution. Combined with functional assays this revealed that dynamics in DNA methylation coincide with changes in regulatory activity and that transcription factors play an important role in shaping methylation patterns. This tightly links DNA methylation with underlying DNA sequence features and suggests that a substantial fraction of methylation changes occur downstream of gene regulation.
In order to create functional genomic binding maps of trans-acting factors involved in reading and writing DNA methylation we use a controlled biotin tagging approach that enables to identify protein domain contribution to chromosomal localization, a system utilized this to map all MBD domain proteins and a set of isoforms and disease causing mutants in order to better understand the readout of DNA methylation by this family of proteins (Baubec et al., Cell 2013).
In order to gain insights into pattern generation we determined the genome-wide distribution and locus-specific activity of the enzymes responsible for de novo DNA methylation in mammals. DNMT3A and B share general binding preference for the CG dinucleotide yet we observe additional distinct binding preferences. Bound regions are preferentially remethylated when reintroducing individual enzymes into cells that lack DNA methylation suggesting that recruitment is the primary level of guiding activity. De novo methylation is excluded from active regulatory regions and is directed to linker regions between positioned nucleosomes.
In case of DNMT3B preferential binding in stem cells occurs at actively transcribed genes and importantly is required to maintain genic hypermethylation. Mutational analysis shows that the PWWP domain is necessary for this targeting suggesting that lysine 36 of histone H3 links histone and DNA methylation at active genes. This work suggests that locus specific targeting of de novo methyltransferases is a highly dynamic process required for maintaining integrity of the methylome.
16.00-16.30 Coffee Break
16.30-18.30 Session 4
Chair: Béla Molnár
Tamas Aranyi, Institute of Enzimology, RCNS
Infection of human cells with lentiviral vector leads to highly reproducible genome-wide DNA methylation changes
Lentiviral vectors (LV) are efficient tools for gene transfer. They are frequently used as vectors in vivo and in vitro in laboratory experiments and clinical trials. After infection LVs integrate the genome and interact with the chromatin. It is therefore of major importance to investigate their effect on the DNA methylation of human cells. In the present study we tested in primary human haematopoietic and Jurkat cells the effect of infection of both wild-type and integration deficient LVs. We used the Infinium Illumina450K BeadChip assay and developed the double average technique (DAT) to detect DNA methylation changes. We demonstrated that independently of the capacity of vectors to integrate soon after infection a great number of CpGs undergo DNA methylation increase. These methylation changes revealed to be highly reproducible in preferentially targeted genomic regions. Interestingly, these regions are found in CpG islands of non-expressed genes. The consequences of these epimutations will be discussed.
Balint L Balint, University of Debrecen
Epigenetic tools for mapping chromatin modifications in clinical samples
Understanding the person-to-person variability in diseases by using the unprecedented amount of available genomic data may be approached as never before. We propose to investigate the impact of individual genetic variations in breast cancers. We performed a meta-analysis of all the publicly available datasets from different breast cancer end endometrial cancer cell lines. In order to improve the reproducibility of ER alpha ChIP in samples with low cell numbers we developed a nanoparticle based spike-in control. With this tool we can not only controll the ChIP reaction but also to measure the binding capacity of an antibody and to compare different ChIP protocolls in a more accurate way than before.
Attila Németh, Biochemistry Center Regensburg
Molecular mapping of nucleolus-associated chromosomal domain dynamics during cellular senescence
Nucleolus-associated chromosomal domains (NADs) were identified genome-wide in human HeLa cervix carcinoma (Németh et al., PLoS Genet 6, e1000889.) and HT1080 fibrosarcoma cells (van Koningsbruggen et al., Mol Biol Cell 21, 3735-3748.) providing a snapshot of genome organization around the nucleolus. Yet, questions about the dynamics of nucleolus-associated chromatin remained to be answered. Since large rearrangements in the nuclear and nucleolar architecture occur during cellular aging we decided to map NAD reorganization in this fundamental biological process. Nucleoli of IMR90 human lung embryonic fibroblasts were isolated from a young, proliferating cell population, as well as from senescent cells. Nucleolus-associated DNA was analyzed on high-resolution microarrays by comparative genomic hybridization. Additionally, RNA was extracted from the same cell populations and subjected to global gene expression analysis to compare genome-wide transcriptome and NAD profiles, further to identify chromatin regulators of cellular aging. The results of genomics analyses are currently validated in single cell 3D-immuno-FISH experiments. In summary, our investigations provide novel insights into the spatial and transcriptional dynamics of the nucleolus-associated genome during cellular senescence.
Judit Balog, Leiden University Medical Center
The role of chromatin repressor SMCHD1 in the development of human disease
Epigenetic screens in different organisms identified known and new epigenetic modifiers of chromatin stability and plasticity. SMCHD1 (Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1) was first identified in a screen for epigenetic modifiers involved in repeat mediated epigenetic repression in mice harboring a multicopy GFP transgene (Ashe et al. 2008). SMCHD1 is a non-canonical member of the SMC family of proteins shown to be involved in epigenetic repression of the inactive X chromosome and a few autosomal loci. Little is known about repeat mediated epigenetic repression in human cells, but we identified SMCHD1 mutations in a subgroup of patients (2%) affected by a rare muscular dystrophy, Facioscapulohumeral Muscular Dystrophy (FSHD) (Lemmers et al. 2012.). A key element in the pathomechanism of FSHD is the germline transcription factor DUX4 which is ectopically expressed in muscle of FSHD individuals but not of healthy controls, leading to cell death. The DUX4 open reading frame resides in the D4Z4 macrosatellite repeat unit, the building blocks of repeat arrays that can consist of up to 100 units at human chromosomes 4q and 10q. Several lines of evidence suggest that DUX4 expression is associated with an epigenetic derepression of the D4Z4 array in somatic cells, either by contraction of the D4Z4 repeat array to a size of 1-10 units (FSHD1) or by mutations in SMCHD1 (FSHD2). The aim of our study was to determine the epigenetic requirements of DUX4 expression in human primary myoblasts and differentiated myotubes. We found that DUX4 expression dynamically changes during myogenic differentiation, being highly increased in myotubes. A series of control and FSHD myoblasts and myotubes were measured for different epigenetic marks at the DUX4 promoter region, including DNA methylation, repressive and permissive histone modifications and the presence of SMCHD1. This, in combination with knock down experiments of D4Z4 chromatin modifiers, identified SMCHD1 as key repressor of DUX4 activity in skeletal muscle.
Ferenc Muller, University of Birmingham
Functional validation of disease associated human enhancer candidates using the zebrafish transgenic embryo as a scalable vertebrate model
Genome wide analyses such as ENCODE and FANTOM predicted a surprisingly large number of non- coding elements with suggested cis-regulatory function in mammals. However, functional validations of the candidate regulatory elements are mostly lacking due to limitations of transgenesis technologies and capacities. We have investigated the utility of the transgenic zebrafish embryo as a scalable in vivo vertebrate model to study the functionality of candidate human enhancers predicted by a combination of chromatin signatures (e.g. H3k4me1, H3K27 Ac, H2AZ), TF binding events and enhancer specific bidirectional transcription. These analyses indicated that despite the evolutionary distance between human and fish, 60% of the candidate enhancers exhibiting sequence conservation lead to reporter expression which recapitulates the expression patterns of either zebrafish or human genes associated with the candidate enhancers. For example, we have demonstrated the activity of enhancer candidates associated with Type 2 diabetes and fasting glycemia in the pancreatic islet of zebrafish larvae. Currently we are developing assays in order to improve the sensitivity of transgenesis tools so as to detect subtle changes in enhancer function caused by sequence variation (SNPs) associated with dysregulation in disease. To improve the reliability of zebrafish enhancer function assays, a targeted integration system mediated by PhiC31 integrase was developed with which we have demonstrated successful elimination of position effect variation commonly found in conventional, transposon-based transgenesis.
Tibor Pankotai, University of Szeged
Mechanistic insights into the transcriptional arrest in the presence of Double Strand Breaks
Double-strand breaks (DSBs) occur frequently in the genome during genome replication or by DNA damaging agents. DNA lesions affect fundamental DNA-dependent nuclear processes such as replication and transcription. We have developed an experimental system where DSBs are induced at coding regions of RNA polymerase II transcribing genes. We have started to study the kinetics of RNA polymerase II transcription inhibition in the presence of DNA breaks. We observed that induction of the break led to transcription inhibition and the restoration of transcription closely followed the dynamics of the repair of breaks. We confirmed by chromatin-immunoprecipitation that the break induction led to displacement of RNA polymerase II affecting both the elongation and the initiation of transcription. Our results show that this is dependent on one of the major kinase in DNA damage repair called DNAPKcs. We also investigated the downstream steps of RNA polymerase II removal and we claimed that it was a multistep process involving additional kinases and ubiquitin ligases NEDD4 and CUL3. At the last step of break dependent transcriptional silencing the RNA polymerase II is targeted for proteasome dependent degradation. These data demonstrate that the DNA damage repair complexes and proteasomal system have a synergistic and active role in transcriptional silencing during the DSB repair by removing the RNA pol II from the transcribing region. We show here that DNA lesions occurring at transcribed regions cause a transient repression until the lesion is repaired. This is probably a cell defense mechanism to avoid production of truncated or mutated transcripts in essential genes whose alterations in their gene expression would endanger cell viability. Understudying the role of DNAPKcs, in preventing RNA pol II bypassing a DSB might be a key in avoiding the production of mutated transcripts that could lead to cancerous phenotypes.
18.35-18.45 Demonstration of world’s fastest qPCR system xxpress (40 PCR cycles in 10 minutes)
18.45- 20.00 Wine and Cheese and Posters
Friday 21. November
Location: Research Center for Natural Sciences
9.00-10.30 Session 5
Chair: Imre Boros
Laszlo Tora, IGBMC, Strassbourg
The SAGA coactivator complex acts on the whole transcribed genome and is required for all RNA polymerase II transcription
The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.
Frank Holstege: Deciphering regulatory circuitry by genome-wide analyses
Iannis Talianidis, Fleming Institute, Athens
Epigenetic mechanisms regulating liver development and function
Histone or DNA modifications are considered as major determinants of epigenetic information for chromatin-templated processes such as transcription. In the liver, similar to other organs, specific histone modification patterns correlate with gene activity and represent important means of regulation of epigenetic states characteristic to different developmental stages, metabolic states or diseases.
However, histone or DNA modifications are not the only epigenetic signals that are responsible for the establishment, maintenance and reversal of metastable transcriptional states during liver development and various metabolic conditions. Trans-epigenetic signals, which eventually accumulate into complex gene networks and gradually emerging “promoter-marking” signals, that determine the potential of gene activation are equally important in the establishment of self-propagating transcription states.
The contribution of the above epigenetic mechanisms in the regulation of liver development and function will be discussed.
10.30-11.00 Coffee Break
11.00-13.00 Session 6
Chair: Petra Hajkova
Erica Watson, University of Cambridge
The transgenerational epigenetic effects of abnormal folate metabolism
Epigenetic changes or ‘epimutations’ accrued in the genome throughout a lifetime in response to environmental stressors may contribute to an increased risk for disease. If an epimutation occurs within the germline, it might be inherited by subsequent generations resulting in developmental defects and disease in their progeny. To study the mechanism of transgenerational epigenetic inheritance, we use a mouse model with a mutation in a key gene involved in folate metabolism (Mtrrgt) that disrupts the folate cycle. Folate is a vitamin important for the one-carbon metabolism and therefore it is necessary for the methylation of cell components (e.g., DNA). Highly controlled genetic pedigrees were used to study the specific effects of the Mtrrgt genotype in either maternal grandparent and revealed developmental abnormalities and widespread epigenetic instability in their grandprogeny at midgestation. This occurred even when the mother and the grandprogeny were genetically wildtype for Mtrr. Some of the abnormalities (e.g., neural tube, heart and placental defects) persisted for up to five generations and after embryo transfer experiments suggesting that the effect was independent of the maternal environment. Together, these data suggest that folate deficiency in humans may lead to transgenerational epigenetic inheritance of disease.
Piroska E. Szabó, Van Andel Institute, Grand Rapids
Epigenetic remodeling between generations
Epigenetic marks are faithfully propagated during cell divisions in the soma. However, the epigenome is thoroughly remodeled between subsequent generations. This is achieved by global remodeling events that affect DNA methylation and chromatin composition during the soma-germline and germline-soma transitions. To understand what dictates the pattern of de novo DNA methylation in the male germ line during soma-germline transition, we mapped DNA methylation, chromatin, and transcription changes in purified fetal mouse germ cells. Our results suggest that the pattern of de novo DNA methylation in prospermatogonia is dictated by opposing actions of broad, low-level transcription and dynamic patterns of active chromatin. Global de novo methylation occurred without any apparent trigger from preexisting repressive chromatin marks but was preceded by broad, low-level transcription along the chromosomes. DNA methylation was excluded only at precisely aligned constitutive or emerging peaks of active chromatin, at most CpG islands and some intracisternal A particles (IAPs). Resetting the maternal-or paternal-specific marking at differentially methylated regions (DMRs) of imprinted genes coincides with global epigenetic changes in the germ lines. We found that DNA methylation emerged in fetal male germ cells by default at each paternally methylated DMR in the presence of transcription-through and in the absence of active chromatin. On the other hand, each maternally imprinted DMR was protected in prospermatogonia from default DNA methylation among highly methylated DNA by an H3K4me2 peak and transcription initiation at least in one strand. We showed earlier that the germline-soma transition in the zygote involves global DNA hydroxymethylation in the paternally inherited genome. Recent studies revealed that certain protected regions resisted this global DNA demethylation. These studies suggest that histone marks play important roles in defining the epigenetic status of soma and germline by protecting specific loci from waves of global DNA demethylation and de novo methylation events.
J. Andrew Pospisilik, MPI for Immunobiology & Epigenetics,
Paternal diet defines offspring chromatin state and intergenerational obesity
The global rise in obesity has revitalized a search to understand genetic, and in particular, epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced Inter-Generational Metabolic Reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as two days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, de-silencing chromatin state-defined transcriptional units in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3 dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system regulates obesity-susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.
Petra Hajkova, MRC Clinical Sciences Centre, London
Dynamics of DNA modifications during epigenetic reprogramming in vivo
The processes of epigenetic reprogramming are characterised by the erasure of epigenetic memory at both DNA methylation and chromatin modifications levels. In vivo, in the context of normal embryonic development, genome-wide erasure of DNA methylation occurs in the zygote following the fertilisation and in the developing embryonic germ line. Recently, a number of studies have linked the DNA demethylation processes with the activity of Tet family of dioxygenases converting 5mC to 5hmC and higher oxidative intermediates (5fC and 5caC). In order to gain mechanistic understanding of developmental reprogramming, we have followed the kinetics of various DNA modifications in both developmental systems. I will discuss our findings on 5mC and 5hmC enrichment and localisation in the context of genome-wide DNA demethylation and locus specific transcriptional regulation.
13.00- 14.30 Buffet Lunch and Poster Session
14.30-16.00 Session 7
Chair: Richard Bártfai
Nabieh Ayoub, Technion Israel Institute of Technology
Dysregulation of KDM4A-D Lysine Demethylases Promotes Chromosomal Instability
Lysine demethylases (KDM), a new and exciting front of biological research, are implicated in multiple cellular processes including transcription, DNA damage response, cell-cycle regulation, cellular differentiation, senescence and carcinogenesis. KDM4A-D members demethylate H3K9 and H3K36 methylation marks. Recent studies show that various types of human cancers exhibit amplification or deletion of KDM4A-D members and thus implicating their activity in promoting chromosomal instability and carcinogenesis. How misregulation of KDM4 members promotes chromosomal instability remains largely unknown. In this meeting, I will present three novel functions of KDM4 family that provide molecular insights into the mechanisms by which KDM4 misregulation leads to chromosomal instability. The first is related to a new role of KDM4 protein in regulating the fidelity of mitotic chromosomes segregation. The second highlights a novel role of KDM4D in the repair of double-strand breaks. The third shows that KDM4 overexpression triggers H3K36me3 demethylation and disrupts the integrity of DNA mismatch repair. Collectively, our findings advance the link between cancer-relevant networks of epigenetic regulation and genome stability.
Maria Fousteri, Fleming Institute, Athens
Deciphering the dynamics of transcription in response to genotoxic stress
DNA damage may impede transcription challenging gene expression and cellular growth. Defects in the way cells restore damage-arrested transcription have been causatively linked to genomic instability as well as cancer, inborn disorders and premature ageing. We study the genome-wide response to genotoxic insults that lead to transcriptional arrest in the 3D chromatin background of the mammalian nucleus in healthy and disease related situations. We have investigated the effect of damage induction on actively transcribed chromatin and gene expression profiles at early and late time points, when all DNA damages and encompassing chromatin sites are expected to be repaired and RNA synthesis is restored in normal but not disease-derived cells. We find a reduction of RNA Polymerase II transcription initiation and an impediment of transcription elongation after damage induction that leads to a 3’->5’ shift in the occupancy of the elongating RNAPII. This increased binding of RNAPII corresponds to retained nascent RNA and lower levels of processed mRNAs. Our approach has provided important insights into the regulatory mechanisms that safeguard gene expression and genome integrity under stressed conditions or specifically promote apoptosis when transcription is permanently stalled and the cellular repair capacity is impaired.
Vincent Géli, Cancer Research Center of Marseille
The many faces of Set1 and of its subunits
In Saccharomyces cerevisiae, all H3K4 methylation is carried out by a single Set1 Complex (Set1C) that is composed of the catalytic (Set1) and seven other subunits (Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1 and Shg1). We recently uncovered that the PHD-containing subunit Spp1, by interacting with H3K4me3 and Mer2, was able to promote the recruitment of potential meiotic DSB sites to the chromosomal axis allowing their subsequent cleavage by Spo11. Therefore, Spp1 emerged as a key regulator of the H3K4 trimethylation catalyzed by Set1C and of the formation of meiotic DSBs. These findings illustrate the remarkable multifunctionality of Spp1, which not only regulates the catalytic activity of the enzyme (Set1), but also interacts with the deposited mark, and mediates its biological effect (meiotic DSB formation) independently of the complex. We will describe new insights illustrating diverse biological functions associated with the Set1-Complex and its subunits.
Tobias A. Knoch, Erasmus Medical Center
The detailed three-dimensional organization and dynamics of the human and mouse genomes
The dynamic three-dimensional chromatin architecture of genomes and the obvious co-evolutionary connection to its function – the storage and expression of genetic information – is still, after ~170 years, a central question of current research. With a systems genomics approach using a novel selective high-throughput chromosomal interaction capture (T2C) technique together with quantitative polymer simulations and scaling analysis of genomic structures and the DNA sequence, we determined the architecture of genomes with unprecedented molecular resolution and dynamic range from single base pair entire chromosomes: for several genetic loci of different species, cell type, and functional states we find a chromatin quasi-fibre exists with 5±1 nucleosome per 11 nm, which folds into 40-100 kbp loops forming aggregates/rosettes which are connected by a ~50 kbp chromatin linker. Polymer simulations using Monte Carlo and Brownian dynamics approaches confirm T2C results and allow to predict and explain additional experimental findings. This agrees also with novel dynamics information from fluorescence correlation spectroscopy (FCS) analysis of chromatin relaxations in vivo by M. Wachsmuth. Beyond, we find a fine-structured multi-scaling behaveour of both the architecture and the DNA sequence which shows for the first time, that genome architecture and DNA sequence organiztion are directly linked – again in detail on the base pair level. Hence, we determined the three-dimensional organization and dynamics for the first time in a consistent system genomics manner from several angles which are all in agreement as well as additionally also with the heuristics of the research of the last 170 years.
Consequently, T2C allows to reach an optimal combination of resolution, interaction frequency range, multi-plexing, and an unseen signal-to-noise ratio at molecular resolution and hence at the level of the “genomic” uncertainty principle and statistical mechanics, this opens the door to architectural sequencing of genomes and thus a detailed understanding of the genome with fundamental new insights with perspectives for diagnosis and treatment.
16.00-16.30 Coffee Break
16.30-18.30 Session 8
Chair: Gábor Szabó
Robert Schneider, IGBMC Strassbourg
Novel Players in Chromatin
One of the major goals of post-genomic biological research is to understand the molecular basis and physiological role of covalent protein modifications. These post-translational modifications (PTMs) can regulate protein interactions and/or stability and thus trigger particular downstream responses.
The best-characterised substrates for multisite PTMs are currently the histone proteins. Two of each of the four core histones (H3, H4, H2A and H2B) form the nucleosomal core particle around which 147 bp of DNA are wrapped. It has been suggested that PTMs of histones constitute a so-called "histone code". Nonetheless the set of characterised histone modifications is far from complete and many modifications are awaiting identification.
PTMs of histones are also clinically very important. Histone modifying enzymes have been found to be rearranged, mutated or deleted in many different types of. In particular the reversible nature of PTMs has led to the emergence of the promising field of epigenetic therapy.
One of key questions in the field is if histone PTMs can be causative for processes like transcription or are just by-products, with limited functional relevance. We recently demonstrated a causative function for lysine acetylation on the lateral surface of the histone octamer. We found that acetylations within the core of the nucleosome at positions that are in contact with the DNA are sufficient to stimulate transcription by modulating histone-DNA binding. Our model is that nucleosome function and signaling to the epigenome is directly regulated by specific lateral surface modifications.
Furthermore, we identified a novel mammalian histone modification and a new histone modifying enzyme that acts as guardian of transposable elements with a key role in maintaining genome integrity.
Imre Boros, University of Szeged
ADA contributions to HAT specificity
ADA2 proteins are components of various GCN5-containing histone acetyltransferase (HAT) complexes, which affect chromatin functions by histone modifications. In Drosophila dADA2a and dADA2b are present in histone H4- and H3-specific dATAC and dSAGA complexes, respectively. Both ADA2a and ADA2b are essential for the HAT catalytic activity of the corresponding complex, and in both complexes a GCN5-ADA2-ADA3 triad is the HAT module. We studied the contribution of different ADA forms to complex specific functions. We found that the functional characteristics of hybrid ADA2 containing HAT complexes depended on the C-terminal region of ADA2 subunit they contained. Our results demonstrate that the ADA2 C-terminal regions play important role in the specific incorporation of ADA2 into SAGA or ATAC type complexes, what than determines H3 or H4 specific histone targeting. ADA2b further contribution to complex variety is that it is expressed in two isoforms at varying levels in different stages of fly development. In the absence of dADA2bL, the dSAGA specific histone H3K9 and H3K14 acetylation is reduced, indicative of the formation of partially active HAT complexes. In concert with that, expression of dADA2bS rescues only partially dAda2b null mutants. Thus, different isoforms of dADA2b contribute to the functional versatility of GCN5 HAT complexes.
Ana Pombo, Institute for Medical Systems Biology, Berlin
Mechanisms of Polycomb repression in mouse embryonic stem cells
Polycomb repressor complexes are important chromatin modifiers with major roles in pluripotency and cancer. Polycomb-mediated gene silencing of developmental regulator genes in embryonic stem cells is accompanied by bivalent chromatin marks, and the presence of an unusual form of RNA polymerase II (RNAPII) at promoters and through coding regions. Polycomb-repressed RNAPII is phosphorylated on Ser5 but not Ser7 or Ser2 residues of the CTD, the later normally associated productive transcription.
To investigate the mechanisms of Polycomb repression in ES cells and the transcriptional activity of the associated RNAPII-S5p complexes, we have combined Chromatin Immunoprecipitation (ChIP) using S5p antibodies with RNA genome-wide sequencing, and investigate the effects of Polycomb knockouts on the transcriptional activity of Polycomb-repressed RNAPII complexes. We show that Polycomb silencing depends on mechanisms of RNA surveillance that actively degrade nascent RNAs produced at Polycomb repressed genes.
Lóránt Székvölgyi, University of Debrecen
Histone mutations driving human disease: a biophysical perspective
Substitution mutation of lysine 27 to methionine in the human H3F3A gene (coding for the histone variant H3.3) has been recently identified as a driver mutation for the brain tumor Pediatric Glioblastome Multiforme. The dominant negative disease phenotype caused the mutant histone is not understood at the molecular level. Here we apply various in vitro and in vivo model systems (E.coli, S. cerevisiae, human cell lines) and state of the art genetic and biophysical approaches to reveal how the histone H3K27M point mutation affect the nanostructure of nucleosome core particles (NCPs) and influence the integrity of chromosomes. We tested the viability and stress tolerance of H3K27M-expressing S. cerevisiae strains under different metabolic (YPD, YPGal, YPG) and stress conditions (H2O2, campthotecin, ethoposide, hidroxyurea, MMS, UV), and we find that the K27M mutation does not cause any growth defect in this model organism. We performed bulk- and single-molecule FRET measurements on in vitro reconstituted nucleosomes and observed a more opened conformation of the H3.3K27M nucleosomes, however the overall stability of the mutant NCPs has been similar to wild-type. Importantly, by FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) we reveal a significantly higher mobile fraction and faster repopulation rate of the H3.3K27M histones. The latter suggests that the disease caused by the H3.3K27M histone is related to the altered mobility and diffusion kinetics of mutant nucleosome core particles.
This work was supported by the European Union (FP7/MCA-CIG) and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1-2012-0001 ‘National Excellence Program’, also by the Hungarian Scientific Research Fund (OTKA-PD) and CRP-ICGEB international research grant.
20.00 - Closing Gala Dinner at the Hungarian Academy of Sciences (HAS)
Bus will leave from the RCNS (MTA-TTK) at 19.30 and from Gellert Hotel at 19.40.
The bus is organized only one way since the HAS is in the very hearth of the city with excellent pubs on the Pest side and nice walks on the Danube bank. More info about the walks and public transport in the MAPS Section.
Please sign up for bus transfer at the registration desk during the Thursday!
Location: Akademia Klub
Magyar Tudományos Akadémia
1051 Budapest, Széchenyi István tér 9.