Project description:Characterization of the H3K9me2 distribution in the C. elegans adult hermaphrodite genome using ChIP-Seq Examination of H3K9me2 in two different nematode strains: fer-1 control and fer-1;him-8 mutant, where the X chromosomes are unsynapsed. The fer-1 mutation prevents production of embryos.
Project description:Chavez2009 - a core regulatory network of OCT4 in human embryonic stem cells
A core OCT4-regulated network has been identified as a test case, to analyase stem cell characteristics and cellular differentiation.
This model is described in the article:
In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach.
Chavez L, Bais AS, Vingron M, Lehrach H, Adjaye J, Herwig R
BMC Genomics, 2009, 10:314
Abstract:
BACKGROUND: The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency.
RESULTS: We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 - 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation.
CONCLUSION: Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies.
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Project description:During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments were enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome were upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.
Project description:Here we report the discovery of a set of potent de-novo cyclic peptides (CPs) targeting different binding sites on KDM7B. One CP (OC9) bound directly to the KDM7 PHD-finger, as supported by bio-layer interferometry (BLI), isothermal calorimetry (ITC), hydrogen-deuterium exchange mass spectrometry (HDxMS) and NMR studies, and was highly selective for KDM7s over other PHD-fingers. OC9 disrupted PHD-finger binding to H3K4me3, and allosterically modulated KDM7 demethylase activity at H3K9me2 site on peptides and histone extracts demonstrating PHD-finger targeting is as JmjC-domain targeted inhibitors, but more selective for specific KDM7 subfamily member and certain combinatorial histone PTM signatures. Proteomic analysis confirmed OC9 to selectively target KDM7 in nuclear lysates demonstrating its high affinity and selectivity against other H3K4me3 reader domains and KDMs.
Project description:Exon array analysis component. Development, differentiation, and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of induced or temporally regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from myriads of potential genomic binding sites is undetermined. Here we show that up to 95% of induced de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligandactivated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cellselective de novo genomic occupancy patterns appear to be comprehensively predetermined by cellspecific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a novel framework for understanding regulatory factorgenome interactions, and provide a molecular basis for the tissueselectivity of steroid pharmaceuticals and other agents that intersect the living genome. Time course of dexamethasone treatment of 2 cell types from a zero hour untreated control, 2 hour, 4 hour and 8hr treatment times. This is part of a larger study: Chromatin accessibility predetermines de novo steroid receptor binding. There are 2 replicates per cell line per time point except in the case of 8hr Dex treated AtT-20 where a second replicate was unavailable.
Project description:Chromatin state influences lifespan and may allow for the epigenetic inheritance of this complex trait. At sites of active transcription, the COMPASS complex methylates histone H3 at lysine 4 (H3K4me). In Caenorhabditis elegans, reductions in COMPASS extend lifespan, and wild-type descendants of COMPASS mutants inherit longevity for four generations. Here we show that the longevity of COMPASS mutants is itself a transgenerational trait caused by gradual changes in the repressive chromatin factor H3K9me2. H3K9me2 is required for longevity in COMPASS mutants and can confer longevity when increased in other chromatin modifier mutants. H3K9me2 levels also correlate with a transgenerational decline in wild-type lifespan after freezing or starvation. We propose that germline transcription-coupled H3K4me encroaches on H3K9me2 to limit lifespan. Loss of COMPASS complex alleviates the burden of H3K4me and therefore extends lifespan. This study suggests a causal role for a single heterochromatin factor in the establishment and inheritance of longevity.
Project description:NGPS is a method for de-novo, full-length protein sequencing in high throughput. The method is based on cleavage of the protein at semi-random sites by microwave-assisted acid hydrolysis (MAAH), enrichment of LC-MS/MS amenable peptides from the hydrolysate by solid-phase-extraction, LC-MS/MS analysis, de-novo long peptide tag sequencing of resulting peptides and assembly of peptide tags into consensus contigs.
Project description:The establishment and maintenance of chromatin domains shape the epigenetic memory of a cell, with histone H3 lysine 9 methylation defining repressed heterochromatin. We show that in C. elegans the SET-25 (SUV39/G9a) HMT that catalyzes H3K9me1-3, is able to establish repressed domains de novo. We identify here two distinct pathways that recruit SET-25 to its targets. One requires LIN-61 (L3MBTL2), a conserved protein with 4 MBT domains that recognizes H3K9me2 deposited by the HMT MET-2 (SETDB1). The second pathway is MET-2-independent and requires a somatic Argonaut NRDE-3 and 22nt small nuclear RNAs. This NRDE-3 pathway targets ~10% of all SET-25-modified loci genome-wide including intact RNA and DNA transposons. Removal of both pathways in the met-2;nrde-3 double mutant synergistically derepresses transposons in early embryos and elevates embryonic lethality. The redundancy of these pathways illustrates the key role played by chromatin-mediated silencing in protecting the genome against inherent threats.