Genome-wide measurement of Nkx-6.1 in mouse islets.
Ontology highlight
ABSTRACT: We perfomed a ChIP-seq experiment to identify the location of binding sites for the transcription factor Nkx6-1 in mouse islets. A single set of islets were extracted from a mouse and Nkx-6.1 and input ChIP-Seq libraries were prepared and sequenced.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells and characterized genome-wide SetDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SetDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SetDB1 solo peaks, particularly those near neural development related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins Jarid2 and Mtf2. Genetic deletion of Setdb1 dramatically reduced Ezh2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SetDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SetDB1 methyltransferase activity in vitro. The currently identified reciprocal action between SetDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation. Examination of 2 different histone modifications in 2 cell status.
Project description:Skeletal muscle tissue shows an extraordinary cellular plasticity, but the underlying molecular mechanisms are still poorly understood. Here we use a combination of experimental and computational approaches to unravel the complex transcriptional network of muscle cell plasticity centered on the peroxisome proliferator-activated receptor M-NM-3 coactivator 1M-NM-1 (PGC-1M-NM-1), a regulatory nexus in endurance training adaptation. By integrating data on genome-wide binding of PGC-1M-NM-1 and gene expression upon PGC-1M-NM-1 over-expression with comprehensive computational prediction of transcription factor binding sites (TFBSs), we uncover a hitherto underestimated number of transcription factor partners involved in mediating PGC-1M-NM-1 action. In particular, principal component analysis of TFBSs at PGC-1M-NM-1 binding regions predicts that, besides the well-known role of the estrogen-related receptor M-NM-1 (ERRM-NM-1), the activator protein-1 complex (AP-1) plays a major role in regulating the PGC-1M-NM-1-controlled gene program of hypoxia response. Our findings thus reveal the complex transcriptional network of muscle cell plasticity controlled by PGC-1M-NM-1. We performed ChIP-Seq experiments to identify all DNA recruitment sites for PGC-1alpha in C2C12 cells on genome-wide scale. The experiment was performed in duplicate and the Whole Cell Extract (WCE; =input DNA) was used as background condition.
Project description:In this study, we compare the binding pattern of CTCF at the T cell receptor alpha locus in different subsets of developing lymphocytes For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina Genome Analyzer IIx.
Project description:In this study, we compare the binding pattern of CTCF at the T cell receptor alpha locus in different subsets of developing lymphocytes For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina Hi-Seq.
Project description:In this study, we compare the binding pattern of CTCF at the T cell receptor alpha locus in different subsets of developing lymphocytes. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina Genome Analyzer IIx.
Project description:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and EM-NM-<, the start site of IM-NM-< germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is EM-NM-<-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-EM-NM-< interactions. ChIP-seq shows high level YY1 binding only at EM-NM-<, but low levels near some antisense promoters. PAIR-EM-NM-< interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to EM-NM-<. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement. ChIP Seq YY1 vs. input control
Project description:It is well-known that embryonic stem cells (ESC) are much more sensitive to replication-induced stress than differentiated cells but the underpinning mechanisms are largely unknown. H2A.X, a minor variant of H2A, constitutes only 1-10% of the mammalian genome. H2A.X plays a well-known for role in the DNA damage response and maintaining stability in the genome, including the regions frequently experiencing replication stress, such as the fragile sites. Intriguingly, several recent studies have reported that H2A.X function is elevated in ESC; and others reported that H2A.X function is provoked during cellular reprogramming (in induced pluripotent stem cells, iPSC), indicating that increased proliferation during iPS may trigger replication stress and the H2A.X DNA damage response. However, several studies of genomic instability in iPSC led to different conclusions on this important issue. For example, frequent copy number variants (CNV) were reported at the genomic regions sensitive to replication stress, such as the fragile sites. On the other hand, another study reported the lack of genomic instability in mouse iPS clones that are able to generate “all-iPS” animals in tetraploid complementation assays (4N+ iPSC), indicative of a potential link between pluripotency and genome integrity. However, whether if high level genomic instability occurs in the 4N- iPSC iPSC clones at replication stress sensitive regions is unknown. Moreover, due to the lack of mechanistic insights on genome integrity maintenance, how pluripotency and genome integrity are connected remains elusive. Here we show that H2A.X plays unexpected roles in maintaining pluripotency and genome integrity in ESC and iPSC. In ESC, it is specially enriched at genomic regions sensitive to replication stress so that it protects genome integrity thereat. Faithful H2A.X deposition is critical for genome integrity and pluripotency in iPSC. H2A.X depositions in 4N+ iPSC clones faithfully recapitulate the ESC pattern and therefore, prevent genome instability. On the other hand, insufficient H2A.X depositions in 4N- iPSC clones at such regions lead to genome instability and defects in replication stress response and DNA repair, reminiscent of the H2A.X deficient ESC. Detect and compare different H2A.X deposition patterns in ES cells and iPS cells, with Illumina HiSeq 2000 and Illumina Genome Analyzer IIx
Project description:We found that Rif1 depletion leads to reduced H3K9me3 levels at 1/3 of the H3K9me3-enriched genomic regions (H3K9me3 peaks), and that reduced H3K9me3 de-represses Zscan4 and other genes that are specific to the 2-Cell stage embryo. Examination of H3K9me3 histone modifications in 2 cell types.