Project description:The mammalian circadian clock relies on the master genes CLOCK (CLK) and BMAL1 and drives rhythmic gene expression to regulate biological functions under circadian control. We recently uncovered a surprising disconnect between the rhythmic binding of CLK:BMAL1 on DNA and the transcription of its target genes, suggesting that they are regulated by as yet uncharacterized mechanisms. Here we show that rhythmic CLK:BMAL1 DNA binding promotes rhythmic chromatin opening. The underlying mechanisms include CLK:BMAL1 binding to nucleosomes and rhythmic chromatin modifications, including the incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLK:BMAL1, suggesting that the activity and the tissue-specific expression of these other transcription factors contribute to the genome-wide CLK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation, well beyond the current confines of developmental/cell specification. H2A.Z ChIP-Seq signal in the mouse liver over 6 time points of the 24h light:dark cycle, in wild-type and Bmal1-/- mice. Libraries containing a mononucleosome insert were sequenced using Ilumina HiSeq2000.
Project description:The mammalian circadian clock relies on the master genes CLOCK (CLK) and BMAL1 and drives rhythmic gene expression to regulate biological functions under circadian control. We recently uncovered a surprising disconnect between the rhythmic binding of CLK:BMAL1 on DNA and the transcription of its target genes, suggesting that they are regulated by as yet uncharacterized mechanisms. Here we show that rhythmic CLK:BMAL1 DNA binding promotes rhythmic chromatin opening. The underlying mechanisms include CLK:BMAL1 binding to nucleosomes and rhythmic chromatin modifications, including the incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLK:BMAL1, suggesting that the activity and the tissue-specific expression of these other transcription factors contribute to the genome-wide CLK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation, well beyond the current confines of developmental/cell specification. Mouse liver CLK ChIP-Seq signal on Mnase-digested or sonicated chromatin, at 2 different time point (ZT22 and ZT06) from the same mice. Libraries were sequenced using Ilumina HiSeq2000. For Mnase-digested chromatin, libraries contained a mononucleosome insert (e.g., ~147bp), whereas the insert was ~150-300bp for sonicated chromatin.
Project description:The mammalian circadian clock relies on the master genes CLOCK (CLK) and BMAL1 and drives rhythmic gene expression to regulate biological functions under circadian control. We recently uncovered a surprising disconnect between the rhythmic binding of CLK:BMAL1 on DNA and the transcription of its target genes, suggesting that they are regulated by as yet uncharacterized mechanisms. Here we show that rhythmic CLK:BMAL1 DNA binding promotes rhythmic chromatin opening. The underlying mechanisms include CLK:BMAL1 binding to nucleosomes and rhythmic chromatin modifications, including the incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLK:BMAL1, suggesting that the activity and the tissue-specific expression of these other transcription factors contribute to the genome-wide CLK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation, well beyond the current confines of developmental/cell specification. Mouse liver nucleosome profile assayed by MNase-Seq over 6 time points of the 24h light:dark cycle (4 wild-type and 4 Bmal1-/- mice per time point). Illumina libraries containing a mononucleosome insert were sequenced using Ilumina HiSeq2000.
Project description:RUVBL2 is most important AAA+ ATPase for RNA polymerase II assembly and transcription regulation, through DNA remodeling or by directly interaction with PIC,this study will comprehensively to study the promiscuous functions of this proteins through the ChIP-MS, ChIP-seq, RNA-seq and nascent RNA seq and biochemistry analysis. Our study would provide more systematic and novel responsibility of this molecule, especially for the development and carcinomas.
Project description:The nucleosome plays a central role in genome regulation. Traditional methods for mapping nucleosomes depend on the resistance of the nucleosome core to micrococcal nuclease (MNase). However, the lengths of the protected DNA fragments are heterogeneous, limiting the accuracy of nucleosome position information. To resolve this problem, we removed residual linker DNA by simultaneous digestion of yeast chromatin with MNase and exonuclease III (ExoIII). Paired-end sequencing of mono-nucleosomes revealed not only core particles (145-147 bp), but also intermediate particles in which ~8 bp project from one side (154 bp) or both sides (161 bp) of the nucleosome core. We term these particles "pseudo-chromatosomes" because they are present in yeast lacking linker histone. They are also observed after MNase-ExoIII digestion of chromatin reconstituted using recombinant core histones. We propose that the pseudo-chromatosome provides a DNA framework to facilitate H1 binding. Comparison of budding yeast nucleosome sequences obtained using micrococcal nuclease (MNase-seq) and MNase + exonuclease III (ExoIII) (MNase-ExoIII-seq): wild type cells and hho1-null cells. Nucleosome sequences from native chromatin and H1-depleted chromatin from mouse liver. Nucleosome sequences from a plasmid reconstituted into nucleosomes using recombinant yeast histones or native chicken erythrocyte histones.
Project description:The Olfactory Receptor (OR) genes are specifically expressed in the MOE in a monogenic and monoallelic fashion. Only 1 out of 2800 alleles is expressed in each olfactory sensory neuron in mice. ChIP-chip from mouse olfactory epithelium (OE) and liver for H3K9me3 and H4K20me3 revealed that the ORs are highly enriched for these modifications in OE but not in liver. 24 samples were analyzed (20 from OE and 4 from liver) with matching inputs as controls. For the OE samples, there were 2-3 replicates for each array.
Project description:Over the past decade, genome-wide assays have underscored the broad sweep of circadian gene expression. A substantial fraction of the transcriptome undergoes oscillations in many organisms and tissues, which governs the many biochemical, physiological and behavioral functions under circadian control. Based predominantly on the transcription feedback loops important for core circadian timekeeping, it is commonly assumed that this widespread mRNA cycling reflects circadian transcriptional cycling. To address this issue, we directly measured dynamic changes in mouse liver transcription using Nascent-Seq. Many genes are rhythmically transcribed over the 24h day, which include precursors of several non-coding RNAs as well as the expected set of core clock genes. Surprisingly however, nascent RNA rhythms overlap poorly with mRNA abundance rhythms assayed by RNA-seq. This is because most mouse liver genes with rhythmic mRNA expression manifest poor transcriptional rhythms, indicating a prominent role of post-transcriptional regulation in setting mRNA cycling amplitude. To gain further insight into circadian transcriptional regulation, we also characterized the rhythmic transcription of liver genes targeted by the transcription factors CLOCK and BMAL1; they directly target other core clock genes and sit at the top of the molecular circadian clock hierarchy in mammals. CLK:BMAL1 rhythmically bind at the same discrete phase of the circadian cycle to all target genes, which not surprisingly have a much higher percentage of rhythmic transcription than the genome as a whole. However, there is a surprisingly heterogeneous set of cycling transcription phases of direct target genes, which even include core clock genes. This indicates a disconnect between rhythmic DNA binding and the peak of transcription, which is likely due to other transcription factors that collaborate with CLK:BMAL1. In summary, the application of Nascent-Seq to a mammalian tissue provides surprising insights into the rhythmic control of gene expression and should have broad applications beyond the analysis of circadian rhythms. CLK and BMAL1 DNA binding profile in the mouse liver at ZT8, sequenced along an Input sample using GAII (ChIP-Seq) Supplementary file ChIPSeq_Mouse_Liver_Processed_data_Table1.txt represents annotated CLK and BMAL1 peaks.
Project description:Stable inheritance of DNA methylation is critical for maintaining the differentiated phenotypes in multicellular organisms. However, the molecular basis ensuring high fidelity of maintenance DNA methylation is largely unknown. Here, we demonstrate that two distinct modes of DNMT1 recruitment, one is DNA replication-coupled and the other is uncoupled mechanism, regulate the stable inheritance of DNA methylation. PCNA-associated factor 15 (PAF15) represents a primary target of UHRF1 and undergoes dual mono-ubiquitylation (PAF15Ub2) on chromatin. PAF15Ub2 specifically interacts with DNMT1 and controls the recruitment of DNMT1 in a DNA replication-coupled manner. Thus, loss of PAF15Ub2 results in impaired DNA methylation at sites replicating during early S phase. In contrast, outside of S phase or when PAF15 ubiquitylation is perturbed, UHRF1 ubiquitylates histone H3 to promote DNMT1 recruitment. Together, we identify replication-coupled and uncoupled mechanisms of maintenance DNA methylation, both of which collaboratively ensure the stable DNA methylation.
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect. Mnase-seq, Mnase-ChIP-seq of Drosophila melanogaster embryo and S2 cells chromatin
Project description:The centromere is a defining feature of eukaryotic chromosomes and is essential for the segregation of chromosomes during cell division. Centromeres are universally marked by the histone variant cenH3 and are restricted to specialized chromatin that most commonly localized to a single position along the chromosome. However, the DNA on which centromeric nucleosomes assemble is not conserved and varies greatly in size and composition. It ranges from genetically defined point centromeres that assemble a single cenH3-containing nucleosome to epigenetically defined regional centromeres embedded in megabases of tandemly repeated DNA to holocentromeres that extend along the length of the entire chromosomes. The organization of regional and holocentric centromeres has so far been elusive, as the precise locations of cenH3-containing sequences could not be determined. Our results show that the point centromere is the basic unit of holocentromeres and provide a basis for understanding how centromeric chromatin is maintained. We use high-resolution mapping of cenH3-associated DNA to show that Caenorhabditids elegans holocentromeres are organized as dispersed but discretely localized point centromeres.