Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Christine Rushlow
PROVIDER: E-GEOD-65441 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:A central question in biology is how enhancers are made accessible. The Drosophila embryo is a good model system to study this question as the gene regulatory networks regulating early developmental events have been well characterized. Zelda (Zld) is a uniformly distributed transcription factor (TF) integral to these networks, acting prior to and in collaboration with the patterning TFs to regulate target enhancers. Here we test the hypothesis that Zld directs TF binding, examplified by Dorsal (Dl) which patterns the dorsoventral axis, across the genome by displacing nucleosomes at enhancers. By performing ChIP-seq and MNase-seq experiments on early embryos with or without Zld, we demonstrated that early enhancers are characterized by an intrinsically high nucleosome barrier, which is overcome by Zld, and that without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that enhancers are initially specified across the genome by the binding of Zld, which locally decreases nucleosome occupancy, thereby assisting TFs in accessing their binding motifs and promoting transcriptional activity.
2015-08-21 | GSE65441 | GEO
Project description:Embryonic stem cells (ESCs) and induced-pluripotent stem cells (iPSCs) self-renew and differentiate into an array of cell types in vitro and in vivo. A complex network of genetic and epigenetic pathways regulates the self-renewal and differentiation of these pluripotent cells, and the structure and covalent modifications of chromatin play a prominent role in this process. We examine nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach that enabled the identification of regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. The majority of changes in nucleosomal signatures that occur in differentiation are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of pluripotency and likely identify key regulatory regions that play a role in determining cell identity. Micrococcal nuclease digestion of chromatin in crosslinked cells was followed by high throughput sequencing. These experiments were carried out in four mouse cell types: embryonic stem cells, induced pluripotent stem cells, somatic tail-tip fibroblasts and liver, and three human cell types: H1-OGN embryonic stem cells, H1-OGN induced pluripotent stem cells, and fibroblasts differentiated from H1-OGN ESCs. At least two replicates performed with each cell type were sequenced.
2014-07-31 | E-GEOD-59062 | biostudies-arrayexpress
Project description:Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the function or regulation of these anti-sense promoters or the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNAs. We find that coupled sense and anti-sense TSSs form the boundaries of an evolutionarily conserved and nucleosome-depleted regulatory region with dramatically enriched transcription factor (TF) occupancy. Notably, as the distance between sense and anti-sense TSSs increases, so does the level of TF binding and signal-dependent gene activation. We further discover a cluster of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Remarkably, this signature identifies promoters that are selectively and rapidly activated during immune challenge. We conclude that anti-sense TSSs can serve as potent, local enhancers of sense-strand gene expression by facilitating TF binding and deposition of activating histone modifications. ChIP-seq, Start-RNA-seq, and MNase-seq from mouse bone marrow-derived macrophages (BMDMs), and Start-RNA-seq from mouse embryonic fibroblasts (MEFs) are included. For ChIP-seq, two biological replicates are included for each of three treatment conditions; untreated, 30 minutes lipopolysaccharide (LPS), and 2 hours LPS. Each biological replicate for all ChIP conditions was sequenced in two lanes for depth, creating two technical replicates for each biological replicate. Raw files for paired technical replicates are labeled as repX.1 and repX.2, where repX.1 is technical replicate 1 and repX.2 is technical replicate 2. For Start-RNA-seq in BMDMs, two biological replicates are included for each of three treatment conditions; untreated, 30 min LPS, and 2 hr LPS. For MNase-seq, data from three biological replicates of untreated BMDMs are included. Four technical replicates of biological replicate 1, two technical replicates of biological replicate 2, and four technical replicates of biological replicate 3 are included. Raw files for paired technical replicates are labeled as repX.1, repX.2, repX.3, etc. For Start-RNA-seq in untreated MEFs, two biological replicates are included. Two technical replicates for each of the MEF Start-RNA-seq biological replicates are included. Paired technical replicates are labeled as repX.1 and repX.2.
2015-06-23 | E-GEOD-62151 | biostudies-arrayexpress
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
2015-09-17 | E-GEOD-69177 | biostudies-arrayexpress
Project description:Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure directly regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) as a new histone modification, occurring on the nucleosomal lateral surface. We show that H4K44ac is specific to yeast sporulation, rising during yeast meiosis and displaying genome-wide enrichment at recombination hotspots in meiosis. The H4K44 residue is required for normal meiotic recombination, for normal levels of double strand breaks during meiosis, and for optimal sporulation. Non-modifiable substitution H4K44R results in reduced MNase digestion and decreased binding of recombination-associated proteins at hotspots. Our results show that H4K44ac creates an accessible chromatin environment for key proteins to facilitate meiotic recombination. Two samples, one WT MNase-seq and one MNase-seq from yeast with a lysine->arginine mutation at H4K44, no replicates Two replicates each of MNase-seq in WT and H4K44->R mutant yeast grown in YPD or YPA.
2015-12-07 | E-GEOD-59004 | biostudies-arrayexpress
Project description:We have combined standard micrococcal (MNase) digestion of nuclei with a modified protocol for construction paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including eviction of nucleosomes. Our protocol and mapping method provide a general strategy for characterizing full epigenomes. We used micrococcal nuclease mapping, chromatin immunoprecipitation and paired-end sequencing to determine the structure of yeast centromeres at single base-pair resolution.
2011-10-26 | E-GEOD-30551 | biostudies-arrayexpress
Project description:During animal development, a fertilized egg is initially under the control of maternal products and only starts zygotic transcription after several cell divisions. In animals such as Xenopus, zebrafish and Drosophila, a massive increase in zygotic transcription occurs during the mid-blastula transition (MBT), when cells shift from rapid, synchronous cell divisions without gap phases to prolonged asynchronous divisions. Before MBT, only a few so-called pre-MBT genes are expressed. How transcription is set up during these early stages is poorly understood. For example, paused RNA Polymerase (Pol II) is frequently found at developmental control genes in mammalian embryonic stem cells and Drosophila embryos but when Pol II pausing is first established in the embryo is unknown. We have analyzed the genome-wide Pol II occupancy during the maternal-to-zygotic transition in hand-staged Drosophila embryos. The results show that massive Pol II recruitment and pausing is established during MBT. The ~100 genes that are transcribed before MBT are particularly short, consistent with a need for rapid transcription during these early cell divisions. Remarkably, most of these genes are transcribed without Pol II pausing and this correlates with a TATA-enriched promoter type. This suggests that distinct strategies are used for activation in the early Drosophila embryo and this may reflect general dynamic properties of promoters used throughout development. Mnase-seq in staged Drosophila embryos
2013-07-26 | E-GEOD-41686 | biostudies-arrayexpress
Project description:Quantitative variation of epigenetic marks, such as histone modifications, can modify gene expression and eventually contribute to inter-individual phenotypic variation. Our goal is to investigate natural inter-individual variation of the epigenome in a quantitative manner. To probe the degree of natural epigenomic diversity in S. cerevisiae, we compared two unrelated wild strains using replicated Mnase-seq and ChIP-seq profiling at mononucleosomal resolution for H3K14 acetylation.
2015-07-22 | E-MTAB-2671 | biostudies-arrayexpress
Project description:A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream of transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from subsets of nucleosomes, rather than the whole array, being present in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes, and affects codon usage and amino acid composition in genes. We propose that these ‘seed’ nucleosomes may aid the AT-rich Tetrahymena genome – which is intrinsically unfavorable for nucleosome formation – in establishing nucleosome arrays in vivo in concert with trans-acting factors, while minimizing changes to the coding sequences they are embedded within. All data are from the macronuclear genome. Datasets: 1) Log-phase cells, fixed chromatin, light MNase digest; 2) Log-phase cells, native chromatin, heavy MNase digest; 3) Starved cells, fixed chromatin, light MNase digest; 4) Starved cells, native chromatin, heavy MNase digest; 5) in vitro reconstituted chromatin, 50ul reaction, 4:10 histone:DNA ratio, light MNase digest; 6) in vitro reconstituted chromatin, 50ul reaction, 7:10 histone:DNA ratio, light MNase digest; 7) in vitro reconstituted chromatin, 150ul reaction, 4:10 histone:DNA ratio, light MNase digest; 8) in vitro reconstituted chromatin, 150ul reaction, 4:10 histone:DNA ratio, heavy MNase digest; Control dataset: 9): MNase-digested naked DNA
2015-07-30 | E-GEOD-64061 | biostudies-arrayexpress
Project description:Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned (phased) in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors influence nucleosome phasing in regions that are not close to genes. We performed a combinational mapping of nucleosome positioning and DNase I hypersensitive sites (DHSs) across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Our results support the barrier model for nucleosome organization as a general feature of eukaryote genomes, including plant genomes, and not limited to TSSs. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome. We propose that genome-wide nucleosome positioning in the eukaryotic genomes is orchestrated by genomic regions associated with regulatory proteins. Rice chromatin was digested by micrococcal nuclease (MNase) into mono-nucleosome size. Mono-nucleosomal DNA was isolated and sequenced (MNase-seq) using Illumina sequencing platforms. We obtained a total of 38 million (M) single-end reads from our first MNase-seq experiment and mapped ~26 M to unique positions in the rice genome. We also conducted pair-end sequencing of an independent MNase-seq library, obtained 274 M paired-end reads, and mapped ~231 M read pairs to unique positions in the rice genome.We applied a strategy of combinational mapping of nucleosome positioning and DHSs (GSE26610) to examine whether nucleosome positioning is associated with all cis-regulatory elements in the rice genome. All datasets used in the analysis were developed using rice leaf tissue in the same developmental stage
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