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Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells (Mnase-seq & ChIP-seq)

ABSTRACT: Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells (Mnase-seq & ChIP-seq)

PROVIDER: PRJNA554664 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9685630_1.fastq.gz	Fastqsanger.gz
	SRR9685630_2.fastq.gz	Fastqsanger.gz
	SRR9685640_1.fastq.gz	Fastqsanger.gz
	SRR9685640_2.fastq.gz	Fastqsanger.gz
	SRR9685650_1.fastq.gz	Fastqsanger.gz

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Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells (Mnase-seq & ChIP-seq)

Project description:We employed an MNase-Transcription Start Site Sequence Capture method to map and determine the accessibility of all nucleosomes, including those that contain H2A.Z, at high coverage for all human Pol II promoters. We uncovered unexpected features of nucleosomal organization in epithelial cells and following transition to a mesenchymal and malignant state. In contrast to the prevailing model, we observe many different types of active and inactive promoter structures that differ in their nucleosome organization and sensitivity to MNase digestion. Further, we found that H2A.Z has an important role in the assembly of repressed promoters into an inaccessible state. Finally, we uncovered a new promoter type in which a stable H2A.Z nucleosome occupies the TSS of an active promoter.

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Regulation of nucleosome architecture and factor binding revealed by nuclease footprinting of the ESC genome

Project description:Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene regulatory factors. Examine three read size footprints from MNase-Seq in EGFP KD, Mbd3 KD, and Smarca4 KD mESCs using ChIP-Seq datasets.

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Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells

Project description:Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells

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Potential Roles of H2A.Z Ubiquitination

Project description:Nucleosome is a highly dynamic macromolecular complex that is assembled and remodeled by the replacement of major histone variant H2A.Z, thereby affecting nucleosome structure and stability.Here we established two cell lines stably overexpressing wild-type H2A.Z and mutant H2A.Z with ubiquitination deficiency to explore the potential effects caused by H2A.Z ubiquitination. Intriguingly, H2A.Z ubiquitination was first revealed to enhance the stability of H2A-H2A.Z heterotypic nucleosomes. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) further revealed that H2A.Z ubiquitination played a crucial role in the transcriptional regulation.

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Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells.

Project description:Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter.

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Project description:While it has been clearly established that well positioned H2A.Z-containing nucleosomes flank the nucleosome depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in trophoblast stem (TS) cells, the level of H2A.Z at promoters decreases during S phase coinciding with homotypic (H2A.Z/H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z/H2A). Surprisingly, these nucleosomes remain heterotypic at M phase. At the TSS, we identify an unstable heterotypic H2A.Z-containing nucleosome in G1 which, strikingly, is lost following DNA replication. These dynamic changes in H2A.Z at the TSS mirror a global expansion of the NDR at S and M which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of H2A.Z at promoters, it is targeted to the centromere when mitosis begins We performed ChIP-Seq experiments (on mouse Trophoblast Stem cells arrested at G1; S and M stages of thecell cycle) using antibodies against histone variant H2A.Z and sequentional ChIP-re-ChIP-Seq experiments using H2A.Z antibody and H2A antibody in sequence. Combining those data sets with microarray gene expression expression data allowed us to see H2A.Z distribution over promoters of mouse coding genes in cell cycle dependant manner. Interestingly, Input also showed cell-cycle dependent effects, but histone H3 could be used as a cell-cycle independent normalisation factor. We also performed ChIP-seq with a CTCF pull-down to investigate its cell-cycle dependent relationship with heterochromatin.

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