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ChIP-seq of H3.3K4A/K36A mutant ESCs and neurons

ABSTRACT: Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. Genomic localization of H3.3 protein was determined by ChIP-Sequencing in ESCs (D0). Histone modifications patterns of H3K4me1, H3K4me3 and H3K27ac were measured by ChIP-Sequencing in ESCs (D0) to assess the impact of the H3.3K4A mutation on the epigenetic landscape. Levels of H3K36me3 were measured by ChIP-Sequencing in WT and H3.3K36A mutant ESCs (D0), NPCs (D8) and neurons (D12) to assess the impact of the H3.3K36A mutation on H3K36me3 levels in development.

ORGANISM(S): Mus musculus

SUBMITTER: Charles Girardot

PROVIDER: E-MTAB-6822 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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ChIP-seq of H3.3K4A/K36A mutant ESCs 2

Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. Genomic localization of H3.3 protein was determined by ChIP-Sequencing in ESCs (D0). Distribution patterns of RNA Polymerase II Phosphorylated on Serine 5 (RNA Pol II Ser5P), of histone modification H3K27me3 and chromatin remodeler components Brg1/Smarca4 (Swi/Snf) and Chd4 (NuRD) were measured by ChIP-Sequencing in ESCs (D0) to assess the impact of the H3.3K4A mutation on the epigenetic landscape. Distribution patterns of H3.3 were assessed by ChIP-Sequencing in HEK293T cells after depletion of Brg1/Smarca4 (Swi/Snf) and Chd4 (NuRD).

2020-01-28 | E-MTAB-8614 | biostudies-arrayexpress

RNA-seq of H3.3K4A/K36A mutant ESCs and neurons

Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. Gene expression profiles were measured by mRNA-Sequencing in undifferentiated ESCs (D0), neurodevelopment (D8) and differentiated neurons (D12) to assess the impact of the mutation on gene expression and development.

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A knockout-first model of H3f3a gene targeting leads to developmental lethality

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Project description:Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9, K27 or K79. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to profile the changes in histone modifications, in the different H3.3 mutant mESC lines. The following histone modifications were profiled: H3K27ac (39685, Active Motif), H3K9ac (C5B11-9649, Cell Signalling Technology), H3K27me3 (C36B11, Cell Signalling Technology), H3K9me2 (D85B4, Cell Signalling Technology), H3K9me3 (D4W1U, Cell Signalling Technology). H3.3 (09-838, Merck-Millipore, purified polyclonal) ChIP-seq was performed in order to profile the deposition of this histone variant in the H3.3 K9A and K27A mESC mutant lines. SUZ12 (D39F6-3737, Cell Signalling Technology) ChIP-seq was performed to profile the genomic distribution of the Polycomb repressive complex 2 (i.e., PRC2). Two to three independent replicates per condition were grown and harvested for each ChIP experiment. Matched input controls were also generated in every experiment. Sequencing libraries were prepared using the NEBNext Ultra II library preparation kit (New England Biolabs) and sequenced on NextSeq500 or NextSeq2000 platforms in single-end mode.

2024-03-09 | E-MTAB-12868 | biostudies-arrayexpress

Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells [ChIP-Seq]

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Project description:We used H3f3b-/- testes to analyze the impact of H3f3b derived H3.3 on spermatogenesis.

2014-09-09 | GSE60207 | GEO

Histone H3.3 regulates dynamic chromatin states during spermatogenesis

Project description:We used H3f3b-/- testes to analyze the impact of H3f3b derived H3.3 on spermatogenesis. Analysis of Histone H3 (Lys 4) tri-methylation in H3f3b WT/WT and H3f3b -/- testes tissue.

2014-09-09 | E-GEOD-60207 | biostudies-arrayexpress

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