MRNA-seq of H3.3 mutant (K9A K27A K79A) and control mESCs
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ABSTRACT:
PROVIDER: PRJEB61021 | ENA |
REPOSITORIES: ENA
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Project description:PRO-seq in control and H3.3 K4A mutant mESCs
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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. To compare the transcriptional profiles of the H3.3 mutant and control mESC lines, three independent replicates per condition were grown and RNA was extracted. After quality assessment of the RNA, messenger RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Preparation Kit for Illumina (New England BioLabs). Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads).
2024-03-09 | E-MTAB-12866 | biostudies-arrayexpress
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 or K27. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. To profile nascent RNA levels in H3.3 mutant and control mESCs, two rounds of PRO-seq experiment were performed. In the first round, three replicates each of H3.3K9A and control (i.e., Ctrl_9) mESCs were used. In the second round, two replicates each of H3.3K27A and control (i.e., Ctrl_27) mESCs were used. After permeabilization of the mESCs (and addition of 5% Drosophila S2 cells spike-in), a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP was carried out to label nascent transcripts. Biotinylated nascent RNA was then enriched as part of the library preparation process. Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads). The experiment allows for an assesment of the changes in the activity of cis-regulatory elements, upon removal of H3.3 K9 or K27 residues.
2024-09-04 | E-MTAB-12869 | biostudies-arrayexpress
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. To assess the effect of the K4A mutation on Pol II activity, nascent RNA levels were mesaured by PRO-seq.
2020-01-28 | E-MTAB-8617 | biostudies-arrayexpress
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
Project description:ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A K27A K79A) and control mESCs
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Project description:PRO-seq (Precision Run-On sequencing) to profile nascent RNA of H3.3 mutant (K9A K27A K79A) and control mESCs
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Project description:Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme Polynucleotide Kinase 3'-Phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.
2024-01-10 | PXD041132 | Pride
Project description:mRNA-seq of iXist-ChrXDom mESCs
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Project description:Reduced Representation Bisulfite Sequencing for mouse in vivo hematopoietic stem cells, 3 conditions: wildtype, expressing dominant negative mutant H3.3 at lysine 9 (K9M) or dominant negative H3.3 at lysine 36 (K36M).
2019-10-07 | GSE123676 | GEO