Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A; K27A; K79A) and control mESCs

ABSTRACT: 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.

INSTRUMENT(S): NextSeq 500, NextSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: Charles Girardot

PROVIDER: E-MTAB-12868 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Missense mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines. In contrast to wild-type p53, the mutant p53 (mutp53) protein has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but retained the property to interact with DNA in a structure-specific fashion. Expression of mutp53 is advantageous for tumor cells, however the molecular mechanism of mutp53 action is still not known. We used the glioblastoma-derived U-251 MG human cell line to analyze DNA binding of mutant p53 (R273H mutation) on a Nimblegen custom 135k tiling array and to correlate mutp53 binding regions with the epigenetic state and occupation by other transcription factors (ETS1 and SP1). We found that mutp53-binding regions are G/C-rich and are located around transcriptional start sites (TSS) of many protein-coding genes, which in most cases are active, but are not always regulated upon transient mutp53 depletion. We propose a model which does not only rely on interactions of mutp53 with diverse transcriptional regulators at active promoters, but primarily is based on a DNA binding activity of mutp53. We designed a Nimblegen custom 135k tiling array that covers a large set of putative and known p53 (wild-type and mutant) target genes in the human genome. For analysis of the epigenetic state of genes covered by the tiling array in control and mutant p53-depleted U251 cells we focused on changes in active histone marks, H3K4me3 and H3K9Ac, and RNA polymerase II recruitment and processivity. H3K4me3 and H3K9Ac marks are enriched in active promoter regions and the phosphorylation of serine 5 (S5-P) and serine 2 (S2-P) in the CTD of RNA polymerase II have been described to define initiated and elongating complexes, respectively. We performed the ChIP-chip experiments for H3K4me3, H3K9Ac, RNA polymerase II (S5-P) and RNA polymerase II (S2-P) from U251 cells transfected with p53-specific siRNA or control siRNA (2 biological replicates each). To analyze binding of mutant p53 to the genes covered by the tiling array we performed mutant p53 ChIP-chip experiments in 4 biological replicates of. In addition, we analyzed the distribution of SP1 and ETS1 binding sites in 3 biological replicates.

Dataset Information

ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A; K27A; K79A) and control mESCs

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