GSM3701065: BT869p21-Rx_cells_H3K27ac; Homo sapiens; ChIP-Seq - SRA

SRX5620611: GSM3701065: BT869p21-Rx_cells_H3K27ac; Homo sapiens; ChIP-Seq
1 ILLUMINA (Illumina HiSeq 4000) run: 28.6M spots, 1.4G bases, 303Mb downloads

Submitted by: NCBI (GEO)

Study: Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas [ChIP-Seq]

PRJNA530223 • SRP190029 • All experiments • All runs

show Abstracthide Abstract

Epigenetic alterations are recurrently observed in cancer and are the subject of active therapeutic investigations. Midline high-grade gliomas (HGGs) are deadly brain tumors characterized by lysine-to-methionine substitutions at position 27 in histone 3 (H3) variants (denoted H3K27M), which are core components of the nucleosome. H3K27M, the first event in midline HGG development, results in a drastic loss of the repressive histone mark H3K27 tri-methylation (H3K27me3), and a notable increase in H3K27 acetylation (H3K27ac), a mark associated with active chromatin and cellular identity. H3K27ac gain was suggested to promote tumorigenesis in H3K27M-HGGs, but how these opposing marks shape oncogenesis remains controversial. We therefore characterized the active regulatory chromatin states in H3.3K27M and H3K27 wild-type HGGs and in H3.3K27M CRISPR/Cas9 knockout tumor-derived cell lines, as an isogenic tumor model of the mutation. We show that H3.3K27M-HGGs have distinct promoter, enhancer, super-enhancer, and core transcription factor circuitries from wild-type HGGs. However, while removal of H3.3K27M restores gross H3K27ac levels to those of wild-type HGGs, we observe minimal disruption of H3K27ac deposition at these active transcriptional elements, suggesting that they are a function of the cell of origin and independent of direct H3K27M mutagenesis and active regulation. Using quantitative ChIP-seq, we show that in H3.3K27M-HGGs, H3K27ac is pervasively deposited across the genome, including at normally silent repeat elements, leading to their increased baseline expression. H3.3K27M cells respond to DNA demethylating agents and histone deacetylase inhibitors, which further increase repeat element expression, including that of specific endogenous retroviral (ERVs) families. Our findings decouple cell lineage programs from H3K27M-dependent pervasive deposition of H3K27 acetylation. De-repression of ERVs may enhance the triggering of innate immune pathways, representing a therapeutic vulnerability in H3.3K27M HGGs. Overall design: We sequenced 17 human HGG tumors (5 H3.3K27WT, 6 H3.3K27M, 6 IDH1 mutants) and 19 patient-derived cell lines and xenografts (8 H3.3K27WT, 11 H3.3K27M) by H3K27ac ChIP-seq. Additionally, we sequenced unedited (2 replicates) and Crispr/Cas9 H3.3K27M-KO clones (4 replicates) for two cell lines (DIPGXIII and BT245).

Sample: BT869p21-Rx_cells_H3K27ac

SAMN11309263 • SRS4566769 • All experiments • All runs

Organism: Homo sapiens

Library:

Instrument: Illumina HiSeq 4000

Strategy: ChIP-Seq

Source: GENOMIC

Selection: ChIP

Layout: SINGLE

Construction protocol: Cells were fixed with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at -80°C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 60 cycles, 10s on 20s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and before ChIP reaction ~5% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing (see below). ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. 25ul Protein A beads were washed and then incubated with 6ug of H3K27ac antibody (Diagenode, C15410196), and 2 million cells of sonicated cell lysate combined with protease inhibitors for 10 hr, followed by 20 min wash cycle with provided wash buffers. Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail (Life Technologies) at 65°C for 30 min followed by 2ul Proteinase K (Thermo Fisher Scientific) at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers' protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. Library preparation was carried out using Kapa HTP Illumina library preparation reagents. Briefly, 25ul of ChIP sample was incubated with 45ul end repair mix at 20°C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50ul buffer enzyme mix for 30°C 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead bound sample was incubated with 45ul buffer enzyme mix and 5ul of different TruSeq DNA adapters (Illumina) for each sample, for 20°C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6x/0.8x ratio of Ampure XP beads (double size selection) set to collect 250-450bp fragments.

Experiment attributes:

GEO Accession: GSM3701065

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 28.6M spots, 1.4G bases, 303Mb

Run	# of Spots	# of Bases	Size	Published
SRR8832311	28,604,830	1.4G	303Mb	2019-06-07

ID:: 7576279

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