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DIPG的H3K27M是通过H3K27ac而不是H3K27me3加重癌症的

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发表于 2017-6-6 17:52:12 | 显示全部楼层 |阅读模式
做了100个测序数据: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE78801
DIPG 这个疾病背景知识:
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise1.
A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M).
Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3)
以前人们认为:H3K27M has been hypothesized to drive gliomagenesis through PRC2 catalytic inhibition

但是作者却发现;H3K27M correlates with H3K27ac and is excluded from PRC2 targets.

实验材料:
SF8628 human DIPG cells—which contain a mutant H3F3A gene that encodes H3.3K27M
SU-DIPG-IV (hereafter called DIPG-IV) cells, a DIPG cell line carrying a point mutation in the HIST1H3B gene that results in the expression of H3.1K27M
SF7761 cells, another H3F3A-mutant DIPG line
HCT116 cells, a human colorectal tumor cell line
其中 SF8628 和 SU-DIPG-IV  细胞系都被JQ1处理了,3/6/12/24/48小时,做了RNA-seq数据。


背景知识:
JQ1, a well-known bromodomain and extra-terminal domain (BET) inhibitor
CDKN2A (also known as p16), a negative cell-cycle regulator and well-established target of PRC2 transcriptional repression
a small-molecule catalytic inhibitor of EZH2 methyltransferase activity, EPZ-6438
a common PRC2 target gene, cyclin D2 (CCND2)

所以作者做了一系列实验反驳这一点:

实验方面的有:
H3K27M-positive DIPG cells treated with EPZ-6438 show reduced proliferation as compared to vehicle-treated cells
DIPG-IV cells tolerate higher doses of EPZ-6438 than SF8628 cells
As for EED and SUZ12 knockdown, EZH2 catalytic inhibition does not elicit p16 upregulation in DIPG-IV cells

Importantly, H3K27ac is the only substantially increased histone acetylation mark induced by H3K27M, as determined by mass spectrometry analysis



ChIP-seq数据方面的有:
首先做了,H3.3,H3K27ac,SUZ12,EZH2,Rna_Pol_II,input,每个两个重复,加上 H3K4me1 的一个重复,的ChIP-seq数据。
结果是它们的peaks差别很大: PRC2 subunits EZH2 and SUZ12, as well as their enzymatic product, H3K27me3, are largely excluded from chromatin on sites containing H3K27M
而且DIPG细胞系里面的PRC2功效并没有丢失,可以找到peaks,而且SUZ12 or EED knockdown cells 的克隆形成能力和生长能力均显著下降。说明DIPG细胞系的PRC2是必须的,不能被破坏。

转录组数据
SUZ12 or EED knockdown resulted in derepression of p16 expression in SF8628 cells, consistent with a role for CDKN2A as a PRC2 target in these cells
DIPG-IV cells do not upregulate p16 expression upon PRC2 depletion  


Many PRC2 targets are shared between SF8628 and DIPG-IV cells (70% and 40%, respectively;),
and are significantly derepressed upon SUZ12 knockdown as compared to non-PRC2 target genes

H3K27M does not sequester PRC2 on chromatin.

但是发现了虽然失去了 H3K27me3 ,却获得了 H3K27ac
Interestingly, upon loss of H3K27me3, these nucleosomes acquire H3K27ac
所以猜测 acetyl-binding bromodomain proteins  应该在其中参与作用, 因此作者做了 BRD2- or BRD4- and H3K27M 的ChIP-seq数据。
都是针对的 SF8628 和 SU-DIPG-IV 细胞系
这些 ChIP-seq 的结果很有趣:
BRD2- or BRD4- and H3K27M-co-occupied loci represent 85% of all BRD2- and BRD4-positive regions in SF8628 cells

然后是JQ1处理的时间序列数据。
To address this point, we treated SF8628 cells and DIPG-IV cells with JQ1, a well-known bromodomain and extra-terminal domain (BET) inhibitor
Fluorescence-activated cell sorting (FACS) analyses also confirmed that the majority of JQ1-treated cells demonstrated upregulation of the mature neuron marker

we performed RNA-seq analysis during a time-course treatment with JQ1.
PRC2 is required for the oncogenic potential of H3K27M DIPG cells.
CDKN2A is unaffected by JQ1 treatment

Surprisingly, we found a modest but consistent increase of MYC transcripts in both the SF8628 and DIPG-IV cell lines upon JQ1 as compared to vehicle treatment, whereas its closest homolog, MYCN, is silenced by PRC2 in those cells
Given that JQ1 treatment causes marked reduction of global H3K27ac levels in DIPG cells
To further support our in vivo data, we used another BET inhibitor (I-BET151) to treat our xenograft DIPG model

Strikingly, H3K27M colocalizes with transcriptionally active chromatin regions, and it is largely excluded from regions that are occupied by PRC2 and H3K27me3.
Moreover, H3K27M-containing nucleosomes are enriched for acetylated H3K27
In our DIPG cells, PRC2 is almost entirely confined to chromatin regions marked with H3K27me3, and this mark is mutually exclusive with H3K27ac

最后的结论是:
H3K27M excludes PRC2 binding, which thus induces aberrant accumulation of H3K27ac at H3K27M-binding sites, consistent with PRC2 loss of function in other systems

although locus-specific reduction of PRC2 activity or binding by H3K27M could promote DIPG formation, the overall PRC2 function is essential for tumor maintenance.



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