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作者: shine_9457 | 来源:发表于2021-02-05 11:36 被阅读0次

The Chaperone FACT and Histone H2B Ubiquitination Maintain S. pombe Genome Architecture through Genic and Subtelomeric Functions

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Pob3 控制5‘末端的基因；
Spt16 控制3’末端的基因；
H2BUb对FACT的影响，促进
H2BUb调控？组蛋白；

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背景介绍：

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组蛋白H2B泛素化修饰与组蛋白伴侣分子FACT
揭示H2Bub如何调节FACT活性，并确定FACT复合物作用(去泛素化酶与染色质调控)
知识点：
组蛋白泛素化(酶： E1s, ubiquitin-activating enzymes; E2s, ubiquitin-conjugating enzymes; E3s, ubiquitin ligases)
H2B 泛素化激活转录，并在组蛋白伴侣分子FACT帮助下在转录延伸中发挥作用。
泛素化(ubiquitination)：泛素(小分子多肽)以单体和多聚体形式，经由酶促反应与底物蛋白结合并发生共价修饰的过程。
去泛素化是指泛素化的底物蛋白在去泛素化
酶(deubiquitylating enzymes，DUB)的作用下移除泛素的反应过程。
去泛素化酶存在使得泛素修饰的调节具有平衡性．
H2Bub1 的泛素化通过促进组蛋白的其他修饰和在组蛋白分子伴侣 FACT 的依赖下改变核小体结构，从而影响基因转录。
文章重点：
1 FACT and H2Bub repress 5‘ and gene body antisense
transcripts, respectively # FACT and H2Bub 抑制5’和反义转录本
2 H2Bub regulates FACT genic distribution, histone binding,
and chaperone activity # H2Bub调节FACT基因分布、组蛋白结合和伴侣活性
3 The S. pombe Spt16 subunit controls 3' end of genes in the
absence of Pob3 # Spt16 亚基(无 Pob3 )控制 3' end
4 FACT maintains highly compacted subtelomeric knobs in
fission yeast # FACT maintains 裂变酵母S.pombe亚端粒highly compacted。
Genic and Subtelomeric Functions
提出问题：
The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA polymerase II (Pol II) passage through chromatin, yet it is not clear how they cooperate mechanistically；it is still not understood how and which genic nucleosomes FACT recognizes and how its activity is modulated in distinct
genomic contexts. Further, whether and how the two subunits of FACT have distinct functions has not been well studied in a genome-wide context.
组蛋白伴侣FACT和组蛋白H2B泛素化(H2Bub)促进RNA 聚合酶II(Pol II)通过染色质,不清楚如何作用；
方法：We used genomics,genetic, biochemical, and microscopic approaches
to dissect their interplay in Schizosaccharomyces pombe（酵母）
实验结果：

We show that FACT and H2Bub globally repress antisense transcripts near the 5‘ end of genes and inside gene bodies, respectively. （结论一）
The accumulation of these transcripts is accompanied by changes at genic nucleosomes and Pol II redistribution.（关系/）

H2Bub is required for FACT activity in genic regions.（结论二）
In the H2Bub mutant, FACT binding to chromatin is altered and its association with histones is stabilized, which leads to the reduction of genic nucleosomes（关系/）

in the absence of Pob3, the FACT Spt16 subunit controls the 30 end of genes（结论三）

FACT maintains nucleosomes in subtelomeric regions, which is crucial for their compaction.（结论四）

关于组蛋白伴侣

Histone chaperones are a diverse family of proteins that sequester histones from DNA and help deposit or remove them during DNA-based processes,including transcription, replication, and DNA repair.
======》FACT (facilitates chromatin transcription) 由两个亚基组成:Spt16和SSRP1/Pob3（FACT由亚基SPT16和SSRP11组成,促进基因转录、DNA复制和DNA损伤修复过程中的核小体解构和重建参考）

Spt16 identified as a suppressor of Tyelements in Saccharomyces cerevisiae(酵母-酪氨酸的抑制剂)
Pob3 was identified by co-purification with DNA polymerase I
The human FACT complex was identified as a factor essential for RNA polymerase II to transcribe through chromatin

转录延伸因子-FACT促进
H2A-H2B dimers are bound by the unstructured C-terminal domains of Spt16 and Pob3 that compete with DNA binding to histones。（H2A-H2B二聚体与Spt16和Pob3的非结构化C端结构域结合-----》与与组蛋白的DNA结合竞争）

FACT is predominantly associated with gene bodies of actively transcribed genes in vivo

FACT is overexpressed in various tumors and stem cells, suggesting a role in the maintenance of undifferentiated cell states
implicated in genome stability and chromosome segregation.

提出问题

已有研究表明

(H2Bub)与 FACT 有协同作用 --->
H2Bub contributes to FACT binding or stabilization at chromatin
report showed that FACT stimulates deubiquitinase activity of Ubp10 in S. cerevisiae ---->
the interplay between FACT and H2Bub ---> complexity
Whether H2Bub has a direct role in FACT binding or chaperoning activity 未知；

实验方法：

used the fission yeast Schizosaccharomyces pombe as a powerful model organism to study FACT and H2Bub functions in genome organization.
1 deletion of the pob3 gene reduces FACT activity

实验结果：

show that within gene bodies, FACT and H2Bub contribute to the proper maintenance of genic nucleosomes and repression of antisense transcription at 50 and genebodies,（FACT和H2Bub有助于维持基因核小体并抑制5‘和基因的反义转录）

RNA-seq(针对两个高度可重复的生物学样本中进行链特异性RNA测序)
解读：PCoA分析主成分分析图；
作图-R 语言；多变量统计学方法PCA 分析和作PCA 图
PC1 表示尽可能最大解释数据变化的主坐标成分
PC2 为解释余下的变化度中占比例最大的主坐标成分
在该图中：可以观察到三个不同颜色的+表示的样本区域是重叠的wild-type (WT); H2BK119R; pob3Δ，

参数解读：log2FC(两组间某基因的差异倍数,fold change)；其中，做差异分析，用edgeR 或者DEseq2等R包分析后产生fold change值；
H2Bub（H2BK119R）和FACT（pob3Δ）都影响基因活性；对反义转录的有抑制效应；

S1B-MA-plots mean expression-log2 Fold Change : 衡量基因表达上下调

参数解读：log2 fold change
差异倍数(fold change) :
假设A基因表达值为1，B表达值为3，那么B的表达就是A的3倍
用了log2 fold change后，差异基因里负数代表下调、正数代表上调
当expr(A) < expr(B)时，B对A的fold change就大于1，log2 fold change就大于0（y轴上半），B相对A就是上调；反之

结合上图，H2BK119R vs 突变体，有2,012个反义转录物显着上调；在pob3D vs 突变体，有2,450个反义转录物显着上调
--> 两组中反义转录本中存在正相关关系

在FACT突变体中， 994个基因表达明显上调，1001个基因表达下调
在H2BK119R突变体中，1,418个基因和1,374个基因分别上调和下调
虽然两个突变体中下调基因的数量相似，但结合下图它们之间的相关性相对较低

比较pob3Δ与野生型（WT）与 H2BK119R与野生型（WT）

Spearman´s R 相关系数
--->提示 FACT和 H2Bub抑制的反义转录本之间存在显著的genes overlap

S1B-MA-plots MA-plot：基因丰度和表达变化之间的关系
antisense：2,012 vs 2,450

S2B-Density colored scatterplots 上图表明受抑制的反义转录物正相关？
结论：
Thus, FACT and H2Bub globally repress antisense transcription in S. pombe

H2B泛素化

H2Bub和FACT抑制反义转录

与转录起始TSS对齐的RNA-seq log2FC的热图

热图包括距TSS上游1 kb和下游5 kb的基因组区域。基因按照长度和分组：I–IV（基因总计= 5,069）
研究---> 受抑制的反义转录物是否影响相应的有义转录；根据有义和反义转录之间的关系对基因clustered
第一组都略有下调(虚线吗？)
第二组：上调
第三组：具有下调基因和反义转录本的组相对较小
第四组：具有上调基因和下调反义转录本
因此，由于H2BK119R和pob3D突变体中反义转录的增加，大量基因可能被潜在地下调。但是，尽管具有类似的反抑制的反义转录，但仍有类似数量的基因上调。这项分析是根据散点图进行的，该散点图没有揭示正义和反义转录本之间的任何关联（与1A与1B)得出的信息一致）
与转录起始位点（TSS）和转录终止位点（TTS）对齐；得到下图