Genome-wide maps of RNA polymerase II in WT and ssu72 mutant cells [8WG16_S2_Y1_ChIPseq]

Deletion of ssu72 gene leads to a transcription elongation defect and the increase in the level of Ser5 and Ser7 phosphorylation in CTD of Rpb1 Phosphorylation of the RNA polymerase II C-terminal domain on heptad Y1S2P3T4S5P6S7 coordinates key events during transcription and when its deregulation leads to defects in transcription and RNA processing. Here we report that histone deacetylase complex activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of the antisense transcription when the C-terminal domain phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, while single hos2/set3 mutants have little effect, loss of hos2 or set3 in addition to ssu72? leads to synergistic increase in antisense transcription globally and correlates with increases genomic instability and formation of deleterious R-loop structures across these regions. We propose that Ssu72 is essential to suppress initiation of cryptic transcription in the 3?end of the RNA polymerase II transcribed regions. In the absence of Ssu72, antisense transcription is supressed by the Hos2/Set3 dependent surveillance mechanism to maintain genome stability. Overall design: Rpb1, Ser2-P CTD, and Tyr1-P CTD ChIP-seq in WT and ssu72?

ssu72基因的缺失会引发转录延伸缺陷，并使RNA聚合酶II（RNA polymerase II）最大亚基Rpb1的羧基末端结构域（C-terminal domain, CTD）的Ser5与Ser7磷酸化水平升高。RNA聚合酶II CTD的七肽重复序列Y¹S²P³T⁴S⁵P⁶S⁷的磷酸化过程协同调控转录过程中的关键事件，当其磷酸化失调时会导致转录与RNA加工过程出现缺陷。本研究发现，裂殖酵母（fission yeast）Hos2/Set3复合物的组蛋白去乙酰化酶（histone deacetylase）活性，在因Ssu72磷酸酶（Ssu72 phosphatase）缺失导致CTD磷酸化失调时，对抑制反义转录的隐秘起始具有重要作用。值得注意的是，尽管单一hos2或set3突变体几乎无明显表型，但在ssu72缺失背景下同时缺失hos2或set3，会在全基因组范围内引发反义转录的协同升高，且该现象与这些区域的基因组不稳定性增加以及有害R环（R-loop）结构的形成相关。我们提出，Ssu72对于抑制RNA聚合酶II转录区域3'端的隐秘转录起始至关重要。在Ssu72缺失的情况下，反义转录会被Hos2/Set3依赖的监视机制所抑制，以维持基因组稳定性。整体实验设计：在野生型（WT）与ssu72Δ菌株中，针对Rpb1、Ser2磷酸化CTD（Ser2-P CTD）以及Tyr1磷酸化CTD（Tyr1-P CTD）开展染色质免疫共沉淀测序（ChIP-seq）。