Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, South Korea.
Graduate School of Cancer Science and Policy, Research Institute, National Cancer Center, Goyang 10408, South Korea.
Theranostics. 2022 Jan 1;12(1):186-206. doi: 10.7150/thno.62274. eCollection 2022.
Reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) is essential for gene expression control. How altering the phosphorylation of the CTD contributes to gene expression in mammalian systems remains poorly understood. Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional mice. To knockout the mouse gene, we infected the cells with adenoviruses of incorporated luciferase and Cre recombinase, respectively. RNA sequencing, ChIP sequencing, ChIP assay, immunoblot analyses, qRT-PCR assay, and immunostaining were performed to gain insights into the functional mechanisms of Ssu72 loss in Pol II dynamics. Using primary cells isolated from Ssu72 conditional knockout and transgenic mice, we found that mammalian Ssu72-mediated transcriptional elongation rather than polyadenylation or RNA processing contributed to the transcriptional regulation of various genes. Depletion of Ssu72 resulted in aberrant Pol II pausing and elongation defects. Reduced transcriptional elongation efficiency tended to preferentially affect expression levels of actively transcribed genes in a tissue-specific manner. Furthermore, Ssu72 CTD phosphatase seemed to regulate the phosphorylation levels of CTD Ser2 and Thr4 through accurate modulation of P-TEFb activity and recruitment. Our findings demonstrate that mammalian Ssu72 contributes to the transcription of tissue-specific actively transcribed gene expression by regulating reciprocal phosphorylation of Pol II CTD.
RNA 聚合酶 II（Pol II）C 端结构域（CTD）的可逆磷酸化对于基因表达调控至关重要。然而，在哺乳动物系统中，CTD 磷酸化如何影响基因表达仍知之甚少。本研究从小鼠的条件性基因敲除模型中分离出原代胚胎成纤维细胞、肝细胞和胚胎干细胞。为了敲除小鼠的基因，我们分别用携带荧光素酶和 Cre 重组酶的腺病毒感染这些细胞。通过 RNA 测序、ChIP 测序、ChIP 检测、免疫印迹分析、qRT-PCR 检测和免疫荧光染色，深入了解 Ssu72 缺失对 Pol II 动力学的功能机制。利用从小鼠 Ssu72 条件性敲除和转基因模型中分离出的原代细胞，我们发现哺乳动物 Ssu72 介导的转录延伸，而不是多聚腺苷酸化或 RNA 加工，有助于各种基因的转录调控。Ssu72 的缺失导致 Pol II 暂停和延伸缺陷。转录延伸效率的降低往往以组织特异性的方式优先影响活性转录基因的表达水平。此外，Ssu72 CTD 磷酸酶似乎通过准确调节 P-TEFb 的活性和募集，调节 CTD Ser2 和 Thr4 的磷酸化水平。我们的研究结果表明，哺乳动物 Ssu72 通过调节 Pol II CTD 的相互磷酸化，促进组织特异性活性转录基因的转录。
