Wani Shotaro, Sugita Ai, Ohkuma Yoshiaki, Hirose Yutaka
Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan and.
Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan and Department of Biochemistry, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
J Biochem. 2016 Aug;160(2):111-20. doi: 10.1093/jb/mvw018. Epub 2016 Feb 26.
The C-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit contains tandem repeats of the heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD is subject to dynamic phosphorylation during transcription, mainly at serine residues (Ser2, Ser5 and Ser7). Regulation of CTD phosphorylation by specific kinases and phosphatases is crucial for coordinating transcription with RNA processing and histone modification. Human small CTD phosphatase 4 (SCP4), also called CTDSPL2 or HSPC129, is a putative CTD phosphatase belonging to the FCP/SCP family and implicated in control of ε- and γ-globin gene expression. Here, we report the biochemical and functional characterization of SCP4. SCP4 exhibited Ser5-preferential CTD phosphatase activity in vitro, while small interfering RNA-mediated SCP4 knockdown in HeLa cells increased phosphorylation levels of Pol II at Ser5 and Ser7, but not at Ser2. Furthermore, cell fractionation, chromatin immunoprecipitation and immunofluorescence assays revealed an exclusive localization for SCP4 in the chromatin, particularly at transcriptionally silenced chromosomal regions. Interestingly, SCP4 was gradually released from the chromatin fraction during hemin-induced erythroid differentiation of K562 cells, with concomitant cytoplasmic accumulation. Therefore, SCP4 is a unique chromatin-associated, Ser5-preferential CTD phosphatase that preferentially distributes to transcriptionally silenced gene regions and may participate in gene regulation during erythroid differentiation.
RNA聚合酶II（Pol II）大亚基的C末端结构域（CTD）包含七肽Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7的串联重复序列。在转录过程中，CTD会发生动态磷酸化，主要发生在丝氨酸残基（Ser2、Ser5和Ser7）上。特定激酶和磷酸酶对CTD磷酸化的调节对于协调转录与RNA加工及组蛋白修饰至关重要。人类小CTD磷酸酶4（SCP4），也称为CTDSPL2或HSPC129，是一种推定的CTD磷酸酶，属于FCP/SCP家族，与ε-和γ-珠蛋白基因表达的调控有关。在此，我们报告了SCP4的生化和功能特性。SCP4在体外表现出对Ser5优先的CTD磷酸酶活性，而在HeLa细胞中通过小干扰RNA介导的SCP4敲低增加了Pol II在Ser5和Ser7处的磷酸化水平，但在Ser2处没有增加。此外，细胞分级分离、染色质免疫沉淀和免疫荧光分析显示SCP4在染色质中具有独特的定位，特别是在转录沉默的染色体区域。有趣的是，在K562细胞的血红素诱导的红系分化过程中，SCP4逐渐从染色质部分释放出来，同时伴有细胞质积累。因此，SCP4是一种独特的与染色质相关的、对Ser5优先的CTD磷酸酶，优先分布于转录沉默的基因区域，并可能参与红系分化过程中的基因调控。
