1. Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, South Korea.
2. Graduate School of Cancer Science and Policy, Research Institute, National Cancer Center, Goyang 10408, South Korea.
3. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, South Korea.
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.
Methods: Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional Ssu72f/f mice. To knockout the mouse Ssu72 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.
Results: 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.
Conclusions: 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.
Keywords: Ssu72 phosphatase, RNA polymerase II, CTD phosphorylation, Transcription, Tissue-selective gene expression, P-TEFb