The effective regulation of RNA Polymerase II (Pol II)-dependent transcription, which typically maintains suitable expression levels of protein-coding genes and non-coding RNAs, is essential to maintain cellular health and prevent diseases. Pol II transcription is rigorously regulated at three primary stages: initiation, elongation, and termination. This regulation involves various factors, such as kinases, phosphatases, chromatin structure, and antisense transcripts. Dysregulation of Pol II elongation and the production of antisense transcripts have been linked to numerous diseases, including cancer, diabetes, cardiac, and neurodegenerative disorders. Consequently, gaining a better understanding of these regulatory processes' fundamentals is vital for improving diagnostic markers and therapeutic treatments. Our research focuses on Pol II transcription regulation in both the fission yeast Schizosaccharomyces pombe and human cells, utilizing an integrated approach that encompasses biochemistry, cellular and molecular biology, classical genetics, and chemical genetics—a technique that sensitizes a kinase to unnatural ATP analogs—in conjunction with genomics and proteomics.
Our research aims to explore three main areas related to RNA Polymerase II (Pol II) transcription:
To gain mechanistic insights into promoter-proximal pausing - Many genes in metazoans (and approximately 20% of genes in fission yeast) are regulated by an early event known as promoter-proximal pausing, where Pol II pauses shortly after initiation, around 20-80 nucleotides downstream of the transcription start site (TSS). The properly regulated release of paused Pol II from the promoter-proximal pause site results in the synthesis of full-length transcripts. Misregulation of this pausing or its release can lead to abnormal gene expression. Given this early event's critical role in regulating Pol II transcription, it's essential to dissect the underlying molecular mechanisms for understanding transcriptional homeostasis and its disruption in human diseases. Emerging studies suggest that distinct kinase-phosphatase switch mechanisms control the phosphorylation of effector proteins, thus modulating pause establishment, maintenance, and release. These vital kinase-phosphatase networks are mostly unknown and need precise identification and characterization. We aim to investigate the regulation of promoter-proximal pausing in fission yeast and human cells to understand how kinases and phosphatases coordinate to ensure pause establishment and synchronized release, beneficial for healthy cells.
To explore the coupling of transcription elongation and co-transcriptional processes - Variations in the rate of Pol II elongation have been implicated in controlling co-transcriptional processes such as 5'- and 3’-end processing, antisense transcription, alternative polyadenylation (APA), and pre-mRNA splicing. However, how the elongation rate controls these processes, and the consequent coupled process, remains largely unknown. The current hypothesis is that normal speeds of Pol II elongation favor the recruitment of factors necessary for executing a particular step, whereas slower Pol II speeds promote aberrant factor recruitment, resulting in premature outcomes. Conversely, faster rates may disrupt the timely execution of specific steps. Our primary objective here is to examine the uncharacterized and unidentified connections among kinase-phosphatase antagonisms, elongation rate, post-translational modifications (PTMs) of histones, pre-mRNA splicing, and transcription polarity.
To understand how spatial and temporal phosphorylation events influence termination - The transition from elongation to termination, a critical stage in transcription, prepares Pol II for efficient and accurate termination through a series of sequential events: (1) Pol II's deceleration as it crosses the cleavage and polyadenylation signal (CPS), leading to (2) the accumulation of Ser2 phosphorylation of Pol II's carboxy-terminal domain (CTD), which in turn facilitates (3) the recruitment of factors involved in pre-mRNA 3’-end formation and termination. A longstanding question has been how this transition from elongation to termination is initiated. We recently identified a novel bistable switch mechanism involving cyclin-dependent kinase 9 (Cdk9) and protein phosphatase 1 (PP1) that rapidly reverses phosphorylation at the CTD of an essential elongation factor, Spt5 (and possibly other Cdk9 substrates) during the elongation machinery's traversal through the CPS, leading to a slowing of Pol II. The phosphorylation of Spt5's CTD is inversely correlated with Pol II's CTD Ser2 and Thr4 phosphorylation at the 3’-end of genes. However, how their reciprocal relationships functionally influence termination is still not fully understood. We aim to assess the spatial and temporal connections of various phosphorylation events and characterize their molecular roles in transcription termination.