Degradation of CDK9 by Ubiquitin E3 Ligase STUB1 Regulates P-TEFb Level and Its Functions for Global Target Gene Expression within Mammalian Cells

Abstract

Positive transcription elongation factor b (P-TEFb) regulates expression of diverse sets of genes within mammalian cells that have implications in several human disease pathogeneses. However, mechanisms of functional regulation of P-TEFb complex through regulation of its stability are poorly known. In this study, we show an important role of C-terminus of Hsc70-interacting protein (CHIP aka STUB1) in regulation of overall level of CDK9 and thus P-TEFb complex within mammalian cells. STUB1 acts as a ubiquitin E3 ligase for proteasomal degradation of CDK9 involving N-terminal lysine 3 (K3) residue. Whereas, overexpression of STUB1 enhances, its knockdown reduces overall CDK9 degradation kinetics within mammalian cells. Interestingly, owing to the same region of binding within CDK9, CyclinT1 protects CDK9 from STUB1-mediated degradation. Factors that cooperatively bind with CyclinT1 to form functional complex also protects CDK9 from degradation by STUB1. Knockdown of STUB1 enhances CDK9 expression and thus P-TEFb complex formation that leads to global increase in RNA polymerase II CTD phosphorylation and transcriptional activation of diverse P-TEFb target genes. Thus, we describe an important functional role of STUB1 in regulation of transcription through modulation of overall level of P-TEFb complex formation within mammalian cells.

Keywords:

• CHIP
• STUB1
• ubiquitination
• CDK9
• P-TEFb complex
• transcription
• RNA polymerase II

ACKNOWLEDGEMENTS

The work as described in this study was supported by a CSIR focused basic research (FBR) project, MLP141 and DST extramural funding (EMR/2016/001593) awarded to DB as well as CSIR-IICB internal funding through P07 budget head. SB is a recipient of UGC Senior Research Fellowship. AN and AG are the recipients of CSIR Senior Research Fellowship. DPM is a recipient of Dept. of Biotechnology Senior Research Fellowship.

AUTHOR CONTRIBUTIONS

SB, AN, and AG performed majority of the experiments in consultation with DB. DPM made important contribution in the P-TEFb overexpression analysis and its effect on target gene expression. AN, AG, and DB consulted and wrote the manuscript. SB, AN, and AG also helped in editing the manuscript.

DATA AVAILABILITY STATEMENT

The entire set of original raw images of Western blots that were used for making figures as described in this study is available through Mendeley data repository through https://data.mendeley.com/datasets/zg89v9sfkz/1.

Additional information

Funding

Council of Scientific and Industrial Research(CSIR) - Indian Institute of Chemical Biology(IICB) internal funding through P07, India.