Abstract 480: CDK9 (a novel BRCA1/BARD1 interaction partner) downregulates BRCA1-mediated transcription

Abstract

Abstract BRCA1 gene encodes a 1863 amino-acid protein comprising a RING finger domain in the N-terminus and two tandem BRCT domains (tBRCT) in C-terminal region. When fused to a heterologous DNA-binding domain, BRCA1 C-terminal region is capable of activating transcription. In addition, BRCA1 was found associated with the RNA polymerase II holoenzyme (RNApolII), a number of transcription factors and regulators. BRCA1 cancer-associated mutations located in the C-terminal region of the protein lead to an impaired transcriptional activation phenotype - the ability to trigger transcription seems to be an important feature of BRCA1 tumor suppression function. BARD1 (BRCA1 associated RING domain 1) also encloses a N-terminal RING finger and a C-terminal tBRCT domains. The BRCA1/BARD1 heterodimer was found to ubiquitinate RNApolII leading to a reduced transcription initiation. Recently, our group described a protein interaction network that identified putative interacting partners of seven different proteins enclosing the tBRCT domain. Among other hits, Cyclin-Dependent Kinase 9 (CDK9) was identified as a common interaction partner of BRCA1 and also BARD1 tBRCTs. CDK9 is a component of the positive transcription elongation factor b complex (P-TEFb), which is involved in co-transcriptional histone modification, mRNA processing, mRNA export and transcriptional elongation. The interaction between constitutive CDK9 and BARD1 was confirmed through co-immunoprecipitation (Co-IP) assay using HEK293FT cells nuclear extracts. We also demonstrated that CDK9 isoforms were able to interact both with BARD1 N- and C-terminal regions in a GST pulldown assay. In addition, the BRCA1/CDK9 constitutive interaction was confirmed by Co-IP assay in HeLa cells. We showed that, differently from BARD1, BRCA1 tBRCT interacts only with the 42KDa isoform of CDK9. In order to investigate CDK9 role in BRCA1 mediated transcription, the kinase expression was downregulated (by shRNA methodology) in HeLa cells, resulting in a two-fold increase in transcription (observed in a luciferase reporter assay). On the other hand, the super expression of CDK9 42KDa isoform leads to the suppression of BRCA1 mediated transcription. These data suggest that the CDK9 42KDa isoform modulates BRCA1 mediated transcription through its tBRCT domain. Now, we are extending our approach to investigate which genes are regulated by this complex. We also ought to investigate the role of BARD1 in the CDK9 regulation function. A better understanding of these interactions will help to unravel the BRCA1 mediated transcription and its tumor suppression function. Citation Format: Thales C. Nepomuceno, Vanessa C. Fernandes, Giuliana De Gregoris, Renato S. Carvalho, Álvaro N. Monteiro, Marcelo A. Carvalho. CDK9 (a novel BRCA1/BARD1 interaction partner) downregulates BRCA1-mediated transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 480. doi:10.1158/1538-7445.AM2014-480