Abstract 3025: Characterization of CDK9/BRCA1 complex in DNA damage response

Abstract

Abstract BRCA1 gene encodes an 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 is associated with the RNA polymerase II holoenzyme (RNA pol II), a number of transcription factors and regulators. As 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 RNA pol II 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 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 with both 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. A cisplatin resistance phenotype was also observed in CDK9 downregulated cells only in a BRCA1 competent context. Now, we are extending our approach investigating the role of CDK9/BRCA1 complex in gene expression control and DNA damage repair. A better understanding of these functions will help to unravel the BRCA1 mediated transcription and its role as a tumor suppressor. Citation Format: Thales Nepomuceno, Vanessa Fernandes, Thiago Gomes, Giuliana De Gregoris, Renato Carvalho, Guilherme Kurtz, Álvaro Monteiro, Marcelo Alex Carvalho. Characterization of CDK9/BRCA1 complex in DNA damage response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3025. doi:10.1158/1538-7445.AM2015-3025