BRCA1 Can Modulate RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Levels

Abstract

A high incidence of breast and ovarian cancers has been linked to mutations in the BRCA1 gene. BRCA1 has been shown to be involved in both positive and negative regulation of gene activity as well as in numerous other processes such as DNA repair and cell cycle regulation. Since modulation of the RNA polymerase II carboxyterminal domain (CTD) phosphorylation levels could constitute an interface to all these functions, we wanted to directly test the possibility that BRCA1 might regulate the phosphorylation state of the CTD. We have shown that the BRCA1 C-terminal region can negatively modulate phosphorylation levels of the RNA polymerase II CTD by the Cdk-activating kinase (CAK) in vitro. Interestingly, the BRCA1 C-terminal region can directly interact with CAK and inhibit CAK activity by competing with ATP. Finally, we demonstrated that full-length BRCA1 can inhibit CTD phosphorylation when introduced in the BRCA1 ؊/؊ HCC1937 cell line. Our results suggest that BRCA1 could play its ascribed roles, at least in part, by modulating CTD kinase components.The tumor suppressor gene BRCA1 encodes a protein of 1,863 amino acids that can interact with a plethora of factors involved in transcription, DNA repair, cell cycle regulation, apoptosis, genome integrity, and ubiquitination (16). Theses interactions underlie the extensive implication of BRCA1 in crucial processes related to tumorigenesis. However, how it can precisely ensure those functions still remains unclear. It has been suggested that BRCA1 could exert its tumor suppressor functions, particularly in DNA repair and cell cycle regulation, via its transcriptional activity (39, 53). Interestingly, various BRCA1 target genes carry out functions that could largely account for its involvement in DNA repair and cell cycle control. BRCA1 induces the expression of the DNAdamage-responsive genes p21 WAF1/CIP1 (54) and GADD45 (24), the nucleotide excision repair (NER) genes DDB2 and XPC (26), and the cell cycle arrest genes p27 (62) and 14-3-3 (5), while it represses the expression of the cell cycle-promoting gene cyclin B1 (35).Transcriptional activators generally possess a DNA binding domain that binds specific sequences on promoters and an activating region known to interact with general transcription factors, the RNA polII holoenzyme, and chromatin remodeling machines to recruit the transcriptional machinery to a target promoter (34,47). In contrast to those activators, BRCA1 has been shown to bind DNA only in a nonspecific fashion (46, 64) and has been shown to be a component of RNA polII holoenzyme (4, 51). Moreover, we have previously shown that at high concentration, the BRCA1 C-terminal region (amino acids 1528 to 1863) can stimulate transcription in vivo and in vitro without the requirement for a DNA-tethering function (41). That evidence suggests that BRCA1 can stimulate transcription by a mechanism alternative to recruitment, for example, by modulating an enzymatic activity. In vitro transcription assays using a highly purified system have demonstrated that t...

Tax Interacts with P-TEFb in a Novel Manner To Stimulate Human T-Lymphotropic Virus Type 1 Transcription

“…It is important, therefore, that the transition from preinitiation to initiation to elongation is regulated to ensure that the proper factors are associated or dissociated, likened to a transcription checkpoint network. Consistent with this concept, it has been proposed that the inhibition of TFIIH kinase activity in PICs by interacting proteins, such as BRCA1, could help maintain RNAP II stability until transcription is initiated and promoter escape is required (48). Similarly, it could be postulated that the inhibition of P-TEFb kinase activity might facilitate transcription by allowing the orderly and timely progression of transcription complexes from initiation to elongation.…”

Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase

“…If U2 genes are indeed sites of low but preferential oxidative damage, one would predict that loss of other remodeling or repair proteins might also cause locus-specific metaphase chromosome fragility. We therefore asked whether defects in BRCA1 or BRCA2, which like CSB are required for repair of oxidative damage (21,52) and for regulation of Pol II transcription (75,79,103), can also cause metaphase fragility of the RNU1, RNU2, and RN5S loci ( Table 2). Unsynchronized, subconfluent cultures of three cell lines-HCC1937 (BRCA1 mutant), MCF7 (BRCA1 low), and CAPAN-1 (BRCA2 null)-were treated with Colcemid for 3 h, and metaphase spreads were examined by fluorescent in situ hybridization using a biotinylated nonrepetitive probe for the U2 snRNA tandem repeat unit (111).…”

Corepressor CtBP and Nuclear Speckle Protein Pnn/DRS Differentially Modulate Transcription and Splicing of the E-Cadherin Gene

“…We adopted the protocol based on the in vitro phosphorylation reaction of the C-terminal domain of Pol II, which utilizes the recombinant CTD as a substrate and nuclear extract as a source of kinase activity (31). We incubated recombinant CTD-GST and Pnn-GST, which served as substrates, with either HeLa or HeLa CtBP-Flag-HA nuclear extracts, which served as sources of kinase activity, in the presence of kinase buffer.…”

“…Kinase reactions were performed as described previously (31), with modifications. Isolated nuclei were resuspended in kinase buffer (20 mM HEPES [pH 7.4], 50 mM NaCl, 10 mM MgCl 2 , 0.02% NP-40, 1 mM dithiothreitol) and briefly sonicated.…”