BRCA1 Associates with Processive RNA Polymerase II

Abstract

The human BRCA1 tumor suppressor interacts with transcriptional machinery, including RNA polymerase II (RNA pol II). We demonstrated that interaction with RNA pol II is a conserved feature of BRCA1 proteins from several species. We found that full-length BRCA1 proteins universally fail to activate transcription in classic GAL4-UAS one-hybrid assays and that the activity associated with the human BRCA1 C terminus was poorly conserved in closely related homologs of the gene. Fractionation studies demonstrated that BRCA1 proteins from all species tested interacted specifically with hyperphosphorylated pol II (IIO), in preference to hypophosphorylated RNA pol II (IIA) expected at promoters. BRCA1-RNA pol II complexes showed evidence of a multiply phosphorylated heptad repeat domain in the catalytic subunit (p220) of RNA pol II, and the complex was highly functional in transcriptional run-off assays. Interestingly, endogenous BRCA1 associated with a large fraction of the processive RNA pol II activity present in undamaged cells, and the interaction was disrupted by DNA-damaging agents. Preferential interaction with processive RNA pol II in undamaged cells places BRCA1 in position to link late events in transcription with repair processes in eukaryotic cells.Mutations in the BRCA1 tumor suppressor gene are associated with an increased risk of breast and ovarian cancer and an elevated incidence of certain other cancers (1-3). Genetic and biochemical data place BRCA1 as a downstream target of ATM/ ATR in cellular responses to genotoxic stress, and the BRCA1 protein has been implicated in chromatin remodeling and homologous recombination functions in mitotic and meiotic cells (4 -8). BRCA1 complexes contain E3 ubiquitin-ligase activity, but the precise target(s) remains an avenue of active study (9, 10). Although BRCA1 proteins show surprisingly low sequence identity, human BRCA1 can replace the mouse gene in genetically engineered animals (11, 12), a result that implies general conservation of important functional motifs. Comparative functional studies are thus likely to play an important role in identifying critical targets of BRCA1 in human cells.A distinct literature has evolved linking BRCA1 to roles in transcription. BRCA1 protein has been shown to associate with RNA polymerase II (RNA pol II) 1 and various transcriptional regulators (13). Early on, several groups demonstrated that the C-terminal domain (CTD; amino acids 1380 -1863) of human BRCA1 scored positively in transcriptional activator trap experiments using various forms of the so-called "one-hybrid" assay (14 -18). While not elucidating a specific function, ectopic expression of full-length human BRCA1 was then shown to increase expression of stress-responsive genes including p21 (19), GADD45 (20), and p27 (21) and decreased expression of other genes, including c-Myc-regulated genes (22) and certain estrogen-regulated genes (23). However, it remains an open question whether endogenous BRCA1 directly mediates recruitment of RNA pol II to promoters of these...

The role of BRCA1 in transcriptional regulation and cell cycle control

“…There are 52 repeats of this heptad in RNA polII CTD and multiple phosphorylations are required to generate the hyperphosphorylated RNA polII form associated with elongation. The association of hypophosphorylated BRCA1 with preferentially the hyperphosphorylated (postinitiation) form of RNA polII suggests that BRCA1 does not affect direct promoter activation but plays roles in transcription related to chromatin remodeling as well as transcription-coupled repair (Krum et al, 2003).…”

Hyperphosphorylation of RNA Polymerase II in Response to Topoisomerase I Cleavage Complexes and Its Association with Transcription- and BRCA1-dependent Degradation of Topoisomerase I

“…2), Top1 degradation is selectively transcription-dependent. 20 Because the DNA-damage responsive protein Brca1 is associated with the elongating Pol II 32 and possesses ubiquitin E3 ligase activity, 33 we examined the possibility that Brca1 could promote Top1 degradation. As expected, 20 CPT induced proteosomal degradation of Top1 in MEF cells (Fig 5a) in a transcription-dependent manner (Fig.…”

BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

“…BRCA1 specifically interacts with a large fraction of hyperphosphorylated, processive polymerase II (IIO), in preference to the hypophosphorylated polymerase II (IIA) found at promoters (17). It has been proposed that BRCA1 binds polymerase IIO complexes as part of a genome scanning function for DNA damage (18).…”

“…Runoff transcription assay. The runoff transcription assay used was described elsewhere (17). Briefly, the runoff template was created by annealing 50 32 P]CTP.…”