BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription

Abstract

The functions of most of the 12 subunits of the RNA polymerase II (Pol II) enzyme are unknown. In this study, we demonstrate that two of the subunits, hRPB2 and hRPB10␣, mediate the regulated stimulation of transcription. We find that the transcriptional coactivator BRCA1 interacts directly with the core Pol II complex in vitro. We tested whether single subunits from Pol II would compete with the intact Pol II complex to inhibit transcription stimulated by BRCA1. Excess purified Pol II subunits hRPB2 or hRPB10␣ blocked BRCA1-and VP16-dependent transcriptional activation in vitro with minimal effect on basal transcription. No other Pol II subunits tested inhibited activated transcription in these assays. Furthermore, hRPB10␣, but not hRPB2, blocked Sp1-dependent activation.R egulation of class II gene expression in eukaryotes involves a myriad of transcription factors and protein complexes that ultimately exert control over the enzymatic activity of the multisubunit RNA polymerase II enzyme (Pol II). Pol II and the general transcription factors bind to template DNA in a sequential fashion before the initiation of transcription to form a preinitiation complex (1-3). The process is more complex because Pol II also exists in a preformed complex known as the Pol II holoenzyme, which contains general transcription factors, mediator and SRB proteins, chromatin remodeling factors, and coactivators such as BRCA1 and CBP (4). Further, regulated transcription requires promoter-specific DNA-binding factors to target stimulation of specific genes and multiple other factors that function in the gene activation process (5, 6).Although hundreds of polypeptides orchestrate gene expression in the nucleus, the mass of a protein required to catalyze a DNA-dependent RNA synthesis is not excessive. Indeed, a single 99-kDa polypeptide, such as the bacteriophage T7 RNA polymerase, is sufficient to catalyze promoter-dependent RNA polymerization (7). Bacterial RNA polymerase consists of an enzymatic core of three subunits (␣ 2 ␤␤Ј) and a variable specificity factor ( ) that selects for various classes of promoters. Promoter-specific activators primarily interact with the ␣ and subunits, although all four subunits can be targets (8-12). By analogy, some of the many subunits of the eukaryotic RNA polymerase would also be targets for activators.Human Pol II consists of 12 subunits (see Table 1 and ref. 14). Several subunits are homologous to prokaryotic subunits, and presumably function similarly. The two largest subunits, hRPB1 and hRPB2, are homologues of the prokaryotic ␤Ј and ␤ subunits that bind DNA and nucleotide triphosphate substrate, respectively (15). hRPB3 contains some sequence similarity to ␣, and likewise contributes to core Pol II assembly. Analysis of individual Pol II subunits in yeast has revealed a role for a number of Pol II subunits in polymerase assembly (16)(17)(18)(19)(20), elongation (21,22), and accurate initiation of transcription (23-25). However, only the C-terminal domain of RPB1 and the full-length polypeptide...

BRCA1 gene in breast cancer

“…Various classes of proteins interact with BRCA1, including: (1) components of the basal transcription machinery [e.g., RNA helicase A and RNA pol II (Anderson et al, 1998;Schlegel et al, 2000a)]; (2) generalized transcriptional coactivators [p300, CBP, Brg1 (Bochar et al, 2000;Pao et al, 2000)] and corepressors [e.g., RbAp46, RbAp48, histone deacetylases-1,2, and CtIP (Yarden and Brody, 1998;Yu et al, 1998)]; (3) tumor suppressors [e.g., p53, RB1, BRCA2 (Chen et al, 1998;Ouichi et al, 1998;Yarden and Brody, 1998;Zhang et al, 1998;Aprelikova et al, 1999;Chai et al, 1999;Fan et al, 2001c)]; (4) steroid hormone receptors, estrogen receptor-a, and androgen receptor (Yeh et al, 2000;Fan et al, 2001a); (5) DNA repair proteins [e.g., Rad51, Rad50, hMSH2 (Scully et al, 1997b;Zhong et al, 1999;Wang et al, 2001a)]; (6) other sequence-specific transcription factors [e.g., c-Myc, Oct-1, and NF-YA Fan et al, 2002b)]; and (7) cell cycle regulatory proteins [e.g., BARD1, E2F1, cyclins (Wu et al, 1996;Wang et al, 1997)]. These interactions are summarized in Figure 1; and the significance of these interactions is discussed in ''Functional Activities of BRCA1''.…”

“…However, the authors provide evidence that this protein is phosphorylated; and nuclear localization may occur by interaction with another nucleoprotein. The mechanism by which BRCA1 regulates G2/M progression is unclear, but it is noteworthy that the truncated BRCA1 protein contains domains that interact with the RNA polymerase II holoenzyme (Scully et al, 1997a;Anderson et al, 1998;Schlegel et al, 2000a;Chiba and Parvin, 2002) as well as a region homologous to Rad9, a protein involved in the G2 damage-responsive cell cycle checkpoint (Weinert and Hartwell, 1988;Hirai and Wang, 2002;Yoshida et al, 2002). Roles for BRCA1 in several DNA damageresponsive checkpoints (including G2/M) have been established and are discussed (see ''Caretaker Role: Maintenance of Genomic Integrity'').…”

“…Subsequently, it was found that BRCA1 regulates a variety of transcriptional pathways. BRCA1 transcription regulatory activity may be mediated, in part, by interaction with the basal transcriptional machinery (RNA helicase A and RNA pol II) (Anderson et al, 1998;Schlegel et al, 2000a) and with transcriptional coactivators [p300, CBP (Pao et al, 2000)] and corepressors [RbAp46/48, HDAC-1/2, and CtIP (Yarden and Brody, 1998;Yu et al, 1998)] (see Fig. 1).…”

Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP)

“…Transient transfection of COS1 cells was carried out as reported previously [1,7,8]. The cells were harvested, washed, and sonicated in LAC buffer (10% glycerol, 20 mM HEPES, pH 7.9, 50 mM KCI, 0.4 M NaCI, 1 mM MgCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 9 mM CHAPS, 0.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin and leupeptin) and then centrifuged.…”

“…The subunits of RNA polymerase II have been the final targets of variety of factors to regulators gene transcription [1][2][3][4]. Pol II consists of 12 subunits of which RPB5 is the common subunit of pol I, II and III [4].…”