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 hRPB10alpha, 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 hRPB10alpha 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, hRPB10alpha, but not hRPB2, blocked Sp1-dependent activation.

Figure 1

BRCA1 binds core Pol II in vitro. Purified calf thymus Pol II was incubated with PinPoint protein (pp; lane 2) or PinPoint-BRCA1 fusion proteins (lanes 3 and 4) immobilized on streptavidin beads. After washing in buffer containing 750 mM KOAc and 0.5% Nonidet P-40, bound proteins were resolved by SDS/PAGE and were visualized by immunoblot analysis using a Pol II RPB1-specific antibody. Lane 1 represents 10% of total Pol II in binding reactions.

Figure 2

Pol II subunits hRPB10α and hRPB2 mediate BRCA1-dependent transcriptional activation. All reactions contain purified basal transcription factors, the coactivator PC4, and core Pol II in the absence or presence of GAL4-BRCA1. Individual Pol II subunits fused to GST were added as indicated. Transcription from the GAL4-dependent template resulted in the accumulation of 380-nucleotide RNA (stim.), and transcription from a control template lacking GAL4 response elements resulted in accumulation of 210-nucleotide RNA (basal). The relative increase in transcription upon addition of activator, quantified by using PhosphorImager analysis, is indicated by black bars under each lane. For comparison, the activation in lane 2 of A (absence of added Pol II subunit) is eight-fold. (A) Inhibition of BRCA1-dependent stimulation of transcription by 300 ng of GST fusion of each hRPB3 to hRPB11 (lanes 3–12). (B) Titration of RPB10α results in an incremental decrease in BRCA1-stimulated transcription. GST-hRPB10α was included in reactions at 75 ng (lane 3), 150 ng (lane 4), and 300 ng (lane 5). (C) Inhibition of BRCA1-dependent transcriptional activation by hRPB2 peptides. Twelve hundred nanograms of each fragment were added as indicated. (D) Titration of hRPB2_(315–606) results in an incremental decrease in BRCA1-stimulated transcription. GST-hRPB2_(315–606) was included in reactions at 300 ng (lane 3), 600 ng (lane 4), and 1,200 ng (lane 5).

Figure 3

Mutant forms of hRPB10α do not mediate BRCA1-dependent activation. All reactions contained the general transcription factors, PC4, and Pol II in the absence or presence of GAL4-BRCA1. (A) Inclusion in reactions of site-specific mutants of hRPB10α that disrupt the zinc finger motif (lanes 4 and 5) or an Rb-binding motif (lane 6) do not inhibit BRCA1-dependent activated transcription. (B) Inclusion in reactions of hRPB10α truncation mutants that disrupt the zinc finger motif (lanes 4 and 5) or a highly basic region (lanes 6 and 7) do not inhibit BRCA1-dependent activated transcription. The hRPB10α_(11–58) truncation mutant does not delete an obvious structural motif, but cannot specifically inhibit BRCA1-dependent activated transcription (lane 8). (C) Amino acid sequence of hRPB10α, with the zinc finger (Cys-X₃-Cys-X₁₃-Cys-X₃-Cys) motif enlarged and the Rb-binding motif underlined.

Figure 4

Pol II subunits hRPB2 and hRPB10α mediate BRCA1-dependent transcriptional activation before initiation. All reactions were performed as in Fig. 2 except that UTP was excluded, stopping synthesis of the nascent transcript after incorporation of seven nucleotides. Individual Pol II subunits in concentrations identical to Fig. 2 were added concurrent with initiation of the reaction (I), or later upon resumption of transcript elongation by the addition of UTP (E).

Figure 5

Pol II subunits hRPB10α and hRPB2 mediate VP16-dependent transcriptional activation. All reactions were performed as in Fig. 2 except that GAL4-VP16 was used as an activator. (A) Effect on VP16-dependent activated transcription by hRPB3 to hRPB11. For comparison, the activation in lane 2 (absence of added Pol II subunit) is 12-fold. (B) Effect on VP16-dependent activated transcription by hRPB2 peptides.

Figure 6

hRPB10α, but not hRPB2, mediates Sp1-dependent activation. All reactions contained the general transcription factors, USA fraction, and Pol II in the absence or presence of Sp1. (A) Effect on Sp1-dependent activated transcription by hRPB3 to hRPB11. For comparison, the activation in lane 2 (absence of added Pol II subunit) is 10-fold. (B) Effect Sp1-dependent transcription by hRPB2 peptides. (C) Titration, as in Fig. 2, of hRPB10α (lanes 7–9), but not hRPB2_(315–606) (lanes 4–6), results in an incremental decrease in Sp1-dependent activated transcription.