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254 nature genetics volume 19 july 1998

BRCA1 protein is linked to the RNA polymerase IIholoenzyme complex via RNA helicase A

Stephen F. Anderson1, Brian P. Schlegel1, Toshihiro Nakajima2,3, Eric S. Wolpin1 & Jeffrey D. Parvin1

1Division of Molecular Oncology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis Street, Boston,Massachusetts 02115, USA. 2Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. 3Institute of Applied Biochemistry,University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki Japan 305-8572. S.F.A. & B.P.S. contributed equally to this study. Correspondence should beaddressed to J.D.P. e-mail: jparvin@bustoff.bwh.harvard.edu

The breast cancer specific tumour suppressor protein, BRCA1(refs 1,2), activates transcription when linked with a DNA-bind-ing domain3,4 and is a component of the RNA polymerase II(Pol II) holoenzyme5,6. We show here that RNA helicase A (RHA)protein7,8 links BRCA1 to the holoenzyme complex. The regionof BRCA1 which interacts with RHA and, thus, the holoenzymecomplex, corresponds to subregions of the BRCT domain ofBRCA1 (ref. 9). This interaction was shown to occur in yeastnuclei, and expression in human cells of a truncated RHA mole-cule which retains binding to BRCA1 inhibited transcriptionalactivation mediated by the BRCA1 carboxy terminus. These dataare the first to identify a specific protein interaction with theBRCA1 C-terminal domain and are consistent with the modelthat BRCA1 functions as a transcriptional coactivator.

The BRCA1 protein and the CREB binding protein (CBP) arecomponents of the Pol II holoenzyme5,6,10. RHA (140 kD) wasoriginally identified as a helicase of unknown function7,8 withhomology to the Drosophila maleless gene, which functions toincrease expression of genes from the male X chromosome11.

RHA is the component of the Pol II holoenzyme which bindsdirectly to CBP12. This CBP-RHA interaction is essential forCREB-dependent transcriptional activation in transfected cellsin culture12. We tested whether BRCA1 similarly bound RHAusing a glutathione-S-transferase (GST) fusion of the C terminus

of BRCA1 (aa residues 1560–1863) and full-length RHA. TheC terminus of BRCA1 promotes transcriptional activation whenfused to a GAL4-DNA binding domain (GBD) in cells3,4, andencompasses the tandem BRCT motifs (residues 1650–1855;ref. 9). Both GST-BRCA1 (1560–1863) and GST-CBP (1805–1890) bound RHA, whereas another fragment of CBP did not(Fig. 1a). Stringent washes (0.75 M KOAc, 0.5% NP 40) wereused to ensure elimination of everything but the most highly spe-cific protein-protein interactions.

Truncation of the C-terminal domain of BRCA1 revealed adomain which inhibits binding to RHA (Fig. 1b). The amount ofRHA bound to BRCA1 (1560–1863) represents only 2% of thetotal input, but by trimming the BRCA1 C-terminal domain toresidues 1650–1800, the specific binding to RHA was increased togreater than 10% of input (Fig. 1b). A parallel result was observedin the purification of the holoenzyme complex when using theseBRCA1 matrices (Fig. 1b), suggesting that a domain whichinhibits BRCA1-holoenzyme interaction resides within excisedregions of BRCA1. Further subdivision demonstrated thatBRCA1 (1650–1800) contains at least three distinct RHA bindingdomains (Fig. 1c, top): BRCA1 (1650–1700), BRCA1 (1701–1750), and BRCA1 (1751–1800), with the latter two domainsapparently binding with higher affinity. We then assessed thesame series of GST-BRCA1 fusion proteins for their ability to

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Fig. 1 RHA binds the C-terminal domain of BRCA1 in vitro. a, Functional organization of BRCA1 domains1 (top). The BRCT domain9 is a tandem repeat from1650–1855; diagram of BRCA1 protein not drawn to scale. Interaction between RHA and BRCA1 (bottom). Full-length in vitro translated 35S-RHA protein wasincubated with CBP and BRCA1 polypeptides. Samples were resolved by SDS-PAGE. Lane 1 represents 2% of total RHA. b, Interaction between RHA and a trun-cated BRCA1 C-terminal domain. Full-length in vitro translated 35S-RHA protein (top row) or purified Pol II holoenzyme (bottom row) was incubated with GST orBRCA1 polypeptides. Holoenzyme was visualized by immunoblot analysis using a pol II CTD specific antibody. Lane 1 represents 10% of total RHA or holoenzyme.c, Mapping of the RHA binding domain of BRCA1. Identical to (b), except further truncated forms of BRCA1 polypeptides were analysed as indicated. Additionalimmunoblots against the SRB10 homolog, cdk8 (ref. 17), and against holoenzyme-bound RHA are shown. Cdk8 is the lowest band in a triplet which appears insome lanes, and RHA is observed as a doublet due to partial proteolysis.

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interact with the Pol II holoenzyme. Immunoblots of stringentlywashed samples revealed an identical binding pattern to theholoenzyme components pol II, cdk8 and RHA (Fig. 1c). TheBRCA1 domains which bind RHA similarly bind holoenzyme,suggesting that RHA is a major determinant of BRCA1 associa-tion with holoenzyme.

RHA has previously been shown to contain discrete CBP and PolII binding domains (CBP bound RHA, 1–250 and Pol II boundRHA, 230-650; ref. 12). We assessed the binding of biotinylatedBRCA1 (1560–1863) fusion protein to GST-RHA polypeptidesimmobilized on glutathione agarose beads and visualized BRCA1using streptavidin-HRP. Only RHA (230–325) bound BRCA1(1560–1863; Fig. 2a). As both polypeptides were recombinant pro-teins expressed in bacteria, the interaction between BRCA1 andRHA is direct. RHA (230–325) also bound full-length BRCA1 pro-tein (Fig. 2b). As shown previously12, CBP bound RHA (1–250)but not the BRCA1-binding domain, RHA (230–325; Fig. 2c).Therefore, the RHA (230–325) domain is specific for BRCA1.

Mutation at residue 1775 of BRCA1 has been observed inbreast cancer patients13, and the specific mutation M1775E isdefective for transcriptional activation3, although this specificchange has not been observed as a germline mutation. Wild-type BRCA1 (1560–1863) was stable when bound to RHA(230–325) and was not eluted even when washing the proteins in1 M salt. The M1775E mutant, however, was less stable whenbound to the RHA and was eluted when washed above 0.25 MKOAc (Fig. 2d). The weaker interaction of the M1775E mutantrelative to wild-type supports the specificity of RHA binding toBRCA1, and suggests a possible biochemical mechanism behindloss-of-function mutations.

BRCA1 (1560–1863) binds specifically to RHA in eukaryoticnuclei in a yeast two-hybrid assay. We transfected GBD-BRCA1(1560–1863) into yeast cells along with a construct consisting ofthe GAL4 transcriptional activation domain (GAD) sequencefused to a fragment of RHA. A standard assay14 for activation of aβ-galactosidase reporter gene revealed that, of the four fragmentsof RHA fused to the GAD, only the RHA (230–625) fragmentinteracted with the BRCA1 C terminus. This interaction was spe-cific and unaffected by reversal of the fusion partners (Table 1).

Similar to the published results using COS7 and 293T cells3,4,GBD-BRCA1 was a weak transcriptional activator of a GAL4 site-

dependent reporter in HeLa cells and approximately threefold lessactive than GBD-Sp1 in the same assay (Fig. 3). Expression by co-transfection of RHA (1–344), a truncated RHA molecule lackingmost of the polymerase binding domain12, resulted in a 2.5-foldreduction in the transcriptional activation by GBD-BRCA1(Fig. 3). This result suggested that an excess of RHA fragmentcompeted with the Pol II holoenzyme for binding to the GBD-BRCA1. Thus, the interaction of BRCA1 and RHA in the nucleusof human cells is key in transcriptional regulation by BRCA1.

As a control, amino acids 1–250 of RHA did not diminish theactivation by BRCA1 C terminus (Fig. 3, centre). In addition,expression of RHA (1–344) had little effect on the transcriptionalactivation of GBD-Sp1 (Fig. 3, right), which was clearly differentthan the results with GBD-BRCA1. Thus, the inhibition of tran-scriptional activation by GBD-BRCA1 specifically requires RHAamino-acid residues 251–344, suggesting that the interactionswhich we have shown in vitro and in yeast cells occur as well inthe nuclei of human cells.

The CBP molecule functions as a transcriptional coactivator, atleast in part, by bridging the Pol II holoenzyme to the regulatoryfactor bound to DNA upstream of the promoter. This interactionbetween CBP and the holoenzyme complex hinges upon the

Table 1• The BRCA1 C-terminus interacts specifically withRHA (230–650) in yeast nuclei

GAL4-BD GAL4-AD β-galfused to fused to units

vector alone vector alone 0.35 ± 0.08BRCA1 (1560–1863) vector alone 1.00 ± 0.21BRCA1 (1560–1863) RHA (1–250) 1.02 ± 0.23BRCA1 (1560–1863) RHA (230–650) 18.32 ± 0.41BRCA1 (1560–1863) RHA (630–1020) 1.56 ± 0.27BRCA1 (1560–1863) RHA (1000–1279) 1.32 ± 0.56RHA (1–250) BRCA1 (1560–1863) 1.20 ± 0.41RHA (230–650) BRCA1 (1560–1863) 15.62 ± 0.15RHA (630–1020) BRCA1 (1560–1863) 1.82 ± 0.26RHA (1000–1279) BRCA1 (1560–1863) 1.09 ± 0.34RHA (1–250) vector alone 1.12 ± 0.08RHA (230–650) vector alone 1.86 ± 0.29RHA (630–1020) vector alone 1.23 ± 0.04RHA (1000–1279) vector alone 1.14 ± 0.15

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Fig. 2 BRCA1 interaction with RHA in vitro. a, Map-ping of the BRCA1 binding domain of RHA. BRCA1(1560–1863) was fused to PinPoint protein and incu-bated with GST or RHA polypeptides. Samples wereprocessed as described in Methods. The fused Pin-Point protein was visualized with a streptavidin-HRPconjugate. Lane 1 represents 2% of total BRCA1.b, Full-length BRCA1 protein binds to RHA (230–325). In vitro translated full-length BRCA1 proteinwas tested for binding to GST control (lane 2) or RHA(230–325; lane 3). Lane 1 represents 2% input.c, Specificity of RHA for CBP versus BRCA1. CBP(1680–1890) was fused to PinPoint protein and incu-bated with GST or RHA polypeptides. Lane 1 repre-sents 2% of total CBP. d, Comparison of wild-type(wt) and mutant BRCA1 binding to RHA. Wild-typeBRCA1 (1560–1863; lanes 2–7) or BRCA1 (1560–1863)containing a M1775E substitution (lanes 9–13) werefused to PinPoint protein and incubated with GST(lane 2) or RHA (230–325; lanes 3–7 and 9–13) fusedto GST and bound to glutathione agarose beads.Lanes 1 and 8 represent 2% of total wild-type andmutant BRCA1, respectively.

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N-terminal domain of RHA (refs 10,12). We show in these exper-iments that BRCA1 also binds to RHA, but via a separate domainthan that which binds CBP. Analogous to CBP, the BRCA1 pro-tein may interact with specific, but as yet unidentified, DNA-bound transcription factors to transmit a regulatory signal to thePol II holoenzyme complex via the RHA protein. Some mutantswhich affect the transcription function of BRCA1 (refs 3,4), suchas Y1853X, are not present in the holoenzyme binding domain,suggesting that the regulation of transcription may require moresignals than just binding to the holoenzyme. Indeed, BRCA1(1600–1800) fused to GBD does not activate transcription intransfected cells (data not shown), whereas BRCA1 (1560–1863)does (Fig. 3). In phospho-CREB regulation of transcription, twosignals were required, one through CBP and the holoenzyme andone via a separate domain of CREB to TFIID (ref. 10). BRCA1has been observed to activate expression of the cell cycle regula-tory protein, p21, dependent upon a 50-bp sequence upstream ofthe p21 promoter15, and BRCA1 also functions as a coactivatordependent upon p53 response elements16 in agreement with themodel presented in this study.

MethodsIn vitro binding assay. TNT rabbit reticulocyte lysate (Promega) generatedfull-length 35S-met labelled RHA using a template containing the RHAsequence downstream of a T7 promoter. Bacterial expression plasmidsencoding BRCA1 and CBP sequences fused to the PinPoint sequence were

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Fig. 3 Transcription activation by GBD-BRCA1 C-terminus is inhibited by trun-cated RHA protein. GBD-BRCA1 activated transcription of a GAL4-site depen-dent reporter in transient transfections in HeLa cells. Expression of truncatedRHA proteins was tested for inhibition of the GBD-BRCA1 specific activation.RHA (1–344), which contains the BRCA1 interaction region, was cotransfected inincreasing amounts into HeLa cells along with GBD-BRCA1 (left panel) or GBD-Sp1 (right panel). The centre panel shows cotransfection of RHA (1–250), whichlacks the BRCA1 interaction region, with GBD-BRCA1. Luciferase reporter activ-ity is normalized in each panel so that activated transcription in the absence ofRHA is 100%. Numbers in the RHA (1–344) and RHA (1–250) rows indicate theratio of RHA plasmid to GBD-fusion plasmid used in the transfection.

1. Miki, Y. et al. A strong candidate for the breast and ovarian cancer susceptibilitygene BRCA1. Science 266, 66–71 (1994).

2. Futreal, P.A. et al. BRCA1 mutations in primary breast and ovarian carcinomas.Science 266, 120–122 (1994).

3. Monteiro, A.N., August, A. & Hanafusa, H. Evidence for a transcriptionalactivation function of BRCA1 C-terminal region. Proc. Natl Acad. Sci. USA 93,13595–13599 (1996).

4. Chapman, M.S. & Verma, I.M. Transcriptional activation by BRCA1. Nature 382,678–679 (1996).

5. Scully, R. et al. BRCA1 is a component of the RNA polymerase II holoenzyme. Proc.Natl Acad. Sci. U.S.A. 94, 5605–5610 (1997).

6. Neish, A.S., Anderson, S.F., Schlegel, B.P., Wei, W. & Parvin, J.D. Factors associatedwith the mammalian RNA polymerase II holoenzyme. Nucleic Acids Res. 26,847–853 (1998).

7. Lee, C.G. & Hurwitz, J. Human RNA helicase A is homologous to the malelessprotein of Drosophila. J. Biol. Chem. 268, 16822–16830 (1993).

8. Zhang, S. & Grosse, F. Domain structure of human nuclear DNA helicase II (RNAhelicase A). J. Biol. Chem. 272, 11487–11494 (1997).

9. Koonin, E.V., Altschul, S.F. & Bork, P. BRCA1 protein products…Functional motifs.Nature Genet. 13, 266–268 (1996).

10. Nakajima, T., Uchida, C., Anderson, S.F., Parvin, J.D. & Montminy, M. Analysis of acAMP-responsive activator reveals a two-component mechanism fortranscriptional induction via signal-dependent factors. Genes Dev. 11, 738–747(1997).

11. Bone, J.R. et al. Acetylated histone H4 on the male X chromosome is associatedwith dosage compensation in Drosophila. Genes Dev. 8, 96–104 (1994).

12. Nakajima, T. et al. RNA helicase A mediates association of CBP with RNApolymerase II. Cell 90, 1107–1112 (1997).

13. Szabo, C.I. & King, M.C. Inherited breast and ovarian cancer. Hum. Mol. Genet. 4,1811–1817 (1995).

14. Aspenstrom, P. & Olson, M.F. Yeast two-hybrid system to detect protein-proteininteractions with Rho GTPases. Methods Enzymol. 256, 228–241 (1995).

15. Somasundaram, K. et al. Arrest of the cell cycle by the tumour-suppressor BRCA1requires the CDK-inhibitor p21WAF1/CiP1. Nature 389, 187–190 (1997).

16. Ouchi, T., Monteiro, A.N.A., August, A., Aaronson, S.A. & Hanafusa, H. BRCA1regulates p53-dependent gene expression. Proc. Natl Acad. Sci. USA 95,2302–2306 (1998).

17. Tassan, J.P., Jaquenoud, M., Leopold, P., Schultz, S.J. & Nigg, E.A. Identification ofhuman cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.Proc. Natl Acad. Sci. USA 92, 8871–8875 (1995).

constructed by inserting the appropriate DNA fragments into PinPointXa-3 (Stratagene). Bacterial expression plasmids encoding BRCA1 andRHA sequences fused to the GST sequence were constructed by insertingthe appropriate DNA fragments into pGEX-2TK (Pharmacia Biotech). Wepurified GST fusion proteins on glutathione agarose beads (20 µl) in bufferH (120 mM KOAc, 20 mM Tris-OAc, pH 7.9, 1 mM EDTA, 20% glycerol).Equal amounts of GST fusion proteins were used in each assay based onCoomassie staining. The beads were then used for binding assays overnightat 4 °C in buffer H (supplemented with 75 mM KOAc, 0.5% NP 40,0.2 mg/ml BSA, 1 mM DTT) with either radiolabelled RHA protein, puri-fied pol II holoenzyme (Biorex fraction5) or bacterial lysate containing Pin-Point fusion protein. We washed samples three times with the same bufferbut containing 0.75 M KOAc and resolved by SDS-PAGE. Samples contain-ing holoenzyme or PinPoint fusion protein were transferred to nitrocellu-lose, probed with specific antibody or streptavidin-horse radish peroxidase(HRP; GibcoBRL), respectively, and visualized by chemiluminescence.

Yeast two-hybrid assay. Two-hybrid assays using indicated GAL4 genefusions to fragments of BRCA1 and RHA were scored in a liquid β-galac-tosidase assay14. We used yeast strain y-190 and plasmids GBT9 andGAD424 (Clontech) in these assays.

BRCA1 in vivo reporter assay. The sequence encoding amino acids1560–1863 of BRCA1 fused to the GAL4 (1–147) DNA-binding domainwas inserted into vector pcDNA3 (Stratagene). HeLa cells were cotrans-fected with these plasmids along with plasmid pFR-luc (Promega), whichcontains five GAL4 DNA-binding sites upstream of a firefly luciferasereporter gene. Transfection efficiency was measured by co-transfectionof control plasmid pRL-TK (Promega), which expresses sea pansyluciferase. HeLa cells in 24-well plates were transfected by standard CaPO4techniques, using activator (1.3 µg) or control DNA-binding domain plas-mid, pFR-luc reporter (0.24 µg), pRL-TK transfection control reporter(0.043 µg) and RHA-containing plasmid per well (up to 1.0 µg). PlasmidpRHA (1–344) was derived from pBK-CMV-RHA (ref. 12). Control trans-fections involving GAL4 (1–147) fusions to the activation domain of Sp1used GAL4-Sp1 DNA (1.3 µg). All reactions were balanced to contain thesame amount of total DNA by the addition of pcDNA3 plasmid. Cells wereharvested 40 h after they were returned to normal medium and lysed inpassive lysis buffer (100 µl; Promega). We assayed samples of lysate (20 µl)for both luciferase activities using the dual-luciferase assay system(Promega). Transfections, performed in triplicate, are presented as a meanand standard deviation.

AcknowledgementsWe thank A. Monteiro and H. Hanafusa for the kind gift of GBD-BRCA1constructs, and we thank M. Montminy, A. Dutta and D. Haile for helpfuladvice during the course of these experiments. This work was supported inpart by NIH grant GM53504, a Junior Faculty Research Award from theAmerican Cancer Society and also a Massachusetts Dept. of Public HealthBreast Cancer Research Program grant to J.D.P. S.F.A. is supported by NRSAGM18829 from the NIH, and T.N. is supported by the Uehara MemorialFoundation and by the Otsuka Pharmaceutical Company.

Received 9 February; accepted 26 May, 1998.

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