It is well established that genetic mutations that impair BRCA1 function predispose women to early onset of breast and ovarian cancer. However, the co-regulatory factors that support normal BRCA1 functions remain to be identified. Using a biochemical approach to search for such co-regulatory factors, we identified hGCN5, TRRAP, and hMSH2/6 as BRCA1-interacting proteins. Genetic mutations in the C-terminal transactivation domain of BRCA1, as found in breast cancer patients (Chapman, M. S., and Verma, I. M. (1996) Nature 382, 678-679), caused the loss of physical interaction between BRCA1 and TRRAP and significantly reduced the co-activation of BRCA1 transactivation function by hGCN5/TRRAP. The reported transcriptional squelching between BRCA1 and estrogen receptor alpha (Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F., Auborn, K. J., Goldberg, I. D., and Rosen, E. M. (1999) Science 284, 1354-1356) was rescued by the overexpression of TRRAP or hGCN5. Histone acetyltransferase hGCN5 activity appeared to be indispensable for coregulator complex function in both BRCA1-mediated gene regulation and DNA repair. Biochemical purification of the hGCN5/TRRAP-containing complex suggested that hGCN5/TRRAP formed a complex with hMSH2/hMSH6, presumably as a novel subclass of hGCN5/TRRAP-containing known TFTC (TBP-free TAF-containing)-type histone acetyltransferase complex (hTFTC, hPCAF, and hSTAGA) (Yanagisawa, J., Kitagawa, H., Yanagida, M., Wada, O., Ogawa, S., Nakagomi, M., Oishi, H., Yamamoto, Y., Nagasawa, H., McMahon, S. B., Cole, M. D., Tora, L., Takahashi, N., and Kato, S. (2002) Mol. Cell 9, 553-562). Unlike other subclasses, the isolated complex harbored a previously unknown combination of components including hMSH2 and hMSH6, major components of the BRCA1 genome surveillance repair complex (BASC). Thus, our results suggested that the multiple BRCA1 functions require a novel hGCN5/TRRAP histone acetyltransferase complex subclass.