BRCA1 interacts with components of the histone deacetylase complex

Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892

^{To whom reprint requests should be addressed at: Genetics and Molecular Biology Branch, National Human Genome Research Institute, 49 Convent Drive, Room 3A14, Bethesda, MD 20892-4442. e-mail:}vog.hin.xileh@ydorbl.

Communicated by Francis S. Collins, National Institutes of Health, Bethesda, MD

Received 1999 Jan 5; Accepted 1999 Feb 25.

Abstract

Germ-line mutations in the BRCA1 tumor-suppressor gene are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 contains a carboxyl-terminal domain (BRCT) that is shared with several other proteins involved in maintaining genome integrity. In an effort to understand the function of BRCA1, we sought to isolate proteins that interact with the BRCT domain. Purified BRCT polypeptide was used as a probe to screen a human placenta cDNA expression library by Far Western analysis. Here we report that BRCA1 interacts in vivo and in vitro with the Rb-binding proteins, RbAp46 and RbAp48, as well as with Rb. Moreover, the BRCT domain associates with the histone deacetylases HDAC1 and HDAC2. These results demonstrate that BRCA1 interacts with components of the histone deacetylase complex, and therefore may explain the involvement of BRCA1 in multiple processes such as transcription, DNA repair, and recombination.

Abstract

More than half of families with inherited breast and ovarian cancer susceptibility are thought to harbor germ-line mutations in the BRCA1 gene. Frequent loss of the wild-type allele in tumors of mutation carriers suggests that BRCA1 acts as a tumor-suppressor gene. Surprisingly, mutations in BRCA1 in sporadic breast and ovarian cancer are extremely rare (1–3). To date, more than 600 different mutations in the BRCA1 gene have been reported (Breast Cancer Information Core: www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/). The majority of these are truncation mutations distributed over the entire length of the gene. Several missense mutations have also been shown to segregate with cancer susceptibility (1, 4, 5).

The BRCA1 gene was isolated and mapped to human chromosome 17q21 (6). The gene encodes an 1,863-aa protein with an apparent molecular mass of 220 kDa. Only a few conserved sequence motifs have been identified in the BRCA1 protein: an amino-terminal RING finger, a carboxyl-terminal region that contains two repeats of a newly identified motif, designated BRCT (BRCA1 carboxyl terminus) domain (7), and three nuclear localization signals in the central portion of the molecule (8). However, much of the biochemical function of BRCA1 is unknown.

BRCA1 is found in nuclear foci that form in a cell cycle-dependent manner (9, 10). Several lines of evidence suggest that BRCA1 expression is cell cycle regulated and plays a role in cell cycle checkpoints. BRCA1 mRNA is highly expressed during embryonic development and is increased in breast epithelia during pregnancy and in adult testis during the final stages of meiosis and spermatogenesis (11, 12), suggesting a role in terminal differentiation. Brca1−/− mouse embryos die early in development from cell proliferation defects, including cell cycle arrest (13, 14). In human cell lines, BRCA1 expression suppresses cell growth (15–17). The presence of the BRCT motif in BRCA1 also links it to cell cycle control. Many other cell cycle checkpoint proteins, such as the p53-binding protein (53BP1), fission yeast replication checkpoint proteins, (RAD9 and RAD4), the DNA repair proteins XRCC1 and XRCC4, and all members of the retinoblastoma protein family (Rbp107 and Rbp130) contain a BRCT motif (7, 18).

A number of observations also link BRCA1 to transcription regulation. The carboxyl-terminal region has an intrinsic trans-activation activity in cells when fused to the GAL4 DNA-binding domain (19, 20). BRCA1 activates transcription of the cell cycle regulators p21^{and MDM2 when cotransfected with p53 (}21, 22), supporting a role in the control of the cell cycle and proliferation. Other evidence suggests that BRCA1 plays a role in DNA repair. BRCA1 colocalizes with the double-strand break-repair, homologous recombination protein, RAD51, the human homolog of Escherichia coli RecA (9, 10, 23, 24). After exposure to ionizing radiation and other DNA-damaging agents, BRCA1 becomes hyperphosphorylated, disperses from nuclear foci, and accumulates in proliferating cell nuclear antigen-containing structures (10). Recently, it was reported that embryonic stem cells lacking BRCA1 are hypersensitive to ionizing radiation and are unable to mediate transcription-coupled repair after DNA damage (25).

Several proteins are reported to bind and interact directly with BRCA1. Among them are components of the nuclear import pathway (8) that bind to the nuclear localization signals; a component of the ubiquitin pathway (26); and a novel RING finger/BRCT domain-containing protein, BARD1 (27), binding to the RING finger motif. Recently, p53, RNA helicase A, and CtIP were reported to bind BRCA1, supporting its role in transcriptional regulation (21, 22, 28–30).

We hypothesized that the carboxyl terminus of BRCA1, harboring a trans-activation function and consisting of two BRCT domains, would interact with other proteins that mediate tumor suppression, transcription regulation and, DNA repair. We screened a human placental cDNA expression library by a Far Western method (31) to identify proteins that interact with the carboxyl terminus of BRCA1. We found that the retinoblastoma-binding protein, RbAp46, interacts in vitro with the BRCT domain as well as with full-length BRCA1 in vivo. RbAp46, together with its homolog RbAp48, were first isolated based on interaction with the carboxyl terminus of the retinoblastoma protein, Rb (32–34). These proteins, members of the WD (Trp-Asp) repeat family, are highly similar in sequence and are functionally related (34). Recently, RbAp48 was found to be one of the three subunits of chromatin assembly factor 1 (35, 36). Both RbAp46 and RbAp48 are components of histone deacetylase complexes and are involved in chromatin remodeling (37, 38). Additionally, we found that the carboxyl terminus of BRCA1 associates with the histone deacetylases HDAC1 and HDAC2, implying that BRCA1 is a component of a histone deacetylase complex.

Acknowledgments

We thank Drs. P. Liu, J. Koh, S. Danoff, and F. Collins for helpful comments on the manuscript. We thank Dr. A. Gazdar for providing the HCC1937 cell line, Dr. P. H. Reddy for the GST-HD protein, M. Erdos for BRCA1 plasmids, and S. King for technical assistance.

Acknowledgments

ABBREVIATIONS

BRCT	BRCA1 carboxyl terminus
GST	glutathione S-transferase

ABBREVIATIONS

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