BRCA1 Regulates GADD45 through Its Interactions with the OCT-1 and CAAT Motifs

Abstract

BRCA1, a breast and ovarian cancer susceptibility gene, has been implicated in gene regulation. Previous studies demonstrate that BRCA1 induces GADD45, a p53-regulated and stress-inducible gene that plays an important role in cellular response to DNA damage. However, the mechanism(s) by which BRCA1 regulates GADD45 remains unclear. In this report, we have shown that BRCA1 activation of the GADD45 promoter is mediated through the OCT-1 and CAAT motifs located at the GADD45 promoter region. Site-directed mutations of both OCT-1 and CAAT motifs abrogate induction of the GADD45 promoter by BRCA1. Both OCT-1 and CAAT motifs are able to confer BRCA1 inducibility in a nonrelated minimal promoter. Physical associations of BRCA1 protein with transcription factors Oct-1 and NF-YA, which directly bind to the OCT-1 and CAAT motifs, are established by biotin-streptavidin pull-down and coimmunoprecipitation assays. Such protein interactions are required for interaction of BRCA1 with the GADD45 promoter because either immunodepletion of Oct-1 and NF-YA proteins or mutations in the OCT-1 and CAAT motifs disrupt BRCA1 binding to the GADD45 promoter. These findings indicate that BRCA1 can up-regulate its targeted genes through protein-protein interactions and provide a novel mechanism by which BRCA1 participates in transcriptional regulation.

Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

“…Since the BRCA1 binding sites within the GADD45 promoter have been characterized, we chose GADD45 promoter constructs, linked to chloramphenicol acetyl transferase (CAT) as a reporter, to evaluate the ability of heregulin β1 to regulate BRCA1 transcriptional activity. BRCA1 binds to two OCT-1 motifs and one CAAT motif within the GADD45 promoter [11]. The level of BRCA1-transactivated GADD45 promoter activity, revealed as CAT catalytic activity, was increased by 150% in cells treated with heregulin β1 relative to the promoter activity in cells treated with vehicle (Fig.…”

“…Mutations were confirmed by DNA sequencing. Oligonucleotides corresponding to DNA sequences in the GADD45 promoter were designed as described [11] and cloned into the Kpn I and Bgl II sites of pCAT3 Enhancer plasmid (Promega) to generate GADD45 promoter constructs. Oligonucleotides were generated by Integrated DNA Technologies (Coralville, IA).…”

“…For example, the abilities of BRCA1 to inhibit cell growth, regulate the cell cycle and induce apoptosis are probably due to its transcriptional co-activation activity. BRCA1 induces the expression of genes involved in these processes, including p21 WAF/Cip 1 [3] and GADD45 [9], which control the cell cycle, and p53 [10] and Bcl-2 [11], which have roles in apoptosis. The ability of BRCA1 to function in DNA repair processes may be coupled to its ability to act as an E3 ubiquitin ligase.…”

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''.…”

“…Several studies suggest that BRCA1 up-regulates the expression of Gadd45 (¼ Gadd45a), in part, by activating the Gadd45 promoter (Harkin et al, 1999;Jin et al, 2000a). BRCA1-mediated activation of the Gadd45 promoter occurred through association with Oct-1 and CAAT sites within the Gadd45 promoter and involved interactions between BRCA1 and the corresponding site-specific transcription factors, Oct-1 and NF-YA, respectively (Fan et al, 2002b). The significance of BRCA1 inducible Gadd45 expression is unclear, but one study suggests that BRCA1 over-expression stimulates the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) pathway of cellular apoptosis via the induction of Gadd45 expression (Harkin et al, 1999).…”