MERIT40 facilitates BRCA1 localization and DNA damage repair

Abstract

The product of breast cancer susceptibility gene 1, BRCA1, plays pivotal roles in the maintenance of genomic integrity. Mounting evidence indicates that BRCA1 associates with many proteins or protein complexes to regulate diverse processes important for the cellular response to DNA damage. One of these complexes, which mediates the accumulation of BRCA1 at sites of DNA breaks, involves the ubiquitin-binding motif (UIM)-containing protein RAP80, a coiled-coil domain protein CCDC98/Abraxas, and a deubiquitinating enzyme BRCC36. Here we describe the characterization of a novel component of this complex, MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd), which together with an adaptor protein BRE/BRCC45, enforces the BRCA1-dependent DNA damage response. MERIT40 is assembled into this RAP80/CCDC98-containing complex via its direct interaction with BRE/BRCC45. Importantly, MERIT40 regulates BRCA1 retention at DNA breaks and checkpoint function primarily via a role in maintaining the stability of BRE and this five-subunit protein complex at sites of DNA damage. Together, our study reveals that a stable complex containing MERIT40 acts early in DNA damage response and regulates damage-dependent BRCA1 localization.

Figure 1.

Identification of MERIT40 as a new component in the RAP80/CCDC98 protein complex. (A) 293T cells stably expressing SFB-tagged (S-tag, Flag epitope tag, and streptavidin-binding peptide tag)-RAP80, CCDC98, BRCC36, MERIT40, or BRE were used for TAP of protein complexes specifically from chromatin fractions isolated from irradiated cells. Tables are summaries of proteins identified by Mass spectrometry analysis. Letters in bold indicate the bait proteins. (B) Immunoprecipitations were performed using preimmune serum or anti-MERIT40 antibodies. Interactions between endogenous MERIT40 and RAP80, BRE, or BRCC36 were assessed by immunoblotting using antibodies as indicated. (C) Myc-tagged MERIT40 interacted strongly with SFB-tagged BRE. 293T cells were transiently transfected with plasmids encoding SFB-tagged RAP80, CCDC98, BRE, BRCC36, or empty vector together with plasmids encoding myc-tagged MERIT40. Cell lysates were precipitated with S-protein beads and immunoblotted with indicated antibodies. (D) CCDC98, BRCC36, or MERIT40 binds to different regions on BRE. 293T cells were transfected with plasmids encoding SFB-tagged full-length (FL), N-terminal part (N; residues 1–200), or C-terminal part (C; residues 201–383) of BRE, together with plasmids encoding Myc-tagged CCDC98, BRCC36, or BRE. Cell lysates were subjected to precipitation (IP) using S-protein beads, and immunoblotting was conducted using antibodies as indicated. (E) MERIT40 associates with BRE and forms IRIF. (Top panel) 293T cells were transfected with plasmids encoding SFB-tagged wild-type MERIT40 or its deletion mutants (Δ1–Δ4) together with plasmids encoding Myc-tagged BRE. Co-IP experiments were performed as described in C. (Bottom panel) 293T cells stably expressing SFB-tagged wild-type (WT) or deletion mutants (Δ1–Δ4) of MERIT40 were irradiated with 10 Gy, allowed to recove for 4 h before fixation and immunostaining with anti-Flag (red) and anti-γH2AX (green) antibodies.

Figure 2.

BRE mediates the interaction between MERIT40 and CCDC98. (A) RAP80, BRE, and BRCC36 bind to different regions on CCDC98. 293T cells were transfected with plasmids encoding SFB-tagged wild-type or internal deletion mutants of CCDC98 together with plasmids encoding Myc-tagged RAP80, BRE, or BRCC36. Cell lysates were prepared, precipitated with S-protein beads, and immnoblotted with indicated antibodies. (B) The CCDC98-interacting region, but not the UIM domains of RAP80 is required for the interaction between RAP80 and BRE, BRCC36, or MERIT40. 293T cells were transiently transfected with plasmids encoding SFB-tagged wild-type RAP80 (WT), UIM deletion mutant (Δ1), or CCDC98-interacting region deletion mutant (Δ3) of RAP80. Cell lysates were subjected to precipitation (IP) with S-protein beads and immunoblotting with indicated antibodies. (C, left panels) BRE and BRCC36 bind directly to CCDC98 in vitro. Bacterially expressed MBP-tagged CCDC98, BRE, MERIT40, or BRCC36 were incubated with GST fusion proteins as indicated. Proteins bound to the beads were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and immunoblotted with anti-MBP antibody. GST fusion proteins used in these pull-down assays were analyzed by Commassie blue staining. (Right panels) BRE associates with MERIT40 in vitro. S-tagged MERIT40 was coexpressed with His-tagged wild-type or deletion mutants of BRE using bicistronic vectors in BL21 cells. Proteins bound to S-beads were eluted and visualized by Commassie blue staining.

Figure 3.

MERIT40 is critical for maintaining the integrity of RAP80/CCDC98-containing protein complex. (A) U2OS cells were transfected with a scrambled control siRNA or siRNAs specific for the indicated genes. Cell lysates were prepared and immunoblotted with indicated antibodies. (B) CCDC98, BRE, and BRCC36 are required for the association of MERIT40 with RAP80. HeLa cells were transfected with control siRNA or siRNAs targeting CCDC98, BRE, or BRCC36. Cell lysates were immunoprecipitated with antibody against MERIT40, and immunoblotting experiments were carried out using antibodies as indicated. (C) CCDC98 and BRCC36 are required for the binding of BRE to RAP80, but are dispensable for BRE/MERIT40 interaction. 293T cells stably expressing SFB-BRE were transfected with control siRNA or siRNAs targeting RAP80, CCDC98, BRCC36, or BRCA1. Cell lysates were immunoprecipitated using anti-MERIT40 antibodies. Immunoblots were performed using antibodies as indicated. (D) Overexpression of BRE, MERIT40, or BRCC36 promotes RAP80/CCDC98 interaction. 293T cells were transfected with plasmids encoding SFB-tagged CCDC98 and Myc-tagged RAP80, together with empty vector or variables amount of plasmids encoding Myc-tagged BRE, MERIT40, or BRCC36 as indicated. Cell lysates were prepared, immunoprecipitated with S-protein beads, and immnoblotted with indicated antibodies. (E) Depletion of BRE, MERIT40, or BRCC36 impaired endogenous RAP80/CCDC98 or RAP80/BRCA1 interaction. HeLa cells were transfected with control siRNA or siRNAs targeting BRE, MERIT40, or BRCC36. Cell lysates were immunoprecipitated using anti-BRCA1 or anti-CCDC98 antibodies, and immunoblotting was carried out using indicated antibodies.

Figure 4.

IRIF formation of RAP80, CCDC98, BRCC36, BRE, and MERIT40 requires all four other components in this protein complex. (A,C–E) U2OS cells or 293T cells stably expressing SFB-tagged BRE (B) were transfected with control siRNAs or siRNAs specifically targeting indicated genes. Cells were irradiated, fixed, and immunostained with anti-MERIT40 (A), anti-Flag (BRE) (B), anti-BRCC36 (C), anti-RAP80 (D), or anti-CCDC98 (E) antibodies with or without costaining with anti-γH2AX antibodies. More than 200 cells were counted to determine the percentages of foci-forming cells in each sample.

Figure 5.

MERIT40 and BRE participate in IR-induced DNA damage response. (A) Defective BRCA1 foci formation was observed in RAP80-, CCDC98-, BRE-, MERIT40-, or BRCC36-depleted cells. U2OS cells were transfected with different siRNAs as indicated. Cells were irradiated, fixed, and immunostained with anti-BRCA1 antibody. More than 200 cells were counted to determine the percentages of cells containing BRCA1 foci. Two independent siRNAs were used for the depletion of CCDC98, BRE, BRCC36, or MERIT40, and similar results were obtained (data not shown). (B) IR-induced G2/M checkpoint is defective in cells depleted of MERIT40, BRE, or other components in the RAP80/CCDC98 complex. HeLa cells were transfected with indicated siRNAs and percentages of mitotic cells before and after radiation were determined by FACS analysis as described in the Materials and Methods. The figure represents the value obtained from three separate experiments. Error bars indicate standard deviations. (C) Cells depleted of MERIT40 or BRE display increased radiation sensitivity. HeLa cells were transfected with control siRNAs or siRNAs specifically targeting indicated genes. Cells were irradiated with various doses of IR. Percentages of surviving colonies were determined 11 d later. Experiments were done in triplicate. Results shown are averages of two or three independent experiments at each dose, and error bars indicate standard deviation. (D) A model describing the assembly of the RAP80/CCDC98/BRCC36/BRE/MERIT40 complex at sites of DNA breaks. See the text for details.