Figure 1.

BRCA1 Interacts with SETX and Is Required for SETX Recruitment to the R-Loop-Associated Termination Pause Region of the Human β-actin Gene

(A) Co-immunoprecipitation (IP) of endogenous BRCA1 and SETX in HeLa cell (top) and in BJ-hTert human fibroblast extracts (bottom), using antibodies against BRCA1 (BRCA1 #1/#2) or SETX (SETX #1/#2). IgG, negative control.

(B) In vitro interaction assay using recombinant GST-BRCA1 fragments performed in HeLa cells. GSH bead-bound proteins were eluted and analyzed by immunoblot. Bottom: relative abundance of each recombinant affinity purified fusion protein (marked with a star) visualized by SDS-PAGE after Coomassie staining. Additional bands are readthrough and/or degradation products of recombinant proteins.

(C) Same as in (B) but with recombinant GST-SETX fragments.

(D) Schematic representation of the human β-actin gene: exons are numbered, and the red box reflects the existence of a transcription pause element. Location of primers used for the ChIP experiments are depicted above the diagram.

(E) Immunoblot that reflects the efficiency of the siRNA-mediated depletion of BRCA1 and SETX. Top: SETX and BRCA1; bottom: Vinculin used as a loading control.

(F) ChIP analyses performed on the β-actin gene from mock-treated (red bars), BRCA1- (green bars), or SETX-depleted cells (gray bars) using BRCA1 Ab #3. Average ChIP values ± SD from three independent experiments are shown.

(G) Same ChIP experiments as in (F) but with SETX Ab #1.

See also Figures S1 and S2.