BRC-1–BRD-1 localization during gametogenesis.

(A) Embryonic viability levels in various mutant and tagged genetic backgrounds. Functionality of fusion proteins was assessed by exposing worms to IR and then scoring the hatching rate of embryos laid in the following 24 hours. Error bars indicate standard deviation. The number of animal analyzed was: WT (0 Gy, n = 11, 75 Gy, n = 27), brc-1(KO) (0 Gy, n = 5, 75 Gy, n = 13), brc-1(tm1145) brd-1(dw1) (0 Gy, n = 12, 75 Gy, n = 18), brd-1(dw1) (0 Gy, n = 5, 75 Gy, n = 10), brd-1(gk297) (0 Gy, n = 5, 75 Gy, n = 19), brc-1::HA (0 Gy, n = 10, 75 Gy, n = 12), GFP::brc-1 (0 Gy, n = 5, 75 Gy, n = 14), brc-1::HA brd-1(gk297) (0 Gy, n = 5, 75 Gy, n = 10), brd-1::HA (0 Gy, n = 5, 75 Gy, n = 11), brd-1::HA brc-1(KO) (0 Gy, n = 5, 75 Gy, n = 13), brd-1::HA brc-1(tm1145) (0 Gy, n = 5, 75 Gy, n = 16). (B) Left: western blot analysis using an anti-HA antibody to monitor BRC-1::HA expression in whole-cell extracts. WT (N2) worms were used as negative control to test specificity of anti-HA antibody. Actin was the loading control. BRC-1::HA is predicted to run at 70KDa, which is consistent with the specific band detected slightly below the 75KDa band in the ladder. Right: protein fractionation showing BRC-1::HA enrichment in the nucleus. Equal amounts of protein were loaded for each fraction. C = cytosol, NS = soluble nuclear pool, CB = chromatin-bound pool. GAPDH and histone H3 were used as loading controls for the cytosolic and chromatin-bound samples, respectively. (C) Top: whole-mount gonad from brc-1::HA worms dissected and stained with DAPI and anti-HA antibody, showing ubiquitous BRC-::HA expression throughout the germline. Note the progressive enrichment on the SC and short arms of bivalents. Bottom: enlarged images of specific regions of the gonad: mitotic tip (red frame), mid-pachytene (yellow frame) and late pachytene/diplotene (magenta frame). Scale bar, 10 μm. (D) Mid-pachytene (light blue frame) and diplotene (light purple frame) nuclei stained with DAPI, anti-HA and BRD-1 antibodies display large co-localization of BRC-1::HA and BRD-1. Scale bar, 5μm.