Loss of BAP111 enhances the phenotype caused by expression of brm^K804R in the developing eye. Representative eyes of the following genotypes are presented: (A) ey-GAL4 UAS-brm^K804R/+. (B) Wild type (Oregon-R). (C) Df(1)18.1.15/+, Pin^88k/+, TM6B, Hu Tb/+. (D) Df(1)18.1.15/+; ey-GAL4 UAS-brm^K804R/+. (E) Df(1)18.1.15/+; P[w⁺, UAS-BAP111] 22–1/+; ey-GAL4 UAS-brm^K804R/+. (F) Df(1)18.1.15/+, P[w⁺, UAS-BAP111^ΔHMG] 42–1/+, ey-GAL4 UAS-brm^K804R/+.

Developmental expression of the BAP111 protein. (A) Ten micrograms of protein extracted from staged embryos (0–2, 2–4, 4–8, 8–12, and 12–22 h), larvae (L), pupae (P), and adult females (♀) or males (♂) was analyzed by Western blotting with anti-BRM and anti-BAP111 antibodies. Blots are overexposed to reveal the signal in larvae. (B) Affinity-purified anti-BAP111 antibodies detect ubiquitous nuclear staining of BAP111 in syncytial blastoderm embryos (Upper) and germ band retracted embryos (Lower). (C) BAP111 is not associated with mitotic chromosomes. Higher magnification (630×) view of a cephalic-furrow stage embryo stained with BAP111 antibodies (green) and propidium iodide to visualize DNA (red). BAP111 is nuclear except in domains of mitotic activity where BAP111 becomes dispersed throughout the cell.

BAP111 is associated with the BRM complex in embryo extracts. (A) Characterization of the BAP111 polyclonal antibody. Western blot of an SDS/8% polyacrylamide gel loaded with 30 μg of 0–12 h embryo extract and probed with affinity-purified anti-BAP111 antibodies. (B) BAP111 coimmunoprecipitates with BRM. Immunoprecipitations were performed by using anti-HA antibodies and either wild-type extract (lanes 1–3) or extract containing HA-tagged BRM (lanes 4–6). Western blotting was performed on 1/10 of the total starting extract (E) and supernatant (S) and 1/5 of the total pellet (P) by using anti-BRM and anti-BAP111 polyclonal antibodies. Arrowheads at right indicate molecular mass markers of 210 kDa and 111 kDa. Note that BAP111 is found only in the pellet when the BRM protein is immunoprecipitated. All proteins in pellet samples show slightly reduced migration relative to S and P samples because of differences in buffer conditions. (C) Western blot of fractions from a Superose 6 gel filtration column loaded with embryo extract and probed with anti-BRM and anti-BAP111 antibodies. Vertical arrows mark the void and elution volumes of native molecular mass markers. Note that all of the endogenous BAP111 coelutes with the 2-MDa BRM complex.

Sequence and predicted domains of the BAP111 protein. (A) Amino acid sequence of the BAP111 protein. The HMG domain is indicated by a single underline, the NHRLI domain is boxed, and the coiled-coil region is indicated by a dashed line. Arrowheads mark the portion of BAP111 used to generate polyclonal antibodies. Brackets enclose the amino acids deleted in BAP111^ΔHMG. (B) Domains of the BAP111 protein. Domains are drawn to scale. Percent amino acid identity to BAP111 is indicated below domains. The proline-rich region of BAP111 (amino acids 360–749) is 30.5% proline. BAF57 is a subunit of BRG1/hBRM complexes (27). g-III-342 is the translation of a Caenorhabditis elegans gene predicted by Genie (48–50) and annotated in Intronerator (43). This sequence is largely confirmed by the 5′ EST yk538e12.5. There is no 3′ EST currently available to confirm the C terminus. (C) Sequence alignment of conserved domains of BAP111. In the NHRLI domain alignment, black indicates identity and gray indicates conservation in two or more residues at a given position. Alignment of the HMG domains is similarly coded but homology is to BAP111. Percent identity to BAP111 is indicated at the right of each HMG-domain sequence. Residues cited in the text as marking distinctions between sequence-specific and nonspecific HMG domains are marked above the alignment. Asterisks specify residues conserved in nonspecific HMG domains; arrows specify residues conserved in sequence-specific HMG domains.