Fig. 1. BAF57 interacts with the SRC1 bHLH–PAS domain in yeast cells. (A) Schematic representation of SRC1e, the LexA chimera used as bait in the two-hybrid screening, the truncated VP16-tagged BAF57 clone, full-length BAF57 and the two BAF57 deletion mutants used in this study. Numbers refer to amino acids in the full-length proteins. The bHLH and the PAS homology region (containing two imperfect repetitions named PAS A and PAS B regions), the nuclear RID, and the activation domains 1 and 2 (AD1 and AD2, respectively) in SRC1e and the HMG domain in BAF57 are indicated. (B) The L40a yeast strain expressing either LexA-DBD or LexA-DBD fused to the SRC1 bHLH–PAS domain (PAS) was transformed with either the empty pASV3 plasmid or pASV3 expressing clone 3.4 fused to the VP16 activation domain. β-galactosidase activity in each yeast extract was measured in duplicate. Data represent the mean + SD of two independent transformants.

Fig. 2. In vitro interaction of BAF57 with p160 coactivators. (A) Binding of GST fusion proteins of truncated BAF57 encoded by clone 3.4 to the SRC1 bHLH–PAS domain, full-length SRC1 and RAC3. GST fusion of amino acids 4–197 of BAF57, coupled to Sepharose beads was incubated with in vitro translated [³⁵S]methionine-labelled SRC1 bHLH–PAS domain (amino acids 1–361), full-length SRC1e or RAC3. After extensive washing, samples were boiled and separated on 10% SDS–PAGE. Gels were fixed and dried, and the labelled proteins were detected by fluorography. (B) Binding of GST–BAF57 to SRC1e, RAC3, TIF2 and ΔPAS-SRC1e. GST fusion proteins of full-length BAF57 were incubated with ³⁵S-labelled full-length SRC1e, RAC3, TIF2 or ΔPAS-SRC1e as described above. (C) Binding of GST–bHLH–PAS to BAF57. GST fusion proteins of the first 450 amino acids of SRC1 were incubated with ³⁵S-labelled full-length BAF57 as described above. In each panel, the input lane represents 10% of the total volume of lysate used in each reaction.

Fig. 3. Effect of BAF57 expression on transcriptional activation by ER in cell lines expressing endogenous BAF57. (A) HeLa cells were transiently transfected with expression vectors for mERα and SRC1e, the 2XERE-pS2-luciferase reporter, different amounts of BAF57 expression vector and an internal control vector (pRL-CMV, providing constitutive expression of Renilla luciferase). (B) COS-1 cells were transiently transfected with expression vectors for mERα and SRC1e, the 2XERE-pS2-luciferase reporter, 20 ng of full-length BAF57, ΔC-BAF57 or ΔN-BAF57 expression vectors and pRL-CMV as an internal control. In each case, after transfection, cells were washed and incubated with vehicle (white bars) or 17β-estradiol (black bars) at 10^–8 M for 24 h. Subsequently, cell lysates were assayed using a dual luciferase reporter system. Normalized values are expressed relative to the activity of mERα alone in the presence of 10^–8 M E2. The results shown represent the average of at least two independent experiments assayed in quadruplicate + SD. The asterisks represent statistical analysis, which shows that the results observed were significant (p < 0.05 for HeLa cells and p < 0.001 for COS-1 cells). (C) Western blotting showing expression levels of BAF57 proteins in HeLa, COS-1 and BT549 cell lines.

Fig. 4. BAF57 is required for ER coactivation by p160 proteins. (A) The BAF57-deficient breast ductal carcinoma cell line, BT549, was transiently transfected with expression vectors for mERα and SRC1e, the 2XERE-pS2-luciferase reporter, different amounts of BAF57 expression vector and pRL-CMV as an internal control. (B) BT549 cells were transiently transfected with expression vectors for mERα and RAC3, the 2XERE-pS2-luciferase reporter, different amounts of BAF57 expression vector and pRL-CMV as an internal control. Data are presented as described in Figure 3. The results shown represent the average of at least two independent experiments assayed in quadruplicate + SD. The asterisks represent statistical analysis, which shows that the results observed were significant (p < 0.001).

Fig. 5. Effect of BAF57 and SRC1e deletion mutants on transcriptional activation by ER. (A) BAF57 deletion mutants do not restore SRC1e coactivation in BAF57-deficient cells. BT549 cells were transiently transfected with expression vectors for mERα and SRC1e, the 2XERE- pS2-luciferase reporter, 20 ng of full-length BAF57, ΔC-BAF57 or ΔN-BAF57 expression vectors and pRL-CMV as an internal control. Data are presented as described in Figure 3. The results shown represent the average of two independent experiments assayed in quadruplicate + SD. (B) BAF57 enhances ER coactivation by ΔPAS–SRC1e in BAF57-deficient cells. BT549 cells were transiently transfected with expression vectors for mERα and ΔPAS–SRC1e, the 2XERE-pS2-luciferase reporter, 20 ng of BAF57 expression vector and pRL-CMV as an internal control. Data are presented as described in Figure 3. The results shown represent the average of two independent experiments assayed in quadruplicate + SD.

Fig. 6. In vitro interaction of BAF57 with ER. (A) Schematic representation of mERα and the deletion mutants used in the GST pull-down assay. Indicated are the ligand-independent activation function (AF1), the DBD, and the ligand-dependent activation function (AF2). Numbers refer to amino acids in the full-length mERα. (B) Binding of GST fusion proteins of BAF57 to ³⁵S-labelled mERα or hERβ. (C) Binding of GST fusion proteins of BAF57 to ³⁵S-labelled mERα AF2 mutants. (D) Binding of GST fusion proteins of mERα deletion mutants to ³⁵S-labelled full-length BAF57. (E) Binding of GST fusion proteins of full-length BAF57 to ³⁵S-labelled AF1-DBD mERα deletion mutant. When required, the assays were performed in the presence of vehicle (–) or 100 nM 17β-estradiol (E2). Bound proteins were visualized as described in Figure 2A. To the right of each panel, the ³⁵S-labelled proteins used in the assay are indicated and the fold induction in the binding observed in the presence of hormone relative to that detected without added hormone. Below each panel, the percentage of the input pulled down for each assay is shown. In each panel, the input lane represents 10% of the total volume of lysate used in each reaction.

Fig. 7. Hormone-dependent interaction between BAF57 and ERα in cellular extracts. (A) Co-immunoprecipitation of endogenous BAF57 and ERα. ZR75.1 cells were treated with anti-estrogens (ICI) or 17β-estradiol for 30 min. Whole-cell lysates were then immunoprecipitated with antibodies against BAF57. The immunoprecipitated material was subjected to western blotting analysis with anti-ERα monoclonal IgG. (B) Whole-cell extracts from SW13 cells (BRG1/BRM-deficient cell line), previously transfected with hERα, were incubated with GST alone or GST–BAF57 bound to glutathione– agarose in the presence of vehicle (–) or 100 nM 17β-estradiol (E2). The associated hERα was detected by western blotting using anti-ERα monoclonal IgG.

Fig. 8. Hormone-dependent association of BAF57 with the endogenous pS2 promoter. (A) ZR75.1 cells were deprived of estrogen for 48 h and then treated with 100 nM 17β-estradiol (E2) or vehicle (–) for 30 min, and fixed immediately using formaldeyde. Soluble chromatin fragments were obtained by sonication and subjected to immunoprecipitation using anti-SRC1 antibodies. (B) Soluble chromatin fragments from ZR75.1 cells treated as described above were subjected to immunoprecipitation using anti-BAF57 antibodies. (C) ZR75.1 cells maintained in estrogenic conditions were treated with anti-estrogens (ICI) or vehicle (–) for 30 min, and soluble chromatin fragments were subjected to immunoprecipitation using anti-acetylated histone H4 antibodies. ChIP assays were quantified by real-time PCR using primers specific to the ERE-containing region of the pS2 promoter or an enhancer region 5′ of β-globin gene. The ChIP assays were repeated several times, and results of a representative experiment are shown.