A novel cDNA clone closely related to yeast, Drosophila and human ADA3. (A) Sequence comparison between yADA3 and mADA3. Degrees of conservation (% identity and similarity) are indicated for the different regions of both proteins. Indicated are the LxxLL motifs. The C-terminal ADA2 interaction domain in yeast ADA3 was also found to encode the NR interface (8). (B) Northern blot analyses of mADA3 expression. A Clontech mouse multiple tissue northern blot was probed with a radiolabeled sonde for mADA3 revealing a broad but uneven expression pattern. Note the high levels of mADA3 RNA in heart and testis and the virtual absence of expression in the spleen. Size markers are on the left.

mADA3, unlike yADA3, does not interact directly with hERα. (A) mADA3 failed to interact with hERα in yeast. Plasmids expressing yeast and mouse ADA3 fused to VP16 were introduced into the L40 reporter strain together with lexA fused to hERα DEF domains (encompassing the C-terminal LBD). Transformants were grown in the presence or absence of estrogen (500 nM). β-Galactosidase activities are expressed as in Figure 2A. (B) mADA3 also failed to interact with hERα in vitro. A GST-based interaction assay similar to that described in Figure 2B was performed using in vitro ³⁵S-labeled yADA3 (lane 1) or mADA3 (lane 5) incubated with unfused GST (lanes 2 and 5) or GST–hERαCDEF (lanes 3 and 6).

Endogenous hADA3 associates with hERα in MCF7 cells. (A) Detection of endogenous hERα in hADA3 immunoprecipitates. MCF7 cells were grown in charcoal-treated and delipidized FCS-containing medium, and treated for 6 h either with OHT, with estrogen (E2) or with vehicle alone (–). Nuclear extracts were prepared and analyzed by western blotting either directly (input) or following immunoprecipitation with a purified polyclonal antibody raised against a mADA3 peptide (α1328) or with an irrelevant antibody raised against the DNA-binding domain of the yeast transcription factor Gal4 (αGal4). Western blots were probed with a monoclonal antibody against hERα (αB10) and subsequently with an anti-light chain peroxidase-coupled secondary antibody (top), or with a mixture of anti-mouse and anti-rabbit heavy chain peroxidase-coupled secondary antibodies (bottom). (B) Detection of TFTC subunits in hERα immunoprecipitates. Immunoprecipitations from MCF7 cell nuclear extracts with a monoclonal antibody against hERα (αF3) co-purify hADA2 (as revealed by polyclonal antibody α1275) and hTAF_II30 (as revealed by monoclonal antibody αTAF_II30) (lanes 5 and 6). Control experiments with the αGal4 antibody confirm the specificity of the interactions (lanes 3 and 4). FT, flow-through from the affinity column (lanes 7 and 8). Note that hERα is quantitatively removed from the extracts.

mADA3 interacts with ADA2 in vivo and in vitro. (A) Yeast and mouse ADA3 interact with yeast and human ADA2 in yeast. cDNAs expressing the indicated VP16 and lexA fusion proteins were transformed into the yeast strain L40, and the activity of the lexA binding site-containing LacZ reporter was quantified by measuring relative β-galactosidase activity. Given are the mean β-galactosidase values (in relative units) for two independent transformations. (B) Yeast and mouse ADA3 interact with hADA2 in vitro. In vitro ³⁵S-labeled yADA3 (lane 1) or mADA3 (lane 4) was incubated with ‘control’ GST (lanes 2 and 5) or GST fused to hADA2 (lanes 3 and 6). An autoradiography of a dried SDS–polyacrylamide gel is shown. The positions of marker proteins relative to the samples are shown on the left. Arrows indicate the positions of mADA3 and yADA3.

hERα associates with mADA3 in vivo and interacts with the mADA3-containing TFTC in vitro. (A) hERα coimmunoprecipitates with FLAG-tagged yADA3 from transfected COS-1 cells. COS-1 cells were transiently transfected with FLAG-yADA3 and hERα cDNAs. Transfected cells were treated with estradiol (E2, +) or vehicle alone (–) for 12 h. Extracts were prepared and subjected to immunoprecipitation with a monoclonal antibody against the FLAG epitope (αM2). Protein eluates were separated by SDS–PAGE and blotted to nitrocellulose membranes. Proteins were identified using the anti-FLAG antibody to detect the FLAG-tagged mADA3 and a monoclonal antibody (αF3) to detect the ERα. Lanes 1–5 show the input controls for the different cellular extracts, lanes 6–10 the immunopurified samples. The different co-transfected cDNA are indicated on top of the panel, size markers are shown to the left. (B) hERα coimmunoprecipitates with FLAG-tagged mADA3 from transfected COS-1 cells. The FLAG-tagged mADA3 cDNA was transfected alone or in combination with the hERα cDNA (as indicated above the panel). Extracts were obtained as in (A) and subjected to immunoprecipitation with the monoclonal anti-FLAG M2 (IBI-Kodak) agarose. Bound proteins (along with input controls) were identified by western blotting. (C) mADA3 can bind to hERα in vitro as part of the human TFTC. About 200 ng of purified human TFTC was incubated with unfused GST (lane 2) or GST–hERα (lanes 3 and 4) in the presence of either OHT or estrogen (E2). Lane 1 represents 40 ng of hTFTC loaded as input control. Shown is a western blot probed with monoclonal or polyclonal antibodies against the TFTC subunits hDDB1L, hGCN5, hPAF65β, hSpt3 and a polyclonal mADA3 antibody that cross-reacts with the human homolog.