TAC, a TBP-sans-TAFs Complex Containing the Unprocessed TFIIAαβ Precursor and the TFIIAγ Subunit  Dimitra J. Mitsiou, Hendrik G. Stunnenberg  Molecular Cell  Volume 6, Issue 3, Pages 527-537 (September 2000) DOI: 10.1016/S1097-2765(00)00052-6

Figure 1 Coexpression of hTBP with hTFIIA Results in the Formation of a Stable TBP-TFIIA-Containing Complex in P19 EC but Not in COS7 Cells (A) Extracts from P19 EC or COS7 cells transfected with plasmids expressing hTBP alone (lanes 1) or with increasing amounts of Myc-tagged hTFIIAαβ and HA-tagged hTFIIAγ (lanes 2–5) were analyzed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39), hTFIIAαβ, hTFIIAα (Myc), and hTFIIAγ (HA). Amounts of extract loaded for each sample were adjusted according to the expression levels of the CAT internal control. (B) Extracts from P19 EC cells transfected with plasmids expressing HA-tagged hTBP, Myc-tagged hTFIIAαβ, and hTFIIAγ as indicated were subjected to immunoprecipitation under high stringency conditions using the Myc antibody. The immunoprecipitates were eluted with an excess of the Myc synthetic peptide. One third of the eluates (lanes 4–6) were analyzed by SDS–PAGE and immunoblotting as in (A), with the exception that the anti-hTFIIAγ polyclonal serum was used for detection of hTFIIAg. Two thirds of the eluates were subjected to a second immunoprecipitation step (lanes 7–9) using the HA antibody against the HA-tagged hTBP and analyzed as described for lanes 4–6. The asterisk indicates a nonrelated protein migrating faster than the endogenous TBP. (C) Extracts from P19 EC cells transfected as described in (B) were subjected to immunoprecipitation using the SL39 antibody against TBP (lanes 4–6) followed by peptide elution and Western blot analysis as above. “endTBP” represents endogenous TBP. Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)

Figure 2 Exogenous TAC Does Not Contain TAFIIs TFIID fractionated from P19 EC cells (phosphocellulose-D fraction) was immunoprecipitated using the SL39 antibody against TBP bound to protein G beads (lane 3) or empty beads as control (lane 2). In addition, extracts from P19 EC cells transfected with plasmids expressing hTBP, Myc-tagged hTFIIAαβ, and HA-tagged hTFIIAγ as indicated were subjected to immunoprecipitation using the Myc antibody. Immunoprecipitates were eluted with the respective synthetic peptides and analyzed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39) and the indicated TAFIIs. The input represents one fourth of the samples analyzed for immunoprecipitation. “endTBP” represents endogenous TBP. Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)

Figure 3 A Native TAC Is Detected in EC Cells (A) Extracts from P19 EC cells were subjected to immunoprecipitation under high stringency conditions using the SL27 antibody against TBP bound to protein G beads (lane 4) or empty beads as control (lane 2). The immunoprecipitates were analyzed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39) and TFIIAαβ (α- and β-specific). Alternatively, the SL39 antibody cross-linked to protein G beads (lanes 3) was used for immunoprecipitation followed by peptide elution and analysis as above. (B) Extracts from P19 EC cells were subjected to immunoprecipitation using the SL39 antibody as in (A). One third of the eluate was analyzed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39) and TFIIAαβ (β-specific) (lane 2). Two thirds of the eluate was subjected to a second immunoprecipitation step using the SL27 antibody (lane 4) or empty beads as control (lane 3) and analyzed as described for lane 2. (C and D) Extracts from Tera2 EC (C), COS7 (D), or P19 EC cells (D) were subjected to immunoprecipitation using the SL27 antibody. Western blot analysis was as described in (B). (E) Extracts from P19 EC cells were subjected to immunoprecipitation under low stringency conditions using the SL39 antibody followed by peptide elution. One third of the eluate was analyzed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39), TFIIAαβ (as in [A]), and the indicated TAFs (lane 2). Two thirds of the eluate was subjected to gel filtration chromatography using a Superose 6 column. Fractions 4–20 were analyzed as described for lane 2 (lanes 3–19). The input (whole-cell extract, WCE) represents one tenth of the extract used for immunoprecipitation in (A)–(E). “IgG” represents immunoglobulin. Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)

Figure 4 Purification of TAC by Ni2+-NTA-Agarose Affinity Chromatography (A) Extracts from P19 EC cells were subjected to affinity chromatography using a Ni2+-NTA-agarose column. Elution of the proteins bound to the column was followed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39), TFIIAαβ (α- and β-specific), TFIIAγ, TRF2, and the indicated TAFs. The whole-cell extract (WCE) and the flowthrough of the column represent one sixth of the eluate. (B) Ni2+-NTA eluate was subjected to immunoprecipitation under low (lanes 2 and 3) or high stringency conditions (lanes 4 and 5) using the SL39 antibody cross-linked to protein G beads (lanes 3 and 5) or empty beads as control (lanes 2 and 4). Elution with the SL39 synthetic peptide was followed by Western blot analysis as in (A). The input represents one fifth of the eluates analyzed for immunoprecipitation. The asterisk indicates a nonrelated protein that was immunoprecipitated. Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)

Figure 5 TAC Binds DNA (A) Whole-cell extracts (WCE, lanes 1–3) or immunopurified TAC (lanes 4–6) from P19 EC cells transfected with plasmids expressing hTBP, Myc-tagged hTFIIAαβ, and HA-tagged hTFIIAγ as indicated were used for electrophoretic mobility shift assay with a synthetic oligonucleotide comprising the adenovirus 2 major late promoter TATA box. (B) Ni2+-NTA eluate (lane 2) or immunopurified TAC (as described in Figure 4B; lanes 3 and 4) were used for electrophoretic mobility shift assay as in (A). E. coli-expressed recombinant hTBP, mTBP, and hTFIIA (refolded from denatured hTFIIAαβ and hTFIIAγ subunits) were used, giving rise to the DA (TBP-TFIIA-DNA) complex (lane 7 in [A] and lane 1 in [B]). Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)

Figure 6 TAC Mediates Transcriptional Activation In Vivo (A–C) P19 EC or COS7 cells were transfected with the tk-luc or RARE-tk-M1-luc reporters and plasmids expressing either hTBP or hTBP-spm3 alone or along with increasing amounts of Myc-tagged hTFIIAαβ and HA-tagged hTFIIAγ and the pSV2-CAT internal control plasmid as indicated. Luciferase values were normalized relative to expression from CAT internal control. (D and E) Total RNA from P19 EC cells transfected with the tk-luc (D) or pGEM3Z(+)-hU1 reporters (E) and plasmids expressing hTBP, Myc-tagged hTFIIAαβ, and HA-tagged hTFIIAγ as indicated was used for the RNase protection assay. Luciferase and U1 probes protect the correctly initiated 137 nt-long (D) and 164 nt-long transcripts (E), respectively. The position of the HinfI-restricted pAT153 fragments of 154 and 145 nt, used as size markers, is shown. Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)

Figure 7 TAC Binds Chromatin In Vivo P19 EC or COS7 cells transfected with the tk-luc reporter and plasmids expressing hTBP, Myc-tagged hTFIIAαβ, and HA-tagged hTFIIAγ as indicated were fixed in vivo with formaldehyde, and the cross-linked chromatin complexes were purified on CsCl gradients. Fractions 1–8 were analyzed by SDS–PAGE and immunoblotting using antibodies against TBP (SL39, [A]) and hTFIIAαβ and hTFIIAα (Myc, [B]). Fraction 1 is from the bottom of the gradient. The asterisk indicates a nonrelated protein migrating roughly at the position of the hTFIIAα subunit. “endTBP” represents endogenous TBP, and “WCE” represents whole-cell extract. Molecular Cell 2000 6, 527-537DOI: (10.1016/S1097-2765(00)00052-6)