Novel cofactors and TFIIA mediate functional core promoter selectivity by the human TAFII150-containing TFIID complex

Abstract

TATA-binding protein-associated factors (TAFIIs) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAFII150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAFIIs within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAFII150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAFII150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAFII-dependent TATA-less promoter. Therefore, TAFII-promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAFII-mediated synergism between TATA and initiator elements (TIC-1) and TAFII-dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC- and TAFII-dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAFIIs through core promoter elements and TAFII-dependent activator function.

FIG. 1

Predicted amino acid sequence of human TAF_II150. Positions identical to those in Drosophila TAF_II150 are marked with asterisks (ClustalW alignment program of MacVector with a blosum matrix; gap distance, 8; open gap penalty, 10; extend gap penalty, 0.1). The underlined sequence (amino acids 90 to 100) corresponds to a Glu-C peptide microsequence from the 135-kDa TAF_II protein of highly purified eTFIID. A mouse TAF_II150 N-terminal EST sequence (GenBank accession no. AA200390) is 100% identical at the amino acid level to the human sequence shown here from position 120 to the first methionine and diverges upstream with no other ATG (data not shown). However, an independent human 5′-end PCR clone has one nucleotide difference, which creates an in-frame upstream but weaker (non-Kozak) initiator ATG and adds the 10 predicted amino acids MPLTGVEPAR at the N terminus. This upstream ATG has also been observed recently in independent CIF150/TAF_II150 cDNAs (12a, 16).

FIG. 2

Human TAF_II150 (hTAF_II150) is a tightly associated component of TFIID. (A) Scheme for fractionation and immunopurification of TFIID. Nuclear extracts (NE) of HeLa cells and derived eTFIID-expressing cell lines were chromatographed as indicated on phosphocellulose P11, DE52 (DE), Mono S (MS), and anti-FLAG (M2)-agarose or anti-HA antibody (Immuno-Affinity) resins. Numbers indicate the KCl molarity of elutions. (B) Immunodetection of human TAF_II150 in a TFIID-containing fraction with anti-human TAF_II150 antiserum and 5 μl of the indicated native fractions (lanes 1 to 5) or of in vitro-translated f:TAF_II150 (Retic-f:TAF150; lane 6) and control (Retic-control; lane 7) lysates. The position of native TAF_II150 and in vitro-translated f:TAF_II150 is indicated by an arrowhead. An asterisk indicates a minor cross-reacting band of unknown origin in lane 5. (C) Stable association of hTAF_II150 in immunopurified eTFIID, determined by Western blot analysis of eTFIID complexes from human cell lines expressing f:TBP, HA:TBP, f:TAF_II100, or f:TAF_II135 and immunopurified directly from nuclear extracts (lanes 2 and 3) or from a two-column-purified TFIID fraction and washed with 0.3 M KCl (lane 1) or with 1.2 M KCl (lane 4; see also text and panel A). The blot first was probed with anti-human TAF_II150 antibodies and then was stripped and reprobed with antisera directed against the other TAF_IIs and TBP as indicated. The TAF_II150 bands on the blot from the first immunoreaction were coincident with the TAF_II135 bands in the second immunoreaction (data not shown), and the relevant portion of the first immunoblot is placed above the second blot in the figure. Similar results were obtained in a side-by-side immunoblot analysis of eTFIID and recombinant TAF_II135 with TAF_II135 and TAF_II150 antibodies (data not shown). (D) Human TAF_II150 is depleted from a two-column (P11 0.85/DE 0.3) native TFIID fraction with specific anti-TAF_II100 antibodies. Lanes 1 and 2 are from an immunoblot analysis of TFIID components present in the supernatant of a control immunodepletion (with protein A resin alone) (mock; lane 1) and in the supernatant after depletion with specific anti-TAF_II100 antibodies bound to protein A resin (Δ TAF100; lane 2). (E) The amounts of TAF_II150 relative to other TFIID subunits are similar in nuclear extracts and in purified TFIID, as determined by Western blot analysis of TBP and TAF_IIs in 5 μl (lane 1) and 1 μl (lane 2) of nuclear extract (NE), 8 μl of immunopurified f:TBP-TFIID (eTFIID; lane 3), and 8 μl of native TFIID (Mono S [MS]) fraction (lane 4). The positions and identities of the different TAF_IIs and TBP are indicated. (F) Recombinant human f:TAF_II150 has an electrophoretic mobility on SDS-PAGE similar to that of human TAF_II135 in eTFIID. Highly purified f:TBP-TFIID (8 μl of eTFIID; lane 1) and two independent preparations of baculovirus-expressed recombinant immunopurified f:TAF_II150 (a and b; lanes 2 and 3) were analyzed on an SDS–10% polyacrylamide gel stained with silver. The positions of TAF_IIs and f:TBP (TBP) are indicated. An arrowhead indicates the position of recombinant f:TAF_II150. An asterisk marks an unspecific (non-TAF) protein band in eTFIID.

FIG. 3

The human TAF_II150-containing TFIID complex is not sufficient for initiator function and TATA-independent transcription in a system reconstituted with highly purified GTFs and pol II. (A) A highly purified transcription system that is dependent on all GTFs and pol II. An arrowhead indicates the position of specific transcripts from the linear INR⁺ template analyzed by primer extension. The complete system A (lane 1) contains Ni-NTA-agarose-purified native TFIIA (A), recombinant TFIIB (B), eTFIID (D), recombinant TFIIE (E), recombinant TFIIF (F), native TFIIH (H), and pol II (Pol). Lanes 2 to 8, transcription reactions lacking single components as indicated. Note that although TFIIA is required for optimal basal TFIID-dependent transcription from this promoter, its requirement is not absolute since low levels of transcription can be seen in lane 2 on overexposures. (B) The highly purified TFIID-dependent system does not support initiator function from a linear TATA-containing promoter. Transcription from linearized TATA-containing INR⁻ (lanes 1 and 3) and INR⁺ (lanes 2 and 4) promoters was analyzed in a HeLa cell nuclear extract (NE; lanes 1 and 2) and in the purified system A (lanes 3 and 4) by primer extension using the same ³²P-labeled primer. (C) TFIID-mediated transcription from supercoiled templates in a highly purified system is independent of TFIIH and does not support TATA-dependent initiator function. Transcription from the supercoiled TATA-containing INR⁻ and INR⁺ templates was analyzed as described above in a nuclear extract (lanes 1 and 2) and in either the complete system A (lanes 7 and 8) or in system A lacking TFIIH (lanes 5 and 6) or TFIIH and TFIID (lanes 3 and 4). (D) The highly purified TFIID-dependent system does not support initiator-directed basal transcription from a TAF_II-dependent TATA-less promoter. Basal transcription from the supercoiled TATA-containing human Hsp70 core promoter (Hsp) and the TATA-less mouse TdT (−41 to +59) core promoter (TdT) was analyzed as described above in a nuclear extract (lane 1) and in the complete system A (lane 2) by transcribing equimolar amounts of templates in the same reaction. Specific transcripts were analyzed by primer extension using a mixture of two ³²P-labeled primers with similar specific activities (see below). (E) Schematic structure of the core promoters used in this study. TdT is the natural murine TdT gene core promoter (positions −41 to +33 or +59 relative to the start site). INR⁺ is the natural TdT core promoter (−41 to +33) with its −30 DNA sequence converted to a consensus TATAAA element. INR⁻ is the same as INR⁺ but without a functional initiator element. ML is the natural AdML core promoter (−45 to +65). Hsp is the natural human Hsp70 gene core promoter (−33 to +99). E1b is a construct containing the AdE1b TATA box sequence (as indicated) cloned in a polylinker sequence context upstream of a CAT reporter gene (see Materials and Methods). β-Pol is the human β-polymerase gene core promoter (−41 to +58). A boxed INR represents a functional initiator element. A boxed DR indicates a promoter with TFIID-dependent DNase I footprints extending downstream of the transcription initiation site (bent arrow). For the TdT promoter, this footprint has been observed only in the presence of a TATA box (i.e., in the INR⁺ construct [20, 26a]). DR? in the β-Pol core promoter indicates that this element has not yet been analyzed. Primer extension analyses of transcripts from the TdT and the INR⁺ and INR⁻ constructs are performed with the same primer, and transcripts from the ML, Hsp, E1b, and β-Pol CAT gene-containing constructs are analyzed with a CAT gene-specific primer (see Materials and Methods).

FIG. 4

TIC-1 is a core promoter-specific cofactor for TAF_II-dependent initiator function. (A) Reconstitution of TAF_II-dependent initiator-mediated transcription in the highly purified system with a TIC-1-containing fraction. Transcription from supercoiled INR⁻ and INR⁺ promoters was analyzed in the complete system A (as for Fig. 3A) but with either 1 μl of eTFIID (lanes 1 to 6) or 10 ng of TBP (lanes 7 to 10) and in the absence (lanes 1 and 2) or presence of 2 μl (lanes 3, 4, 7, and 8) or 4 μl (lanes 5, 6, 9, and 10) of the TIC-1/3 (P11 0.85/DE 0.12) fraction. (B) Two separate fractions, TIC-1 and TIC-1/3, can restore initiator function in the highly purified eTFIID-dependent system. Transcription from linear INR⁻ and INR⁺ promoters was analyzed as described above in a nuclear extract (NE; lanes 1 and 2) and in the highly purified system A (lanes 3 to 8) either alone (lanes 7 and 8) or with 4 μl of either the P11 0.5/DE 0.12 fraction (TIC-1; lanes 3 and 4) or the P11 0.85/DE 0.12 fraction (TIC-1/3; lanes 5 and 6). (C) Purified TIC-1 fractions with initiator sequence-dependent activity. Transcripts from the linear INR⁻ (open bars) and INR⁺ (hatched bars) promoters were quantitated in the purified system A alone (−) and in system A complemented with either partially purified TIC-1 (HS) fraction (0.5 and 1.0 μl; black triangle) or the activity peak of glycerol gradient fraction 8 (GG#8; 5 μl; see also below). Closed bars are initiator activities calculated as the ratio of INR⁺ to INR⁻ (INR⁺/INR⁻) transcripts and indicated by the scale on the right side (as fold initiator-mediated stimulation). (D) Core promoter-selective activity of TIC-1 on natural core promoters. Transcription from linear AdML (ML) and human Hsp70 (Hsp) core promoter templates (analyzed by primer extension) was compared in the highly purified system A alone (lane 1) and with either increasing amounts (0.5 and 2.0 μl) of the TIC-1 (HS) fraction (lanes 2 and 3) or with fractions of a subsequent glycerol gradient (5 μl of each; lanes 4 to 10). Peak TIC-1 activity sediments at fraction GG#8 (lane 7). (E) TAF_II-dependent core promoter-selective activity of the most purified TIC-1 fraction. Transcription from the Hsp and ML linear templates was performed in system A with either 2 μl of eTFIID (lanes 1 and 2) or 10 ng of TBP (lanes 3 and 4) and in either the absence (lanes 1 and 3) or presence (lanes 2 and 4) of the Ni²⁺-NTA-agarose TIC-1 fraction. (F) Purification scheme for TICs and TFIID used in the transcription systems. HeLa cell nuclear extracts (NE) were chromatographed as indicated (see also Materials and Methods) on phosphocellulose P11, DE52 (DE), Q Sepharose (QS), heparin-Sepharose (HS), Ni²⁺-NTA-agarose (Ni), dsDNA-cellulose (dsDNA), Mono S (MS), and anti-FLAG (M2)-agarose (Immuno-Affinity) resins.

FIG. 5

TIC-1 and TIC-2 are different from TFIID, TFII-I, YY1, and USF. (A) Immunoblot analysis of TIC fractions (see the legend to Fig. 4F for definitions) for the presence of TFIID components. Specific antisera against human TAF_II150, TAF_II80, and TAF_II31 were used to probe a Western blot containing 8 μl of eTFIID (lane 1), 8 μl of native TFIID (MS fraction; lane 2), and 5- to 10-fold excess (over amounts used in transcription) of TIC-1 (5-μl HS fraction; lane 3), TIC-2 (5-μl HS fraction; lane 4), and TIC-1/3 (5-μl HS/USA fraction; lane 5). (B) Immunoblot analysis of purified native TIC and GTF fractions for the presence of TFII-I, YY1, and USF. Specific antisera against TFII-I, YY1, and USF1 were used to probe a Western blot containing a 5- to 10-fold excess (over transcription amounts, as above) of the TIC fractions as indicated (5 μl of each; lanes 1 to 3), and of purified native TFIIA (6-μl Ni fraction; lane 4), TFIIH (3-μl Q2 fraction; lane 5), and pol II (4-μl DE fraction; lane 6) used in system A, as well as 1 μl (lane 7) and 5 μl (lane 8) of nuclear extract (NE). (C) Nuclear extracts immunodepleted of TFII-I. Western blot analysis with specific antiserum against recombinant TFII-I (anti-TFII-I/A1) of 9 μg of each of normal nuclear extract (NE; 1.4 μl; lanes 1 and 4), nuclear extract mock depleted twice on protein A-Sepharose (mock; 2.8 μl; lane 2), and nuclear extract depleted twice with affinity-purified anti-TFII-I antibodies (anti-TFII-I/A1) cross-linked to protein A-Sepharose (Δ TFII-I; 2.8 μl; lane 3). By scanning densitometry, about 98% of TFII-I was depleted from the extract. (D) Specific immunodepletion of TFII-I from nuclear extracts does not affect transcription from the AdML and TdT promoters. Transcription from the supercoiled AdML (−257 to +33) (ML; 5 fmol; lanes 1 and 2) and TdT (−1300 to +59) (TdT; 75 fmol; lanes 3 and 4) promoters was analyzed in control mock-depleted (mock; lanes 1 and 3) and TFII-I-immunodepleted (Δ TFII-I; lanes 2 and 4) extracts by primer extension as described in the legend to Fig. 3. Autoradiography was about 10 times shorter for lanes 1 and 2 than for lanes 3 and 4.

FIG. 6

TIC-1 is not sufficient for initiator-directed transcription from a TATA-less promoter; a novel TIC-3 activity present in the TIC-1/3 fraction is required. (A) TIC-3 activity is different from TIC-1 and PC4 and absent in P11 0.1 and P11 0.3 fractions. Transcription from a supercoiled TdT (−41 to +59) core promoter was analyzed in 5 μl of nuclear extract (lane 1) and in system B (lanes 2 to 12), containing purified native TFIIA (Mono Q fraction), recombinant TFIIB, eTFIID, recombinant TFIIE, recombinant TFIIF, and a two-column (P11 0.5/DE 0.3) TFIIE/F/H/pol II fraction (see text), either alone (lane 2) or complemented with recombinant PC4 (100, 200 and 400 ng; lanes 3 to 5), 500 ng of P11 0.1 (lane 6), P11 0.3 (lane 7), P11 0.5/DE 0.12 (1 μl of TIC-1; lane 8), and P11 0.85/DE 0.3 (lane 12) fractions or with 500 ng (4.5 μl; lane 9), 220 ng (2 μl; lane 10), and 110 ng (1 μl; lane 11) of TIC-1/3 (P11 0.85/DE 0.12) fraction. (B) TIC-3 activity does not correlate with the concentration of PARP/PC1, YY1, and TBP in the TIC-1/3 fraction. Western blot analysis with specific antisera against human PARP/PC1, YY1, and TBP of 5 μl (lane 1), 1 μl (lane 2), and 0.2 μl (lane 3) of nuclear extract (NE) and 4 μl of TFIIE/F/H/pol II (P11 0.5/DE 0.3) fraction (the amount used for system B in panel A; lane 4) and 4.5 μl of TIC-1/3 (P11 0.85/DE 0.12) fraction (the highest amount of TIC-1/3 added to system B in panel A; lane 5).

FIG. 7

TIC-2, a TATA-less promoter-specific cofactor required together with TIC-1/3 for both basal and activated transcription from the TdT core promoter. (A) TIC-2 and TIC-1/3 are required for basal and activated transcription from the TATA-less TdT core promoter. Transcription from supercoiled TATA-less G5-TdT and TATA-containing G5-E1b promoters (80 fmol) was analyzed in system C, containing purified native TFIIA (Mono Q fraction), recombinant TFIIB, native TFIID (Mono S fraction), a four-column native TFIIE/F/H fraction, and pol II (see Materials and Methods). System C was used alone (lane 1) or complemented with either 2 μl of TIC-2 (HS) fraction (lane 2), 1 μl of TIC-1/3 (USA) (HS fraction; lane 3), or 3 pmol of Gal4-VP16 (lane 4) independently and in different combinations (lanes 5 to 8) as indicated. The bottom and middle panels are identical exposures (1×) of experiments run on the same gel. The upper panel is a fivefold-longer exposure (5×) of the middle panel. (B) Reduced TIC-2-dependent transcription from the TdT promoter by deletion of natural TdT downstream promoter sequences between +33 and +59. Transcription from the supercoiled Hsp, G5-TdT (−41 to +59), and G5-TdT+33 (−41 to +33) constructs was performed in the reconstituted system C complemented with Gal-VP16 and the TIC-1/3 (USA) fraction, in either the absence (−) or presence (+) of TIC-2 (HS) fraction as for panel A. (C) TIC-2 is a TATA-less promoter-specific cofactor. Transcription from the supercoiled TATA-less G5-β-Pol and G5-TdT+33 constructs and from the TATA-containing G5-INR⁻ and G5-INR⁺ promoters (see text) was analyzed in the absence (lane 1) and presence (lane 2) of TIC-2 (HS) fraction as for panel B. Positions of specifically initiated transcripts are indicated for each promoter.

FIG. 8

TFIIA is essential for both basal and activator-dependent transcription from a TIC- and TAF_II-dependent TATA-less core promoter. (A) Specific immunodepletion of TFIIA from nuclear extracts (NE) inhibits basal transcription from the TATA-less TdT promoter. Transcription from the supercoiled TdT core promoter (−41 to +59) either in a mock-depleted nuclear extract (mock; lane 1) or in a nuclear extract depleted of TFIIA with specific antibodies against TFIIA α/β-p55 subunit (7) (Δ TFIIA; lanes 2 and 3) was analyzed by primer extension; 1.5 μl of TFIIA (Mono Q fraction) was added to the TFIIA-depleted extract in lane 3. (B) TFIIA has a general function in activation and an essential core promoter-selective role in basal transcription from a TIC- and TAF_II-dependent TATA-less promoter. Transcription from the supercoiled TATA-less G5-TdT and TATA-containing G5-E1b promoters was analyzed as for panel A, in the complete system C (lanes 1 to 3) or in system C lacking TFIIA (lane 4) and in the presence (+) or absence (−) of Gal-VP16 activator, the TIC-1/3 (USA) fraction, and the TIC-2 (HS) fraction, as indicated.