Synergistic Transcriptional Activation by TATA-Binding Protein and hTAF_II28 Requires Specific Amino Acids of the hTAF_II28 Histone Fold

Abstract

Coexpression of the human TATA-binding protein (TBP)-associated factor 28 (hTAF II 28) with the alteredspecificity mutant TBP spm3 synergistically enhances transcriptional activation by the activation function 2 of the nuclear receptors (NRs) for estrogen and vitamin D 3 from a reporter plasmid containing a TGTA element in mammalian cells. This synergy is abolished by mutation of specific amino acids in the ␣2-helix of the histone fold in the conserved C-terminal region of hTAF II 28. Critical amino acids are found on both the exposed hydrophilic face of this helix and the hydrophobic interface with TAF II 18. This ␣-helix of hTAF II 28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1 ␣-helix of TBP either reduces or increases interactions with hTAF II 28. The mutations which reduce interactions with hTAF II 28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAF II 28 is defective in its ability to synergistically enhance activation by NRs. However, this TBP mutant supports activation by other activators and is thus specifically defective for its ability to synergize with hTAF II 28.The RNA polymerase II transcription factor TFIID is a multiprotein complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAF II s) (3, 7). Not only are TAF II s components of transcription factor TFIID, but distinct subsets of TAF II s are also components of the SAGA, PCAF, and TFTC complexes (13,14,21,31,40). For human TFIID (hTFIID), cDNAs for 11 hTAF II s have been characterized (10,16,18,24,25).Genetic and biochemical experiments show that some TAF II s are important for promoter recognition and expression of a subset of promoters (15,38,39), while others are more generally required for transcription in Saccharomyces cerevisiae (2,26,27,29 Expression of hTAF II 28 also potentiates ligand-dependent activation by the AF-2s of many NRs, the most dramatic effects being seen with the receptors for 9-cis retinoic acid (retinoid X receptor), estrogen (estrogen receptor [ER]), and the VDR (22). Deletion analysis showed that coactivator activity required amino acids 150 to 179 in the C-terminal domain of hTAF II 28. Subsequent determination of the three-dimensional structure of the hTAF II 28/hTAF II 18 heterodimer at 2.6-Å resolution by X-ray crystallography indicated that these two proteins interact via a histone fold motif present in the C-terminal domain of hTAF II 28 and in the central region of hTAF II 18 (4). Amino acids 150 to 179 required for coactivator activity form the amphipathic ␣2-helix of the hTAF II 28 histone fold. In the hTAF II 28/hTAF II 18 heterodimer, residues on the hydrophobic face of the ␣2-helix make intermolecular contacts with hTAF II 18, while the residues on the mainly hydrophilic solvent-exposed face are available for mediating interactions with other proteins.Although the ability of hTAF II 28 to act as a transcriptional coactivator did no...

A functional interaction between ATF7 and TAF12 that is modulated by TAF4

“…The activity of the chimeric product (pG4-ATF7, Figure 1) was assayed in cotransfection experiments with a luciferase reporter gene whose promoter contained five Gal4-binding sites and a TGTA motif in place of the original TATA element. Cos-1 cells were transfected with the following recombinant plasmids: (i) the pG4-ATF7 vector, (ii) a vector encoding a TBP mutant (TBP-spm3) that selectively recognizes the TGTA derivative of the TATA box (Lavigne et al, 1999), (iii) the luciferase reporter and (iv) a vector expressing each of the hsTAFs tested or the corresponding empty vector. The results of a typical experiment are shown in Figure 1.…”

“…The (17m5)-TK TGTA-Luc reporter (Lavigne et al, 1999) contains the luciferase gene, driven by the thymidine kinase promoter, with an altered TATA box (TGTA, only recognized by the mutant TBP, TBP-spm3 (Lavigne et al, 1999)) and five Gal4-binding sites (see Figure 1).…”

“…The series of hsTAF vectors encoding the human TAF II polypeptides have been described (Mengus et al, 1995;Lavigne et al, 1999;Gangloff et al, 2000). The p-hsTAF12wt recombinant encodes both TAF12-1 and TAF12-2 proteins, through alternative initiation codon usage, whereas the deleted p-hsTAF12-2 version only encodes the shorter form.…”

The Human Transcription Factor IID Subunit Human TATA-binding Protein-associated Factor 28 Interacts in a Ligand-reversible Manner with the Vitamin D3 and Thyroid Hormone Receptors

“…Coexpression of TBP and hTAF II 28 shows that they act synergistically to potentiate activation by the VDR or estrogen receptor. This synergism requires specific amino acids of the hTAF II 28 histone fold domain located in the conserved C-terminal half of the protein and can also be abolished by a mutation in the H1Ј helix of TBP (31,32).…”

“…Coexpression of TBP and hTAF II 28 shows that they act synergistically to potentiate activation by the VDR or estrogen receptor. This synergism requires specific amino acids of the hTAF II 28 histone fold domain located in the conserved C-terminal half of the protein and can also be abolished by a mutation in the H1Ј helix of TBP (31,32).Despite its ability to act as a transcriptional coactivator for several NRs, we previously reported that no ligand-dependent interactions with the RXR could be observed (29). In this study, we show that hTAF II 28 does selectively interact with the VDR and thyroid hormone receptor (TR␣) but does so in a ligandreversible manner.…”