TBP-associated factor 1

TFIID components and interaction with promoters

Four regions within three promoters (hsp70, hsp26 and histone H3) have been found to be important for TFIID binding: the TATA element, the initiator (transcriptional start site), and two regions located from 18 to 28 nucleotides downstream of the transcriptional start site. The initiator appears to contribute as much to the affinity as the TATA element. There is weak conservation of the sequence in this region. To determine whether a preferred binding sequence exists in the vicinity of the initiator, the nucleotide composition of this region within the hsp70 promoter was randomized and then subjected to selection by TFIID. After five rounds of selection, a preferred sequence motif emerged. This motif is a close match to consensus sequences that have been derived by comparing the initiator region of numerous insect promoters. Selection of this sequence demonstrates that sequence-specific interactions downstream of the TATA element contribute to the interaction of TFIID on a wide spectrum of promoters (Purnell, 1994).

Three promoter sequences influence the access of Drosophila HSF transcription factor to its binding sites: the GAGA element, sequences surrounding the transcription start site, and a region in the leader sequence downstream of the hsp70 start site where RNA polymerase II arrests during early elongation. The GAGA element has been shown to disrupt nucleosome structure. Because the two other critical regions include sequences that are required for stable binding of TFIID in vitro, the in vivo occupancy of the TATA elements in the transgenic promoters was examined. TATA occupancy correlates with HSF binding for some promoters. However, in all cases HSF accessibility correlates with the presence of paused RNA polymerase II. It is proposed that a complex promoter architecture is established by multiple interdependent factors, including GAGA factor, TFIID, and RNA polymerase II, and that this structure is critical for HSF binding in vivo (Shopland, 1995).

It is thought that occupancy of sites within the basal promoter by Even-skipped protein inhibits subsequent TFIID binding resulting in repressed transcription, a mechanism termed cooperative blocking. The C-terminal 236 amino acids of the Even-skipped protein (region CD) repress transcription. A fusion protein was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein represses transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors; repression is dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression is prevented when the promoter DNA is preincubated with TFIID or TBP, whereas preincubation of the template DNA with the fusion protein prevents TFIID binding. Together, these results strongly imply that the EVE fusion protein represses transcription by inhibiting TFIID binding. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional fusion protein molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites has been shown to contribute to repression. Further experiments suggested that the full-length EVE protein also represses transcription by the same mechanism (Austin, 1995).

Antennapedia P2 and many other promoters of Drosophila contain a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), which is located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including Antennapedia P2. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).

The binding of TAFs to core promoter elements and the differential ability of TFIID to mediate basal transcription directed by wild type promoters suggests that one or more of the TAFs might serve as a promoter selectivity factor. The transcription properties of holo-TFIID were compared with those of various partial TBP-TAF complexes, as well as with TBP alone. Two different dimeric complexes (TBP-TAFII250 and TBP-TAFII150) and a trimeric complex (TBP-TAFII250-TAFII150) were assembled. These were assayed for their ability to discriminate between wild-type and mutant hsp70 core templates, in which the downstream core elements from -3 to +43 have been deleted. In vitro transcription reactions supplemented with the dimeric TBP-TAF complexes behave essentially like TBP alone and are unable to mediate the function of the downstream core promoter elements. In contrast, the trimeric complex directs the transcription of wild type hsp70 template 10 to 20 fold more efficiently than the mutated template. The differential transcription activity displayed by the trimeric complex is very similar to that observed with holo-TFIID. Both TAFII250 and TAFII150 are essential for promoter discrimination, while the presence of other TAFs fail to contribute significantly to core promoter recognition. Neither holo-TFIID nor the trimeric complex bind in a very stable manner to the truncated promoter. When in complex with TBP, TAFs may actually destabilize the interaction between TBP and a template lacking downstream sequences that bind the TAFs (Verrijzer, 1996).

Reconstituted transcription reactions reveal the ability of TFIID versus TBP to discriminate between distinct core promoters. A comparison of different partial TBP-TAF assemblages establishes that a trimeric TBP-TAFII250-TAFII150 complex is minimally required for efficient utilization of the initiator and downstream promoter elements. Depending on the promoter structure, TAFs can increase or decrease the stability of TFIID-promoter interactions. These findings suggest that TAFs play a critical role in promoter selectivity and transcription regulation through direct contacts with core promoter elements (Verrijzer, 1995).

The transcriptional potential of the hsp70 heat shock gene promoter is established prior to induction by stress. The TBP subunit of TFIID is associated with the TATA element; RNA polymerase II is paused downstream from the transcription start site. In order to identify new interactions involved in establishing this potentiated state, a detailed analysis of the molecular architecture of a single copy of the hsp70 promoter was performed. Genomic footprinting using DNase I reveals two previously unidentified interactions. First, the GAGA element (binding the Trithorax-like transcription factor) located at -120 is protected by protein. Second, the pattern of DNase I cleavage in the vicinity of the transcription start is found to bear significant similarity to the pattern associated with binding of purified TFIID. Noting that purified GAGA factor (Trithorax-like) and TFIID interact similarly with the hsp70 and H3 promoters, the architecture of the endogenous H3 promoter was analyzed to determine what interactions might be needed to establish a potentiated state containing a paused polymerase. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase was evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing (Weber, 1995).

GAGA factor, TFIID, and paused polymerase are present on the hsp70 promoter in Drosophila melanogaster prior to transcriptional activation. In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed, on P elements, into flies. One construct lacks the TATA box and the other lacks the upstream regulatory region where GAGA factor binds. Transcription of each mutant during heat shock is at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters are found to adopt a DNase I hypersensitive state that includes the region downstream from the transcription start site. High-resolution analysis of the DNase I cutting pattern identifies proteins that could be contributing to the hypersensitivity. GAGA factor footprints are clearly evident in the upstream region of the TATA deletion construct, and a partial footprint possibly caused by TFIID is evident on the TATA box of the upstream deletion construct. Permanganate treatment of intact salivary glands was used to further characterize each promoter construct. Paused polymerase and TFIID are readily detected on the normal promoter construct, whereas both deletions exhibit reduced levels of each of these factors. Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation. In contrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase. Nevertheless GAGA factor could not be detected on the downstream region of the TATA deletion, and thus there is no direct proof that the GAGA factor interacts with the core promoter region in vivo. Nevertheless, purified GAGA factor is found to bind near the transcription start site; the strength of this interaction is increased by the presence of the upstream region. It is concluded that GAGA factor alone might be capable of establishing an open chromatin structure that encompasses the upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID (Weber, 1997).

Protein Interactions between TAFs

The recombinant TFIID subunit (p230) interacts directly with the TATA box-binding subunit of TFIID (TBP) from Drosophila, human and yeast. Recombinant p230 inhibits the TATA box-binding activity and function of TFIIDtau (the TBP subunit), suggesting that p230 interactions with TFIIDtau, and its possible modulations by other factors may play an important role in TFIID function (Kukubo, 1993).

Subunit p230 contains at least two sites of interaction with TBP. The N-terminal domain not only inhibits TATA-box binding of TBP but also dissociates prebound TBP from the TATA box. This inhibitory activity resides in the N-terminal 80 amino acids. N-terminally truncated p230 also interacts with TBP, indicating that there is a second TBP binding site (Kokubo, 1994a).

Subunit p230 can directly interact with p110, the third largest subunit of TFIID (Kokubo, 1994b).

Of the four well-characterized TAFII110 binding partners within the initiation complex, TAFII250, TAFII30alpha, TAFII150, and the large subunit of TFIIA, only TAFII150 is bound by N-terminal sequences. Sequences within the C-terminal 126 amino acids of TAFII110 are required for binding of TAFII250, TAFII30alpha and TFIIA-L, the large subunit of TFIIA (Sauer, 1996).

TAFII250 contains two distinct kinase activities, one associated with the N-terminal region (NTK) and another associated with the C-terminal region (CTK). Both purified recombinant Drosophila and human TAFII250 become efficiently autophosphorylated on serine residues in vitro (Dikstein, 1996)

Drosophila TAFII250 possesses a histone transacetylase activity that targets histone H3 and histone H4. The dual H3 and H4 specificity of Drosophila TAFII250 differs from the strong preference for only H3 displayed by human GCN5 (the human homolog of the yeast transcriptional adaptor-protein GCN5). Lysine 14 of histone H3 is the preferred site for both human GCN5 and Drosophila TAFII250. This same residue is the preferrred site of H3 acetylation of yeast GCN5 in vitro (Mizzen, 1996).

A minimal complex containing TBP and TAFII250 (two of the eight or nine proteins constituting TFIID) directs basal but not activator-responsive transcription. In contrast, reconstituted holo-TFIID supports activation by an assortment of activators. The Drosophila activator Grainyhead (also known as NTF-1), which binds TAFII150, stimulates transcription with a complex containing only TBP, TAFII250, and TAFII150, whereas mammalian Sp1 binds and additionally requires TAFII110 for activation. Interestingly, TAFII150 enhances Sp1 activation even though this subunit does not bind directly to Sp1. These results establish that specific subcomplexes of TFIID can mediate activation by different classes of activators and suggests that TAFs perform multiple functions during activation (Chen, 1994).

TAFII250 ubiquitinates histone H1

Ubiquitination of histones has been linked to the complex processes that regulate the activation of eukaryotic transcription. However, the cellular factors that interpose this histone modification during the processes of transcriptional activation are not well characterized. A biochemical approach has identified the Drosophila coactivator TAFII250, the central subunit within the general transcription factor TFIID, as a histone-specific ubiquitin-activating/conjugating enzyme (ubac). TAFII250 mediates monoubiquitination of histone H1 in vitro. Point mutations within the putative ubac domain of TAFII250 abolish H1-specific ubiquitination in vitro. In the Drosophila embryo, inactivation of the TAFII250 ubac activity reduces the cellular level of monoubiquitinated histone H1 and the expression of genes targeted by the maternal activator Dorsal. Thus, coactivator-mediated ubiquitination of proteins within the transactivation pathway may contribute to the processes directing activation of eukaryotic transcription (Pham, 2000).

Polyubiquitination represents a mark on proteins that identifies them for degradation and requires the involvement of three enzymes: (1) ubiquitin-activating enzymes (E1), which mediate the adenosine triphosphate (ATP)-dependent conjugation of E1 with ubiquitin via a covalent thioester linkage; (2) ubiquitin-conjugating enzymes (E2), which mediate the transfer of ubiquitin from E1 to E2, conjugate ubiquitin via thioester bonds and, (3) together with ubiquitin-protein ligase (E3), link ubiquitin to target proteins via isopeptide bonds. Polyubiquitination requires all three enzymes, whereas monoubiquitination of proteins requires E1 and E2 activities only. Unlike polyubiquitination, monoubiquitination of histones has been correlated with activation of gene expression. However, the functional connections between histone ubiquitination and activation of gene expression remain unknown. Thus, as a first step toward understanding the role of histone ubiquitination for transcriptional regulation, attempts were made to identify enzymes that ubiquitinate histones in Drosophila embryonic nuclear extract using an activity gel assay (Pham, 2000).

Nuclear extract was separated in SDS-polyacrylamide gels containing histones. After electrophoresis (SDS-PAGE), gel-bound proteins were subsequently denatured, renatured, and, to monitor enzymatic activities, incubated with ³²P-labeled ubiquitin. By using this assay, a protein was identified with a molecular mass of approximately 200 kD that mediates ubiquitination of histones. The 200-kD activity coincides with TAF_II250, suggesting that TAF_II250 may ubiquitinate histones (Pham, 2000).

TAF_II250 most likely does not interact with E1, E2, or E3 enzymes. Since mono-ubiquitination requires at least E1 and E2 activities, these results imply that TAF_II250 may have intrinsic E1 and E2 activities. The ubiquitin/H1 conjugates resisted reducing agents, suggesting that TAF_II250 may mediate a covalent bond between ubiquitin and H1 by means of isopeptide linkages. Since this enzymatic reaction is characteristic for E2 enzymes, TAF_II250 may have intrinsic E2 activity. The E1 enzyme requires ATP to conjugate with ubiquitin by means of thioester bonds. Therefore, to explore whether TAF_II250 has E1 activity, the capability of TAF_II250 for conjugating with ubiquitin by means of thioester bonds was investigated. TAF_II250 conjugated with ubiquitin in an ATP-dependent manner in the absence, but not in the presence, of reducing agents, suggesting that TAF_II250 and ubiquitin form a covalent bond by means of a thioester linkage. Thus, TAF_II250 may have both E1 and E2 activities and may therefore be a ubac (Pham, 2000).

To provide supporting evidence that TAF_II250 mediates ubiquitination of H1, solution assays were used. Reactions containing TAF_II250, ³²P-labeled ubiquitin, H1, and ATP mediate the formation of a 39-kD protein that is recognized by antibodies to both ubiquitin and H1. These results suggest that the 39-kD protein represents a conjugate composed of one ubiquitin moiety (7 kD) and H1 (32 kD). By contrast, TAF_II250 does not ubiquitinate other histones, H2A/H2B dimers, H3/H4 tetramers, or core nucleosomes. Thus, TAF_II250 mediates monoubiquitination of H1 (Pham, 2000).

To determine the portion of TAF_II250 that mediates monoubiquitination of H1, TAF_II250 mutants truncated at the COOH-terminal were used. Membrane assays indicate that full-length TAF_II250 and 250deltaC850 (lacking the 850 amino acids closest to the COOH-terminal), but not 250deltaC1300 (lacking the 1300 amino acids closest to the COOH-terminal) ubiquitinate H1. Thus, the H1-specific ubac activity is likely to reside between amino acids 768 and 1218 (Pham, 2000).

Two Drosophila TAF250 alleles, TAF250^XS-2232 and TAF250^S-625, have been described that contain single-amino acid point mutations that reside within the putative TAF_II250 ubac domain. TAF250^XS-2232 contains a valine-1072 to aspartic acid change, and TAF250^S-625 an arginine-1096 to proline change. To investigate the effect of these mutations on TAF_II250 ubac activity, the middle region of TAF_II250 containing amino acids 612 to 1140 (TAF250-M), TAF_II250-M-V1072D (containing the V¹⁰⁷² to D mutation), and TAF_II250-M-R1096P (containing the R¹⁰⁹⁶ to P mutation) were subjected to membrane assays. Although TAF250-M ubiquitinates H1, the mutants do not. Wild-type and mutant TAF_II250-M proteins have histone acetyltransferase activity. The TAF250-M proteins (used for the membrane assays) acetylate histones; this suggests that the lack of ubac activity seen with TAF250-M-V1072D and TAF_II250-M-R1096P is most likely not due to a general functional inactivity of the mutant proteins (Pham, 2000).

In Drosophila, TFIID mediates transcriptional activation by the maternal activator Dorsal. Dorsal activates the expression of the mesoderm-determining genes twist (twi) and snail (sna), which are transcribed in 20 and 18 of the ventral-most cells of cellularizing embryos, respectively. To investigate the functional relevance of TAF_II250 ubac activity for Dorsal-dependent transcriptional activation in vivo, in situ hybridization was used to monitor twi and sna expression in Drosophila embryos containing reduced levels of Dorsal and expressing TAF_II250^XS-2232 or TAF_II250^S-625, which lack ubac activity in vitro. Both twi and sna expression are severely reduced in dl-sensitized, TAF250^XS-2232 embryos and dl-sensitized, TAF250^S-625 embryos, but not in control embryos. Weak twi mRNA levels were detectable in 10 to 12 cells, and sna expression was restricted to 4 to 12 ventral-most cells and disrupted by gaps. Analyses of cuticular preparations revealed that dl-sensitized TAF250^XS-2232 mutants or dl-sensitized TAF250^S-625 mutants, but not control embryos exhibit a dorsalized and twisted body pattern. These results indicate that Dorsal-dependent activation of transcription is impaired in embryos lacking TAF_II250 ubac activity (Pham, 2000).

To investigate whether H1 may represent a target for TAF_II250 ubac activity in Drosophila, H1 was purified from nuclei prepared from 0- to 3-hour-old wild-type and TAF_II250 mutant embryos. Western blot analyses indicate that antibodies to both H1 and ubiquitin detect a monoubiquitin/H1 conjugate. This result indicates that at least a fraction of H1 present in early Drosophila embryos is monoubiquitinated. Moreover, Western blot analyses indicate that compared with wild-type embryos, mutant embryos that lack TAF_II250 ubac activity contain a significantly reduced level of monoubiquitinated H1. These results suggest that TAF_II250 ubac activities may contribute to monoubiquitination of H1 in Drosophila (Pham, 2000).

How coactivators convert activation signals from activation domains of transcription factors into enhanced levels of mRNA synthesis lies at the heart of transcriptional regulation. These results suggest that one coactivator, TAF_II250, may use intrinsic ubiquitin-activating/conjugating activities to mediate activation of transcription. Multiple-alignment analysis and comparison with protein database sequences reveal that TAF250-M exhibits similarities to E1 and E2 enzymes. Thus, the result that TAF_II250 mediates monoubiquitination of H1 in vitro is in agreement with other results suggesting that E1 and E2 activities are sufficient to mediate monoubiquitination of proteins. As point mutations that abrogate TAF_II250 ubac activity in vitro also reduce gene expression in the Drosophila embryo, TAF_II250 ubac activity may play an important role for the activation of gene expression in Drosophila. Although the in vivo targets of TAF_II250 ubac activity remain unknown, the results that H1 is monoubiquitinated in Drosophila and that the level of monoubiquitinated H1 is significantly reduced in embryos lacking TAF_II250 ubac activity imply that H1 may represent one in vivo target of TAF_II250. Thus, ubiquitination of H1 or other proteins within the transcription machinery, or both, by TAF_II250 may constitute an important coactivator function of TAF_II250 and, hence, may allow TFIID to direct events during the processes of transcriptional activation (Pham, 2000).

TAFII250 interaction with Adf-1

Many transcriptional activators have been shown to interact with the TAF subunits of the TFIID complex, and this interaction has been correlated with transactivation in several cases. Tests were carried out to discover whether TAFs are important in Adf-1-directed transcription and, if so, whether there is any correlation between activation and potential Adf-1-TAF interactions. An Adf-1-dependent in vitro transcription system was depleted of TFIID by using antibodies against both TAF_II250 and TBP. In the absence of an exogenous source of TFIID, this transcription system is inactive. The addition of either purified TFIID or purified recombinant TBP to the depleted transcription system restores basal transcription levels. The addition of purified Adf-1 to these reaction mixtures leads to a high level of activation when TFIID is present but has no effect on the reaction mixture containing only TBP. These results suggest that Adf-1, like many Drosophila activators, requires the presence of TAFs in order to activate transcription in vitro (Cutler, 1998).

To determine if the transcriptional requirement for TAFs is due to direct interactions between one or more of these proteins and Adf-1, in vitro binding experiments were conducted. Beads containing anti-TAF_II250 monoclonal antibodies were used to immunoprecipitate TFIID from partially purified TFIID fractions. Silver staining and Western blotting using anti-TAF_II80 antibodies has confirmed that an intact holo-TFIID complex is immunoprecipitated. Purified wild-type or mutant Adf-1 protein was incubated with these TFIID beads or with negative-control beads. Anti-Adf-1 antibodies were used to visualize the amounts of bound and input Adf-1 proteins on Western blots. Wild-type Adf-1 binds strongly to beads containing TFIID. In contrast, the two carboxy-terminal transcriptionally inactive mutants, 5A214 and N228, failed to bind the TFIID beads, suggesting that the carboxy terminus of Adf-1 is necessary for TFIID binding (Cutler, 1998).

The Drosophila TFIID complex includes TBP and approximately eight major TAFs. To determine which of these are bound by Adf-1, each of the eight TAFs and TBP were produced by in vitro transcription and translation in the presence of ³⁵S-labeled methionine. The lysates were then incubated with glutathione beads bound to either GST fused to the carboxy terminus of Adf-1 (C92; amino acids 162 to 253) or to GST alone. The C92 fragment was used since the TFIID-binding data suggested that TAF-binding activity may reside in the carboxy terminus of Adf-1. After extensive washing, bound and input TAFs were visualized by autoradiography. Of the nine proteins, only TAF_II110 and TAF_II250 binds at significant levels to Adf-1. The similar amounts of TAF binding observed with full-length Adf-1 and with proteins having amino-terminal deletions of Adf-1 further confirm that the carboxy terminus of Adf-1 is the major TAF-binding determinant in the protein. Smaller fragments of TAF_II110 and hTAF_II250 were also tested for their ability to bind Adf-1. The carboxy-terminal 240 amino acids of TAF_II110 are sufficient for binding Adf-1, while binding with regions at both the amino and carboxy termini of hTAF_II250 is seen. The central portion of hTAF_II250 also binds Adf-1, though to a lesser extent. This binding also demonstrates that the carboxy-terminal region of Adf-1 is sufficient for TAF binding (Cutler, 1998).

To verify that the in vitro binding observed reflects events that can occur inside of a cell, the interaction between Adf-1 and TAF_II110 and hTAF_II250 was tested in S. cerevisiae. Several portions of both TAFs were fused to the GAL4 DBD, and the carboxy terminus of Adf-1 was fused to the GAL4 AAD. These vectors were cotransformed into S. cerevisiae containing GAL4 sites upstream of the gene for beta-galactosidase. The regions of TAF_II110 and hTAF_II250 that bind Adf-1 in vitro also interact efficiently in this 'in vivo' two-hybrid assay (Cutler, 1998).

Several of the mutations in the carboxy-terminal region of Adf-1 decrease its ability to activate transcription. Some of these carboxy-terminal Adf-1 mutants are also deficient for binding to the holo-TFIID complex. Similar experiments using individual TAFs and other Adf-1 mutants were performed to examine these results in more detail. Paralleling the TFIID-binding results, mutants 5A214 and N228 bind with low efficiency to full-length hTAF_II250 and TAF_II110 as well as to an amino-terminal fragment of hTAF_II250 (amino acids 1 to 414). These results provide a good correlation between TAF binding and transcriptional activation by the carboxy-terminal region of Adf-1. However, because activation by Adf-1 requires sequences outside of the carboxy-terminal region, it is suspected that binding may be necessary but not sufficient to stimulate transcriptional activation by Adf-1. Instead, these results suggest that sequences outside of the TAF interaction region contribute additional activities essential for transactivation (Cutler, 1998).

A distinct DBD is located in the amino-terminal 100 amino acids of Adf-1. This domain contains homologies to the Myb DNA-binding motif and Drosophila protein Stonewall. Adf-1 dimerizes in solution through a domain found near the carboxy terminus. Although dimerization increases the affinity of Adf-1 for DNA, its activity is not essential for binding, as mutants unable to dimerize retain sequence-specific DNA-binding activity. Since most Myb proteins contain two or three tandem repeats of the Myb motif, it is possible that the dimerization by Adf-1 compensates for the presence of only one DNA-binding Myb motif in the protein. Although monomeric forms of Adf-1 can still bind DNA, they do so with a lower affinity. In addition to being required for dimerization, the carboxy terminus of Adf-1 possesses TAF-binding activity. TFIID cannot be replaced by TBP for Adf-1-directed transcriptional activation, consistent with the notion that TAF binding is likely to be functionally important for the activity of Adf-1. All mutations that disrupt Adf-1 dimerization also disrupt TAF binding, while mutants of Adf-1 that are competent for dimerization are also competent for TAF binding. While these data suggest that dimerization may be required for TAF binding, the observation that a carboxy-terminal fragment of Adf-1 containing the dimerization domain is sufficient for TAF binding indicates that dimerization and TAF binding activities are likely due to closely adjacent or overlapping sequences within Adf-1 (Cutler, 1998).

TAF1 activates transcription by phosphorylation of serine 33 in histone H2B

TAF1 contains two kinase domains, an N-terminal (NTK, amino acids 1 to 496) and a C-terminal (CTK, amino acids 1496 to 2132) domain (Dikstein, 1996). In vitro, the NTK and the CTK autophosphorylate and the NTK transphosphorylates the RAP74 subunit of the GTF TFIIF. In contrast to the NTK, the CTK did not phosphorylate RAP74 but strongly phosphorylated H2B, indicating that the CTK possesses H2B-specific kinase activity. Protein kinases contain two essential functional motifs, an adenosine triphosphate (ATP) binding motif and an amino acid–specific kinase motif. Computational sequence comparison analyses identified a putative serine and threonine (S/T) kinase motif (amino acids 1534 to 1546) and two tandem ATP binding domains (amino acids 1747 to 1780) in the CTK. Interestingly, the S/T kinase motif is located in the first bromodomain of the double bromodomain module (DBD), which binds acetylated lysines. However, CTK(D1538A) retains the ability to bind acetylated H4 in vitro, indicating that the introduced mutation disrupts kinase activity rather than acetylated lysine-based substrate recognition. In addition to kinase domains, TAF1 has a histone acetyltransferase (HAT) domain that acetylates H3 Lys¹⁴ (H3-K14) and unidentified lysines in H4 in vitro (Maile, 2004).

Dynamic changes in chromatin structure, induced by posttranslational modification of histones, play a fundamental role in regulating eukaryotic transcription. Histone H2B is phosphorylated at evolutionarily conserved Ser³³ (H2B-S33) by the carboxyl-terminal kinase domain (CTK) of the Drosophila TFIID subunit TAF1. Phosphorylation of H2B-S33 at the promoter of the cell cycle regulatory gene string and the segmentation gene giant coincides with transcriptional activation. Elimination of TAF1 CTK activity in Drosophila cells and embryos reduces transcriptional activation and phosphorylation of H2B-S33. These data reveal that H2B-S33 is a physiological substrate for the TAF1 CTK and that H2B-S33 phosphorylation is essential for transcriptional activation events that promote cell cycle progression and development (Maile, 2004).

Transcription initiation in eukaryotes involves dynamic changes in chromatin structure that permit assembly of the transcription machinery at a gene promoter. The fundamental structural unit of chromatin is the nucleosome, which contains 146 base pairs of DNA wrapped around a histone octamer composed of two copies each of histones H2A, H2B, H3, and H4. Distinct patterns of histone modifications (e.g., acetylation, phosphorylation, and methylation) may act as 'modification cassettes' that facilitate DNA-dependent events. For example, in vertebrates phosphorylation of H2B Ser¹⁴ is associated with apoptotic chromatin, and in all eukaryotes phosphorylation of H3 Ser¹⁰ is associated with transcriptionally active and mitotic chromatin. Although all histones are phosphorylated in vivo, the function of many of these modifications and the kinases that carry them out are not known (Maile, 2004).

With the use of an in vitro kinase assay, it was found that the Drosophila general transcription factor (GTF) TFIID phosphorylates histone H2B but not H1, H2A, H3, or H4. TFIID is a multiprotein complex composed of the TATA box–binding protein (TBP) and numerous TBP-associated factors (TAFs). TFIID functions during transcription initiation by nucleating assembly of GTFs and RNA polymerase II at the promoter. TAF1 (formerly TAF_II250) is the only TFIID subunit that possesses kinase activity, suggesting that it phosphorylates H2B (Wassarman, 2001). In fact, recombinant TAF1 and denatured and renatured recombinant TAF1 phosphorylated H2B in vitro, demonstrating that TAF1 has intrinsic, H2B-specific kinase activity. Collectively, these results indicate that TAF1 alone and in the context of TFIID phosphorylates H2B (Maile, 2004).

TAF1 contains two kinase domains, an N-terminal (NTK, amino acids 1 to 496) and a C-terminal (CTK, amino acids 1496 to 2132) domain (Dikstein, 1996). In vitro, the NTK and the CTK autophosphorylate and the NTK transphosphorylates the RAP74 subunit of the GTF TFIIF. To determine which domain phosphorylates H2B, NTK and CTK were assayed separately in vitro. In contrast to the NTK, the CTK did not phosphorylate RAP74 but strongly phosphorylated H2B, indicating that the CTK possesses H2B-specific kinase activity (Maile, 2004).

Protein kinases contain two essential functional motifs, an adenosine triphosphate (ATP) binding motif and an amino acid–specific kinase motif. Computational sequence comparison analyses identified a putative serine and threonine (S/T) kinase motif (amino acids 1534 to 1546) and two tandem ATP binding domains (amino acids 1747 to 1780) in the CTK. To test whether the identified motifs mediate H2B phosphorylation, in vitro kinase assays were performed with the use of CTK polypeptides lacking the S/T kinase motif (CTKDelta1600) or the ATP binding motifs (CTKDeltaATP). Relative to the wild-type CTK, CTKDelta1600 and CTKDeltaATP weakly phosphorylated H2B. To confirm the role of the S/T kinase motif, a catalytically important aspartic acid was mutated to an alanine (D1538A) in the motif. Like CTKDelta1600, CTK(D1538A) exhibited weak autophosphorylation and H2B transphosphorylation activities. Interestingly, the S/T kinase motif is located in the first bromodomain of the double bromodomain module (DBD), which binds acetylated lysines. However, CTK(D1538A) retains the ability to bind acetylated H4 in vitro, indicating that the introduced mutation disrupts kinase activity rather than acetylated lysine-based substrate recognition. Thus, the identified S/T kinase and ATP binding motifs of the TAF1 CTK are essential for H2B phosphorylation (Maile, 2004).

To identify H2B residue(s) phosphorylated by the CTK, whether the CTK phosphorylates the N-terminal tail of Drosophila H2B (amino acids 1 to 39, H2BT) or the tailless H2B core domain (amino acids 40 to 123) was examined; the CTK phosphorylated H2BT but not the H2B core domain. Next, to pinpoint which residue(s) in H2BT is phosphorylated, mutant H2BT peptides were generated in which alanines replaced all or individual serines or threonines. The CTK did not phosphorylate peptides lacking all serines, suggesting that it phosphorylates either Ser⁵ (H2B-S5) or Ser³³ (H2B-S33). To test this, H2BT peptides were used as substrates that contained alanines in place of H2B-S5, H2B-S33, or both (H2BT-S5A, H2BT-S33A, and H2BT-S5/33A, respectively). The CTK phosphorylated H2BT-S5A but not H2BT-S33A or H2BT-S5/33A, indicating that H2B-S33 is the target of the CTK (Maile, 2004).

To investigate whether H2B-S33 is phosphorylated in vivo, a polyclonal antibody was raised recognizing phosphorylated H2B-S33 (H2B-S33P). On Western blots, the antibody recognized H2BT containing H2B-S33P but not recombinant, unphosphorylated H2B or an H3 peptide (amino acids 1 to 32) containing phosphorylated Ser¹⁰ and Ser²⁸. In addition, the H2B-S33P antibody recognized H2BT and recombinant H2B that was phosphorylated in vitro by the CTK or TFIID, indicating that the antibody specifically recognizes phosphorylated H2B-S33. The H2B-S33 antibody also recognized a protein with a molecular weight similar to that of H2B from histone preparations from Drosophila embryos or S2 cells, providing evidence that H2B-S33 is a target for phosphorylation in vivo. To determine whether TAF1 mediates H2B-S33 phosphorylation in vivo, RNA interference (RNAi) was used to eliminate TAF1 expression in S2 cells. As shown by Western blot analysis, both TAF1 expression and H2B-S33 phosphorylation were reduced in TAF1 RNAi cells compared with mock RNAi cells, suggesting that TAF1 is a major H2B-S33 kinase in vivo (Maile, 2004).

Flow cytometry analysis of TAF1 RNAi cells revealed that loss of TAF1 results in G2-M phase cell cycle arrest. To test the hypothesis that TAF1 controls the transcription of genes whose activities contribute to G2-M progression, microarray expression profiling and reverse transcription polymerase chain reaction (RT-PCR) were used to monitor transcription in mock and TAF1 RNAi cells. Both methods showed that transcription of string (stg), which encodes a Drosophila homolog of yeast Cdc25, was reduced. The Stg protein phosphatase is predominantly expressed during G2 and activates the cell cycle by dephosphorylating Cdc2. Because loss of stg from S2 cells by RNAi causes G2-M arrest, TAF1 may regulate G2-M progression by activating stg transcription (Maile, 2004).

Chromatin immunoprecipitation (XChIP) was used to establish whether there is a direct correlation between transcriptional activation of stg and TAF1-mediated phosphorylation of H2B-S33 at the stg promoter. Cross-linked chromatin was isolated from mock and TAF1 RNAi S2 cells and immunoprecipitated with TAF1 or H2B-S33P antibodies. Immunoprecipitated DNA was purified and used as a template for PCR to detect the stg promoter or coding region and actin5C promoter. In contrast, TAF1 is not essential for actin5C transcription, and H2B-S33P antibodies do not precipitate the actin5C promoter. Thus, the transcriptional dependence of a gene for TAF1 is correlated with H2B-S33 phosphorylation, not with TAF1 association (Maile, 2004).

To distinguish whether loss of H2B-S33 phosphorylation at the stg promoter is due directly to loss of TAF1 or indirectly to G2-M arrest, XChIP analysis was performed on S2 cells arrested in G2-M by RNAi of the SIN3 transcriptional corepressor. Stg transcription is repressed in SIN3 RNAi cells, yet the stg promoter remains associated with H2B-S33P and TAF1, indicating that loss of H2B-S33 phosphorylation in TAF1 RNAi cells is because of elimination of TAF1 rather than G2-M arrest (Maile, 2004).

In addition to kinase domains, TAF1 has a histone acetyltransferase (HAT) domain that acetylates H3 Lys¹⁴ (H3-K14) and unidentified lysines in H4 in vitro (Mizzen, 1996). XChIP analysis detected acetylated H3-K14 and H4 at the transcriptionally active stg promoter in mock RNAi cells but not at the inactive stg promoter in TAF1 RNAi cells. In contrast, TAF1-independent histone modifications did not correlate with activation of stg in mock and TAF1 RNAi cells. Taken together, these results indicate that TAF1-mediated phosphorylation of H2B-S33 and acetylation of H3 and H4 potentiate transcriptional activation in Drosophila cells (Maile, 2004).

To investigate the role of TAF1-mediated phophorylation of H2B-S33 during fly development, a recessive lethal TAF1 allele, TAF1^CTK, was used which contains a nonsense mutation at amino acid 1728 that truncates the CTK downstream of the DBD was used. The corresponding protein (TAF1DeltaCTK) is expressed in Drosophila but presumably does not have CTK activity, because it does not phosphorylate H2B in vitro. In situ hybridization was used to monitor transcription in embryos homozygous mutant for TAF1^CTK and heterozygous mutant for the maternal activator Caudal (Cad). In this genetic background, transcription of the gap gene giant (gt) was reduced. Gt is transcribed in two domains along the anterior-posterior axis of blastoderm-stage embryos. Transcription of the posterior gt domain (pgt) is Cad-dependent, whereas transcription of the anterior gt domain (agt) is Cad-independent. Relative to controls (cad/+ or TAF1^CTK), pgt transcription was reduced in cad/+;TAF1^CTK embryos (Maile, 2004).

XChIP analysis was used to examine whether TAF1-mediated phosphorylation of H2B-S33 contributes to pgt transcription. Cross-linked chromatin was isolated from the posterior halves of cad/+;TAF1^CTK and control embryos and immunoprecipitated with antibodies to H2B-S33P, acetylated histones, or TAF1. PCR analysis detected H2B-S33P at the transcriptionally active gt promoter in control embryos, but not at the transcriptionally repressed promoter in cad/+;TAF1^CTK embryos. To monitor TAF1 binding, two antibodies, TAF1-M and TAF1-C, where used that recognize the middle domain and the CTK of TAF1, respectively. Both antibodies precipitated the gt promoter from control embryos, indicating that TAF1DeltaCTK and maternally contributed, wild-type TAF1 are present at the gt promoter in the pgt. In contrast, although the TAF1-M antibody precipitated the gt promoter from cad/+;TAF1^CTK embryos, TAF1-C did not. Because TAF1DeltaCTK is present at a higher concentration in cad/+;TAF1^CTK embryos than maternal TAF1, this result indicates that TAF1DeltaCTK is preferentially recruited to the gt promoter in the pgt. This result is supported by the presence of TAF1-mediated histone acetylation at the transcriptionally silent gt promoter. Thus, TAF1-mediated phosphorylation of H2B-S33 contributes to transcriptional activation during Drosophila embryogenesis (Maile, 2004).

Ser³³ is the only evolutionarily conserved serine or threonine in the N-terminus of metazoan H2Bs. In the crystal structure of the Xenopus laevis nucleosome, the equivalent serine links the H2B DNA-binding N-terminal tail to the histone fold domain. Thus, replacing the hydroxyl group on Ser³³ with a bulkier, negatively charged phosphate group may drastically affect H2B tail interactions with DNA. This is important because the H2B tail regulates nucleosome mobility. Deletion of the tail bypasses the requirement for the SWI/SNF nucleosome-remodeling complex in yeast, and the tail is critical for maintaining the position of histone octamers in in vitro sliding assays. These findings support a model in which TAF1-mediated phosphorylation of H2B-S33 disrupts DNA-histone interactions, resulting in local decondensation of chromatin. Decondensation may trigger chromatin remodeling and formation of a chromatin structure that facilitates assembly of other GTFs at a promoter, a function that is primarily attributed to TFIID (Maile, 2004).

These data indicate that the S/T kinase motif of the CTK is located in the DBD. In the crystal structure of the DBD, the position of the S/T kinase motif does not overlap with the acetylated lysine-binding surface of the DBD, suggesting that it is an independent functional unit of the DBD. Members of the fsh/RING3 (BET) family of DBD proteins have kinase activity, suggesting that TAF1 is a member of a kinase family whose catalytic motif resides within the DBD (Maile, 2004).

Phosphorylation of H2B-S33 by TAF1 is essential for transcriptional activation of stg/cdc25 and, consequently, cell cycle progression. Similarly, depletion of yeast TAF5, human TAF2, or a twofold reduction in chicken TBP results in G2-M arrest. Like TAF1, TBP regulates stg/cdc25 expression, providing support for the finding that the H2B-S33 kinase activity of TAF1 occurs in the context of TFIID. Interestingly, depletion of yeast TAF1, which does not possess a CTK, and inactivation of TAF1 HAT activity induce G1 arrest because of reduced transcription of B- and D-type cyclins, respectively (Apone, 1996; Dunphy, 2001). Thus, loss of all TAF1 activities causes G2-M arrest whereas loss of TAF1 HAT activity causes G1 arrest, suggesting gene-specific requirements for TAF1 CTK and HAT activities. In contrast, the presence of phosphorylated H2B-S33 and acetylated H3 and H4 at the stg and gt promoters implies that TAF1 CTK and HAT activities can cooperate in transcriptional activation of some genes. This proposal is supported by the finding that loss of H2B-S33P from the gt promoter results in reduced transcription, despite the presence of TAF1-mediated histone acetylation. Thus, TAF1-mediated phosphorylation of H2B-S33 may work in concert with other TAF1-mediated histone modifications, H1 ubiquitination, and H3 and H4 acetylation to contribute to the chromatin-based mechanisms underlying transcription activation of eukaryotic genes (Maile, 2004).

DNA binding properties of TAF1 isoforms with two AT-hooks

TATA-binding protein-associated factor 1 (TAF1) is an essential component of the general transcription factor IID (TFIID), which nucleates assembly of the preinitiation complex for transcription by RNA polymerase II. TATA-binding protein and TAF1.TAF2 heterodimers are the only components of TFIID shown to bind specific DNA sequences (the TATA box and initiator, respectively), raising the question of how TFIID localizes to gene promoters that lack binding sites for these proteins. Drosophila TAF1 protein isoforms TAF1-2 and TAF1-4 directly bind DNA independently of TAF2. DNA binding by TAF1 isoforms is mediated by cooperative interactions of two identical AT-hook motifs, one of which is encoded by an alternatively spliced exon. Electrophoretic mobility shift assays revealed that TAF1-2 binds the minor groove of adenine-thymine-rich DNA with a preference for the sequence AAT. Alanine-scanning mutagenesis of the alternatively spliced AT-hook indicates that Lys and Arg residues made essential DNA contacts, whereas Gly and Pro residues within the Arg-Gly-Arg-Pro core sequence are less important for DNA binding, suggesting that AT-hooks are more divergent than previously predicted. TAF1-2 binds with variable affinity to the transcription start site of several Drosophila genes, and binding to the hsp70 promoter is reduced by mutation of a single base pair at the transcription start site. Collectively, these data indicate that AT-hooks serve to anchor TAF1 isoforms to the minor groove of adenine-thymine-rich Drosophila gene promoters and suggest a model in which regulated expression of TAF1 isoforms by alternative splicing contributes to gene-specific transcription (Metcalf, 2006; Full text of articel).

Binding of core promoters by the general transcription factor TFIID correlates with transcription activity for most RNA polymerase II genes. In metazoans, core promoters for RNA polymerase II genes comprise ~70 bp surrounding the transcription start site (TSS) and are made up of ~8-bp elements. Core promoter elements include the TATA box located 26-31 bp upstream of the TSS, the initiator element located at the TSS, and the downstream promoter element located 28-33 bp downstream of the TSS. Multiple subunits of TFIID engage in DNA binding at the core promoter. TATA-binding protein binds the TATA box and TATA-binding protein-associated factors (TAFs) bind downstream elements. Several lines of evidence point to the importance of TAF1-core promoter DNA interactions. Studies in yeast and mammalian cells indicate that core promoters, not activator-binding sites, render genes TAF1-dependent; cross-linking studies in Drosophila place TAF1 in close proximity to the TSS of the hsp70 gene; studies with purified human TFIID reveal TAF1 as the major TAF species that can be cross-linked to the downstream core element of several promoters; and TAF1·TAF2 heterodimers preferentially bind initiator sequences in vitro. However, TAF1 domains required for core promoter interactions have not been identified, and direct DNA binding by TAF1 on its own has not been observed (Metcalf, 2006 and references therein).

TFIID is a family of complexes with different TAF components. In Drosophila and humans, germline homologs of general TAFs are necessary for transcription during germ cell differentiation. In addition, changes in the TAF composition of TFIID result from alternative splicing of general TAF pre-mRNAs. For example, the human TAF6 pre-mRNA is alternatively spliced in response to apoptotic signals, and the encoded TAF6 isoform, TAF6Δ, modulates the transcription of pro-apoptotic genes. Thus, regulated expression of TAF isoforms is an important determinant of gene-specific transcription (Metcalf, 2006 and references therein).

In Drosophila, alternative splicing of the TAF1 pre-mRNA generates four TAF1 mRNA isoforms that differ by the inclusion of two small exons 12a and 13a (Chen, 2002). The functional importance of TAF1 alternative splicing is suggested by the fact that TAF1 isoform levels differ between Drosophila tissues and are regulated in response to genotoxic stress. TAF1-1 and TAF1-3 encode one predicted AT-hook motif, whereas TAF1-2 and TAF1-4 encode two predicted AT-hooks. The DNA-binding AT-hook motif contains a central core of Arg-Gly-Arg-Pro residues flanked by Lys and Arg residues. AT-hooks were first described in the high mobility group non-histone chromosomal protein HMGA (also known as HMG-I(Y)) and have been shown to bind DNA through contacts with the minor groove of adenine-thymine (A-T) tracts. The AT-hook motif is found in one or more copies in a large number of transcription factors and components of chromatin remodeling complexes. AT-hooks may serve as accessory DNA-binding domains that anchor chromatin-associated proteins to particular DNA sequences or participate in cooperative DNA-binding with other proteins (Metcalf, 2006).

This study demonstrates direct DNA binding by the two AT-hook containing TAF1-2 and TAF1-4 proteins at the TSS of several Drosophila genes. Lys and Arg residues of the AT-hook are critical for DNA binding, whereas Ala substitutions are tolerated at highly conserved Gly and Pro residues. TAF1 binds the minor groove of DNA with a preference for A-T tracts, especially an AAT sequence element. Direct DNA binding by TAF1 isoforms whose expression is regulated by developmental and stress signals has implications for the mechanism of gene-specific transcription initiation (Metcalf, 2006).

ATM and ATR pathways signal alternative splicing of Drosophila TAF1 pre-mRNA in response to DNA damage

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, the splicing of the Drosophila TAF1 pre-mRNA was analyzed. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells is mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing is mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing (Katzenberger, 2006; Full text of article).

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. This study used RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, it was found that TAF4 (TBP associated factor 110) rather than TBP or TAF1 (TBP-associated factor 250kD) plays the most critical role in maintaining stability of the complex. This analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on these results, it is proposed that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID (Wright, 2006; Full text of article).

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