A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNAcontacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.Transcription of protein-encoding genes in eukaryotes involves the assembly of RNA polymerase II and general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). TFIID is the major DNA-binding GTF. It is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TAFs regulate transcription at multiple levels. Certain TAFs interact with activators to facilitate PIC formation (2, 18) and transcription reinitiation (1). TAFs also have a role in recognition and binding to core promoter elements. DNase I footprinting revealed direct contact of TAFs with sequences upstream and downstream of the TATA box (11,19,23,26,29,35). TAF1-TAF2 (TAF II 250-TAF II 150) binds the Initiator element (7,33,34), multiple TAFs were cross-linked to the adenovirus major late (AdML) promoter (24), and Drosophila melanogaster TAF6 (dTAF6) and dTAF9 (dTAF II 60-dTAF II 42) were cross-linked to the downstream promoter element (DPE) (5). Some TAFs are TFIID specific, but others are shared by other transcription regulatory complexes (3,4,15,22,25,28).One common feature found in 9 out of the 14 TAFs is the histone fold motif (for a review see reference 13). This motif has been established as an essential protein-protein interaction domain that facilitates assembly of TFIID in a manner analogous to that for histones (17,30). TAF6 and TAF9 are structurally related to histones H4 and H3, respectively (38). TAF12 is similar to H2B, TAF4 contains an H2A-like domain, and both interact with each other via the histone fold domain (HFD) (14,16,30). In vitro Saccharomyces cerevisiae TAF6 (yTAF6)-yTAF9 can assemble with yTAF12-yTAF4 to form a histone octamer-like structure (30).The nucleosome-like interaction of TFIID with DNA (24) and the presence of histone fold TAFs within this complex have led to the proposal that a nucleosome-like octamer within TFIID may be involved in direct DNA binding (17). However, the issue has remained elusive. First, there is no experimental evidence for D...

The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b⅐TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is ϳ70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes.Two types of DNA elements regulate transcription of protein-encoding genes in eukaryotes. Enhancer elements, which may be localized proximally or distally relative to the transcription initiation site, are the binding sites for gene-specific transcription factors. A core promoter, situated close to the transcription start site (TSS), 2 serves as the site on which RNA polymerase II and the general transcription factors bind and assemble into a pre-initiation complex (1, 2). Enhancer-bound transcription factors activate transcription by modulating chromatin structure or by recruiting the transcription machinery to the core promoter.The major core promoter-binding factor within the general transcription apparatus is TFIID, a large complex composed of the TATA-binding protein (TBP) and about 14 TBP-associated factors (TAFs) (for recent reviews see Refs. 3,4). Within TFIID TBP is responsible for recognition and binding of TATA-containing promoters. The TAFs are also important for core promoter recognition, and they bind primarily to non-TATA-box elements, interacting with sequences upstream and downstream to the TATA box (5-13). In addition certain TAF subcomplexes have been reported to specifically bind different core promoter elements. The TAF1⅐TAF2 complex binds to the Initiator element (14) and Drosophila TAF6 and TAF9 crosslinked to the downstream promoter element in the context of TFIID (15), and, as a reconstituted complex, these were shown to associate with a downstream promoter element-containing promoter (16).A feature common to 9 of the 14 TAFs is the histone-fold domain (HFD) (17)(18)(19)(20). The presence of histone-fold TAFs within TFIID led to the proposal that there is a nucleosomallike interaction between HFD TAFs and DNA (21). Recently we reported that the H4-H3-like TAF6...

TAF4b is a cell type-specific subunit of the general transcription factor TFIID. Here, we show that TAF4b is highly expressed in embryonic stem cells (ESC) and is down-regulated upon differentiation. To examine the role of TAF4b in ESC, we applied a knockdown (KD) approach. TAF4b depletion is associated with morphological changes and reduced expression of the selfrenewal marker alkaline phosphatase. In contrast, KD of TAF4, a ubiquitously expressed TAF4b paralog, retained and even stabilized ESC stemness. Retinoic acid-induced differentiation was facilitated in the absence of TAF4b but was significantly delayed by TAF4 KD. Furthermore, TAF4b supports, whereas TAF4 inhibits, ESC proliferation and cell cycle progression. We identified a subset of TAF4b target genes preferentially expressed in ESC and controlling the cell cycle. Among them are the germ cell-specific transcription factor Sohlh2 and the protein kinase Yes1, which was recently shown to regulate ESC self-renewal. Interestingly, Sohlh2 and Yes1 are also targets of the pluripotency factor Oct4, and their regulation by Oct4 is TAF4b-dependent. Consistent with that, TAF4b but not TAF4 interacts with Oct4. Our findings suggest that TAF4b cooperates with Oct4 to regulate a subset of genes in ESC, whereas TAF4 is required for later embryonic developmental stages.

The proximal promoter consists of binding sites for transcription regulators and a core promoter. We identified an overrepresented motif in the proximal promoter of human genes with an Initiator (INR) positional bias. The core of the motif fits the INR consensus but its sequence is more strict and flanked by additional conserved sequences. This strict INR (sINR) is enriched in TATA-less genes that belong to specific functional categories. Analysis of the sINR-containing DHX9 and ATP5F1 genes showed that the entire sINR sequence, including the strict core and the conserved flanking sequences, is important for transcription. A conventional INR sequence could not substitute for DHX9 sINR whereas, sINR could replace a conventional INR. The minimal region required to create the major TSS of the DHX9 promoter includes the sINR and an upstream Sp1 site. In a heterologous context, sINR substituted for the TATA box when positioned downstream to several Sp1 sites. Consistent with that the majority of sINR promoters contain at least one Sp1 site. Thus, sINR is a TATA-less-specific INR that functions in cooperation with Sp1. These findings support the idea that the INR is a family of related core promoter motifs.