The transcription factor TFIID, a central component of the eukaryotic RNA polymerase II (Pol II) transcription apparatus, comprises the TATA-binding protein (TBP) and approximately ten TBP-associated factors (TAFs). Although the essential role of TBP in all eukaryotic transcription has been extensively analysed in vivo and in vitro, the function of the TAFs is less clear. In vitro, TAFs are dispensable for basal transcription but are required for the response to activators. In addition, specific TAFs may act as molecular bridges between particular activators and the general transcription machinery. In vivo, TAFS are required for yeast and mammalian cell growth, but little is known about their specific transcriptional functions. Using conditional alleles created by a new double-shutoff method, we show here that TAF depletion in yeast cells can reduce transcription from some promoters lacking conventional TATA elements. However, TAF depletion has surprisingly little effect on transcriptional enhancement by several activators, indicating that TAFs are not generally required for transcriptional activation in yeast.

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA- The regulation of transcription of mRNA-encoding eukaryotic genes is a complicated process involving the modulation of chromatin structure, activities of upstream activators and repressors, and the concerted action of multiple components of the basal transcription machinery, including RNA polymerase It itself (1, 2). It is thought that the interaction of the TATA-binding protein (TBP), with the TATA-box promoter element is the first step in the formation of the RNA polymerase II preinitiation complex (PIC), and numerous studies have shown that PIC formation is subject to modulation by a variety of transcriptional regulators. However, the mechanisms by which these factors exert their effects are not yet fully understood. In metazoan systems, one basal factor that has been shown to be directly involved in mediating activation by upstream activators is the transcription factor TFIID, which is composed of TBP and TBP-associated factors (TAF11s

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST) Transcription initiation by RNA polymerase II (Pol II) requires assembly of a large complex consisting of Pol II and general transcription factors (GTFs) at the promoter. It has been proposed that assembly of this complex begins when TFIID, consisting of TATA box-binding protein (TBP) and its associated factors (TAF II proteins), binds to the core promoter, followed by sequential binding of other GTFs and Pol II itself (9). In another scenario, Pol II, certain GTFs, and coactivator proteins bind to the promoter as a preformed holoenzyme complex (46). Transcriptional activators bind to the promoter, generally upstream of the TATA element, and stimulate the assembly or function of the transcription initiation complex. Binding of TFIID to the core promoter appears to be rate limiting for initiation (12,43,88), and certain activators stimulate this step in initiation complex formation (3,11,21,39,40,50,91). Several activators bind TBP in vitro in a manner that depends on amino acids in the activation domain that are critical for transcriptional activation in vivo (7,11,26,35,38,51,(61)(62)(63), suggesting that direct interactions between the activator and TBP are involved in recruiting TFIID to the core promoter. Certain activation domains also bind TFIIB in vitro in a sequence-specific manner (4,7,14,41,56,91) and may stimulate recruitment of this GTF to the initiation complex (15,41,55,56).-Other studies suggest that activator function is mediated by one or more of the TAF II coactivator proteins associated with TBP in TFIID. Different activators may require specific TAF II proteins for activation (13,(74)(75)(76), and indeed, certain activation domains bind preferentially to specific TAF II proteins in vitro (24,37,57,83). The interactions between activators and TAF II proteins may serve primarily to recruit TFIID to the promoter (75). The human TAF II 250 subunit (and its Saccharomyces cerevisiae homolog yTAF II 130) has histone acetyltransferase (HAT) activity that may also promote initiation complex formation by destabilizing a repressive nucleosome structure at the promoter (64). A yeast Pol II-TAF II complex was shown to be required for transcriptional activation of a Gcn4p-regulated promoter in vitro (44); however, recent studies indicate that yTAF II proteins are not essential for transcriptional activation in vivo by Gcn4p and by several other yeast activator proteins (65,85).

TFIID, one of the multiple eukaryotic general transcription factors (GTFs), plays a key role in DNA-dependent RNA polymerase II (RNAP II)-mediated transcription initiation and regulation. The form and function of TFIID have been extensively studied by using in vitro approaches (see references 7, 22, and 57 for recent reviews). TFIID exists as a stable multisubunit complex in a variety of distinct eukaryotic systems (7,17,22,47,57,92), and in vitro transcription assays have shown that one subunit of TFIID, the TATA-binding protein (TBP), is sufficient for TATA element DNA recognition and subsequent incorporation of the other GTFs and RNAP II into the preinitiation complex (PIC). A TBP-assembled PIC can catalyze basal transcription in vitro (6, 67). However, these same in vitro assays also demonstrate that activation of transcription by sequence-specific DNA binding transactivator proteins can be observed only from a PIC formed by utilizing the multisubunit TBP-containing complex, TFIID, and not with TBP (32, 64). This observation suggested that the other subunits of TFIID are essential for its regulatory functions and ultimately led to the identification of the TBP-associated factors (TAFs), which in combination with TBP comprise the TFIID complex (17,47,64,92).At least 8 to 10 RNAP II-specific TAFs (or TAF II s) associate with TBP to form eukaryotic TFIID. These TAF II s exhibit molecular masses ranging from 250 to 15 kDa, depending on the organism analyzed (human, Drosophila melanogaster, and Saccharomyces cerevisiae) (17,47,61,66,92; see reference 7 for a recent review). A comparison of the amino acid sequences of TAF II s from these evolutionarily divergent organisms shows that there is a striking conservation of TAF II sequences (7). The results of various biochemical analyses have led to the hypothesis that TAF II s participate in a variety of protein-protein and protein-DNA interactions. These interactions range from the facilitation of the formation and/or stabilization of the PIC by TAF II binding to DNA (78,82,83) to TAF II s interacting with each other (7, 29, 30, 33, 34, 37, 41-46, 53, 71, 79, 80, 83, 86, 87, 89, 91) or with basal transcription factors (7,23,26,30,42,90) to the direct interaction of TAF II s with the activation domains of transcriptional regulatory molecules (9, 11, 12, 19-21, 23, 31, 37, 42, 51, 72, 75, 80, 81, 86). Clearly, TAF II s are involved in a large number of critical regulatory events in RNAP II transcription, and detailed analyses of TAF II molecules will shed light on the molecular mechanisms of RNAP II transcriptional regulation.The largest subunit of metazoan TFIID has been termed either hTAF II 250 or dTAF II 250, depending on whether it is a human or Drosophila protein. The human protein, hTAF II 250, contains an acidic N terminus, a central region including a high-mobility-group (HMG) homology box, and two bromodomain-like direct repeats, as well as a glycine-and serine-rich C terminus (29, 71). The Drosophila homolog, dTAF II 250, has Ͼ90% similarity and Ͼ50% iden...

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.