Basal transcription by human RNA polymerase II requires the coordinate action of several ancillary factors (TFIIA-J) and can be regulated by various promoter-specific DNA binding proteins. An additional class of factors, called coactivators, are dispensable for basal transcription but are indispensable for regulation by transcriptional activators. Biochemical studies established that some coactivators are associated with the TATA-binding protein (TBP) to form the TFIID complex. We therefore set out to define the relationship between TBP and these TBP-associated factors (TAFs). Here we describe the cloning, expression and properties of the first human TAF, hTAFII250. The hTAFII250 gene is identical to a gene, CCG1, (ref 7,8), implicated in cell-cycle progression. Recombinant hTAFII250 binds directly to TBP both in vitro and in yeast, and participates in the formation of the TFIID complex. This largest TAF may therefore play a central role in TFIID assembly by interacting with both TBP and other TAFs, as well as serving to link the control of transcription to the cell cycle.

The TAFII250 subunit of the human transcription factor IID (TFIID) rescues the temperature-sensitive hamster cell line ts13 and overcomes a G1 arrest. Investigation of the transcriptional properties of ts13 nuclear extracts in vitro showed that activation by the site-specific regulators Sp1 and Gal4VP16 is temperature sensitive in ts13 extracts, whereas basal transcription remains unaffected. This transcriptional defect can be rescued by purified human TFIID or by expression of wild-type TAFII250 in ts13 cells. Expression from the cyclin A but not c-fos promoter is temperature sensitive in these mutant cells. Thus, the mutation in TAFII250 appears to have gene-specific effects that may lead to the ts13 cell cycle phenotype.

We describe an X-linked genetic syndrome associated with mutations in TAF1 and manifesting with global developmental delay, intellectual disability (ID), characteristic facial dysmorphology, generalized hypotonia, and variable neurologic features, all in male individuals. Simultaneous studies using diverse strategies led to the identification of nine families with overlapping clinical presentations and affected by de novo or maternally inherited single-nucleotide changes. Two additional families harboring large duplications involving TAF1 were also found to share phenotypic overlap with the probands harboring single-nucleotide changes, but they also demonstrated a severe neurodegeneration phenotype. Functional analysis with RNA-seq for one of the families suggested that the phenotype is associated with downregulation of a set of genes notably enriched with genes regulated by E-box proteins. In addition, knockdown and mutant studies of this gene in zebrafish have shown a quantifiable, albeit small, effect on a neuronal phenotype. Our results suggest that mutations in TAF1 play a critical role in the development of this X-linked ID syndrome.

A specific mutation in TAF II 250, the largest subunit of the transcription factor TFIID, disrupts cell growth control in the temperature-sensitive mutant hamster cell line ts13. Transcription from the cyclin A and D1 but not the c-fos and myc promoters is also dramatically reduced in ts13 cells at the nonpermissive temperature. These findings provide an intriguing link between TAF-mediated transcriptional regulation and cell cycle progression. Here we report the mapping of an enhancer element in the cyclin A promoter (TSRE) that responds to mutations in TAF II 250. An analysis of chimeric promoter constructs reveals that the cyclin A TSRE can confer TAF II 250 dependence to the core promoter of c-fos. In addition, reciprocal hybrid promoter constructs suggest that TAF II 250 also contributes to the transcriptional properties of the cyclin A core promoter. We have purified and identified cellular activators that specifically bind to the TSRE and mediate transcription in a TAF II 250-dependent manner. By micropeptide sequencing, we determined that TSRE-binding proteins include members of the activating transcription factor (ATF) family. These results suggest that the ts13 mutation of TAF II 250 has compromised the ability of TFIID to mediate activation of transcription by specific enhancer factors such as ATF, as well as to perform certain core promoter functions. These defects in TAF II 250 apparently result in the down-regulation of key molecules, such as cyclin A, which may be responsible for the ts13 cell cycle arrest phenotype.