TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.

The transcription factor E2F-1 plays a key role in regulating cell cycle progression. Accordingly, E2F-1 activity is itself tightly controlled by a series of transcriptional and post-transcriptional events. Here we show that the E2F-1 activation domain interacts with a kinase activity which phosphorylates two sites, Ser403 and Thr433, within the activation domain. We demonstrate that TFIIH is responsible for the E2F-1 phosphorylation observed in cell extracts and that endogenous E2F-1 interacts in vivo with p62, a component of TFIIH, during S phase. When the two phosphorylation sites in E2F-1 are mutated to alanine, the stability of the E2F-1 activation domain is greatly increased. These results suggest that TFIIH-mediated phosphorylation of E2F-1 plays a role in triggering E2F-1 degradation during S phase.

A connection between transcription and DNA repair was demonstrated previously through the characterization of TFIIH. Using filter binding as well as in vitro transcription challenge competition assays, we now show that the promoter recognition factor TATA box-binding protein (TBP)/TFIID binds selectively to and is sequestered by cisplatin- or UV-damaged DNA, either alone or in the context of a larger protein complex including TFIIH. Computer-assisted 3D structural analysis reveals a remarkable similarity between the structure of the TATA box as found in its TBP complex and that of either platinated or UV-damaged oligonucleotides. Thus, cisplatin-treated or UV-irradiated DNA could be used as a competing binding site which may lure TBP/TFIID away from its normal promoter sequence, partially explaining the phenomenon of DNA damage-induced inhibition of RNA synthesis. Consistent with an involvement of damaged DNA-specific binding of TBP in inhibiting transcription, we find that microinjection of additional TBP in living human fibroblasts alleviates the reduction in RNA synthesis after UV irradiation. Future anticancer drugs could be designed with the consideration of lesion recognition by TBP and their ability to reduce transcription.

PC4 is a nuclear DNA-binding protein that stimulates activator-dependent class II gene transcription in vitro. Recent biochemical and X-ray analyses have revealed a unique structure within the C-terminal domain of PC4 that binds tightly to unpaired double-stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA-binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator-dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single-stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. We now show that Myc recruits TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the pre-initiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. We suggest that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes.

The trimeric Cdk7-cyclin H-Mat1 complex comprises the kinase subunit of basal transcription factor TFIIH and has been shown to function as a cyclin-dependent kinase (Cdk)-activating kinase. Herein we report that disruption of the murine Mat1 gene leads to peri-implantation lethality coincident with depletion of maternal Mat1 protein. In culture, Mat1(-/-) blastocysts gave rise to viable post-mitotic trophoblast giant cells while mitotic lineages failed to proliferate and survive. In contrast to wild-type trophoblast giant cells, Mat1(-/-) cells exhibited a rapid arrest in endoreduplication, which was characterized by an inability to enter S phase. Additionally, Mat1(-/-) cells exhibited defects in phosphorylation of the C-terminal domain (CTD) of RNA polymerase II on both Ser5 and Ser2 of the heptapeptide repeat. Despite this, Mat1(-/-) cells demonstrated apparent transcriptional and translational integrity. These data indicate an essential role for Mat1 in progression through the endocycle and suggest that while Mat1 modulates CTD phosphorylation, it does not appear to be essential for RNA polymerase II-mediated transcription.

The metazoan cyclin-dependent kinase Cdk7 was purified originally as part of a biochemical activity called CAK (Cdk-activating kinase) capable of phosphorylating and activating in vitro the Cdks that promote the different cell cycle transitions. Cdk7 is also found in the transcription factor complex TFIIH, suggesting that it participates in vivo in the control of RNA polymerase II. We have examined the physiological role of Cdk7 during the course of Drosophila development. By expressing dominant-negative forms of the kinase, we were able to alter Cdk7 function at given developmental stages. Expression of Cdk7 mutants severely delayed the onset of zygotic transcription in the early embryo, but did not alter the timing of the first 13 embryonic nuclear cycles. These results implicate Cdk7 in the control of transcriptional machinery in vivo. While cell cycle regulation is not sensitive to our manipulations of Cdk7 activity, it suggests that a distinct pool of CAK activity that is unaffected by expression of the cdk7(DN) mutants is present in these embryos.