TFII-I down-regulates a subset of estrogen-responsive genes through its interaction with an initiator element and estrogen receptor alpha.

TFII-I was initially identified as the general transcription factor that binds to initiator (Inr) elements in vitro. Subsequent studies have shown that TFII-I activates transcription of various genes either through Inr elements or through other upstream elements in vivo. Since, however, most studies so far on TFII-I have been limited to over-expression and reporter gene assays, we reevaluated the role of TFII-I in vivo by using stable knockdown with siRNA and by examining the expression of endogenous genes. Contrary to the widely accepted view, here we show that TFII-I is not important for cell viability in general but rather inhibits the growth of MCF-7 human breast cancer cells. MCF-7 cells are known to proliferate in an estrogen-dependent manner. Through analysis of TFII-I's cell-type specific growth inhibitory effect, we show evidence that TFII-I down-regulates a subset of estrogen-responsive genes, only those containing Inr elements, by recruiting estrogen receptor (ER) alpha and corepressors to these promoters. Thus, this study has revealed an unexpected new role of TFII-I as a negative regulator of transcription and cell proliferation.

In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets.

Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington's disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. Here, we report the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. We demonstrate that both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. Our results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus.

Inhibition of TFII-I-dependent cell cycle regulation by p53.

The multifunctional transcription factor TFII-I is tyrosine phosphorylated in response to extracellular growth signals and transcriptionally activates growth-promoting genes. However, whether activation of TFII-I also directly affects the cell cycle profile is unknown. Here we show that under normal growth conditions, TFII-I is recruited to the cyclin D1 promoter and transcriptionally activates this gene. Most strikingly, upon cell cycle arrest resulting from genotoxic stress and p53 activation, TFII-I is ubiquitinated and targeted for proteasomal degradation in a p53- and ATM (ataxia telangiectasia mutated)-dependent manner. Consistent with a direct role of TFII-I in cell cycle regulation and cellular proliferation, stable and ectopic expression of wild-type TFII-I increases cyclin D1 levels, resulting in accelerated entry to and exit from S phase, and overcomes p53-mediated cell cycle arrest, despite radiation. We further show that the transcriptional regulation of cyclin D1 and cell cycle control by TFII-I are dependent on its tyrosine phosphorylation at positions 248 and 611, sites required for its growth signal-mediated transcriptional activity. Taken together, our data define TFII-I as a growth signal-dependent transcriptional activator that is critical for cell cycle control and proliferation and further reveal that genotoxic stress-induced degradation of TFII-I results in cell cycle arrest.

Inhibition of host RNA polymerase II-dependent transcription by vesicular stomatitis virus results from inactivation of TFIID.

During infection with vesicular stomatitis virus (VSV), host-cell mRNA synthesis is inhibited due to shut off of host-cell transcription. The transcriptional activity of nuclear extracts prepared from VSV-infected cells was compared to the activity of nuclear extracts from uninfected cells. An exogenous DNA template was used which contained an adenovirus major late promoter (AdMLP) but lacked upstream activating sequences, so that only basal transcription activity was assayed in these experiments. AdMLP-initiated transcription was decreased by 75% in nuclear extracts from infected cells as early as 3 h p.i. and by >90% by 6 h p.i. Mixing nuclear extracts from uninfected and VSV-infected cells revealed that the inhibition was caused by lack of an active form of a host factor involved in basal transcription rather than by the presence of an excess of inhibitory factor. To determine which transcription factors were lacking from nuclear extracts of infected cells, host transcription initiation factors isolated from uninfected cells by ion-exchange chromatography were added separately to nuclear extracts inactivated by VSV infection. A phosphocellulose column fraction from uninfected cells eluted with 0. 8 M KCl, which contained transcription factor IID (TFIID), overcame the inhibition. The corresponding fraction from infected cells had no detectable activity in a TFIID-dependent in vitro transcription assay. TATA-binding protein (TBP) is the DNA-binding subunit of TFIID and has been shown previously to substitute for TFIID in basal transcription. Purified recombinant TBP also reconstituted the transcription activity of nuclear extracts from infected cells, supporting the idea that TFIID is the target of virus-induced inhibition. Western blot analysis showed that the level of TBP in nuclear extracts or in the 0.8 M KCl column fraction was not changed by VSV infection. These results indicated that VSV infection leads to an inhibition of host transcription by inactivation of TFIID rather than reduction in the level of TFIID.