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R Tjian

Publications and source records attributed to R Tjian.

At least 37 records · Page 2Linked to original sources
PubMed1998

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription.

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Binding Sites↗
PubMed1998

Functional analysis of the human TAFII250 N-terminal kinase domain.

The largest subunit of the human transcription factor TFIID, TAFII250, was previously reported to contain serine/threonine kinase domains that can autophosphorylate and transphosphorylate the large subunit of the basal factor TFIIF. Here, we identify the regions of the N-terminal kinase domain (amino acids 1-414) necessary for kinase activity and examine its function in vivo. Point mutations within two patches of amino acids in the kinase domain decrease both autophosphorylation and transphosphorylation activities. Importantly, we find that TAFII250-bearing mutations within the N-terminal kinase domain exhibit a significantly reduced ability to rescue ts13 cells that express a temperature-sensitive TAFII250. Moreover, transcription from the cyclin A and cdc2 promoters becomes impaired when cotransfected with hTAFII250 containing inactive forms of the N-terminal kinase domain. Our results suggest that the TAFII250 kinase activity is required to direct transcription of at least some genes in vivo.

Cell Line↗
PubMed1998

Adf-1 is a nonmodular transcription factor that contains a TAF-binding Myb-like motif.

Adf-1 is an essential Drosophila melanogaster sequence-specific transactivator that binds the promoters of a diverse group of genes. We have performed a comprehensive mapping of the functional domains of Adf-1 to study the role of transactivators in the process of gene activation. Using a series of clustered point mutations and small deletions we have identified regions of Adf-1 required for DNA binding, dimerization, and activation. In contrast to most enhancer-binding factors, the Adf-1 activation regions are nonmodular and depend on an intact protein, including the Adf-1 DNA-binding domain, for activity. Like many transcriptional activators, Adf-1 contains a TFIID-binding domain that can interact with specific TAF subunits. Although TAFs are required for Adf-1-directed activation, TAF binding is not sufficient, suggesting that Adf-1 may direct multiple essential steps during activation. Interestingly, both the TAF-binding domain and the DNA-binding domain contain sequences homologous to those of the Myb family of DNA-binding domains. Thus, Adf-1 has evolved an unusual structure containing two versions of the Myb motif, one that binds DNA and one that binds proteins.

Amino Acid Sequence↗
PubMed1997

TAFII250-dependent transcription of cyclin A is directed by ATF activator proteins.

A specific mutation in TAFII250, 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 TAFII250. An analysis of chimeric promoter constructs reveals that the cyclin A TSRE can confer TAFII250 dependence to the core promoter of c-fos. In addition, reciprocal hybrid promoter constructs suggest that TAFII250 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 TAFII250-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 TAFII250 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 TAFII250 apparently result in the down-regulation of key molecules, such as cyclin A, which may be responsible for the ts13 cell cycle arrest phenotype.

Animals↗
PubMed1997

Transcription properties of a cell type-specific TATA-binding protein, TRF.

Eukaryotic cells are thought to contain a single TATA-binding protein (TBP) that directs transcription by cellular RNA polymerases. Here we report a cell type-specific TBP-related factor (TRF) that can form a stable TRF/IIA/IIB TATA DNA complex and substitute for TBP in directing RNA polymerase II transcription in vitro. Transfection studies reveal that TRF can differentially mediate activation by some enhancer proteins but not others. Like TBP, TRF forms a stable complex containing multiple novel subunits, nTAFs. Antibody staining of embryos and polytene chromosomes reveals cell type-specific expression and gene-selective properties consistent with the shaker/male sterile phenotype of trf mutants. These findings suggest TRF is a homolog of TBP that functions to direct tissue- and gene-specific transcription.

Animals↗
PubMed1997

Repression of p53-mediated transcription by MDM2: a dual mechanism.

The oncoprotein MDM2 binds to the activation domain of the tumor suppressor p53 and inhibits its ability to stimulate transcription. This same region of p53 is able to bind several basal transcription factors that appear to be important for the transactivation function of p53. It has therefore been suggested that MDM2 acts to inhibit p53 by concealing its activation domain from the basal machinery. Here we present data suggesting that MDM2 possesses an additional inhibitory function. Our experiments reveal that in addition to a p53-binding domain, MDM2 also contains an inhibitory domain that can directly repress basal transcription in the absence of p53. By fusing portions of MDM2 to a heterologous DNA-binding domain to allow p53-independent promoter recruitment, we have localized this inhibitory domain to a region encompassing amino acids 50-222 of MDM2. Furthermore, the function of this inhibitory domain does not require the presence of either TFIIA or the TAFs. Of the remaining basal factors, both the small subunit of TFIIE and monomeric TBP are bound by the MDM2 inhibitory domain. It is possible that MDM2 inhibits the ability of the preinitiation complex to synthesize RNA through one of these interactions. Our results are consistent with a model in which MDM2 represses p53-dependent transcription by a dual mechanism: a masking of the activation domain of p53 through a protein-protein interaction that additionally serves to recruit MDM2 to the promoter where it directly interferes with the basal transcription machinery.

Cell-Free System↗
PubMed1997

Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1.

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.

Amino Acid Sequence↗
PubMed1997

Mechanisms of transcriptional activation: differences and similarities between yeast, Drosophila, and man.

Activation of transcription requires an interplay between enhancer-binding factors and components of the general transcription machinery. New developments within the past few years suggest that novel cofactors are required for relaying specific activation signals to the RNA polymerase II transcription complex in order to achieve enhanced levels of mRNA synthesis. The role of these different cofactors in mediating activation and potential differences in their utilization by divergent organisms, however, raise new questions about the mechanisms of transcriptional regulation.

Animals↗
PubMed1996

TAF(II)s mediate activation of transcription in the Drosophila embryo.

Mutations in the genes for two highly conserved TAFs, TAF(II)60 and TAF(II)110, reduce transcription of Bicoid-dependent target genes in vivo. By means of several distinct genetic test systems, specific activator-TAF interactions are shown to support both simple and synergistic enhancement of transcription in the embryo. These studies provide in vivo evidence that TAFs can serve as coactivators to receive gene-specific transcriptional activation signals. This genetic system also presents the opportunity to study the function of basal transcription components in regulating development of complex organisms.

Alleles↗
PubMed1996

SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein.

The sterol regulatory element binding proteins (SREBP-1 and -2) activate transcription of genes whose products are involved in the cellular uptake and synthesis of cholesterol. Although considerable effort has been exerted to define the events regulating the levels of active SREBP, little is known about the transcriptional cofactors mediating SREBP function. In an unbiased search for potential coactivators of SREBP, we isolated a protein of 265 kD from HeLa cells that directly bound SREBP-1 and SREBP-2. Peptide sequencing and Western blot analysis established that the 265-kD protein was CBP (CREB-binding protein), a recently identified transcriptional coactivator. The putative activation domain of SREBP was shown to bind specifically to amino-terminal domains of recombinant CBP and p300 (a CBP-related protein). Moreover, transfection studies demonstrated that CBP enhances the ability of SREBP to activate transcription of reporter genes in HeLa cells. Together, these data suggest that CBP mediates SREBP transcriptional activity, thus revealing a new step in the biochemical pathway regulating cholesterol metabolism.

Animals↗
PubMed1996

Human TAFII 105 is a cell type-specific TFIID subunit related to hTAFII130.

We previously characterized Drosophila and human TAF subunits that make up the core TFIID complex found in all cells. Here, we report that differentiated B cells contain a novel substoichiometric TAF of 105 kDa not found associated with TFIID isolated from other cell types. The cDNA encoding hTAFII105 reveals a highly conserved C-terminal domain shared by hTAFII130 and oTAFII110, while the N-terminal coactivator domain has diverged significantly. All cells tested express TAFII105 mRNA, but only B cells contain significant levels of protein associated with TFIID. Transient overexpression of hTAFII105 selectively squelches the transcription of some genes in B cells. These properties suggest that TAFII105 is a cell type-specific subunit of TFIID that may be responsible for mediating transcription by a subset of activators in B cells.

Amino Acid Sequence↗
PubMed1996

The biochemistry of transcription in eukaryotes: a paradigm for multisubunit regulatory complexes.

The control of gene expression during development, differentiation and maintenance of cellular function is governed by a complex array of transcription factors. We have undertaken a molecular dissection of the regulatory factors that direct transcription of protein coding genes by RNA polymerase II. Our early studies identified sequence-specific transcriptional activators that bind to enhancer and promoter sequences to modulate the transcriptional initiation event. However, the mechanism by which activators enhance transcription and mediate promoter selectivity remained unknown. Combining biochemical purification and in vitro assays, we have recently identified an essential class of transcription factors called TAFs that are tightly associated with the basal factor TBP (TATA-binding protein). We have found that TAFs are responsible for at least two regulatory functions. Some TAFs serve as coactivators capable of binding activators and mediating enhancing function. Other TAFs have been shown to confer template selectivity by binding directly to core DNA elements of the promoter. Thus different subunits of TBP/TAF complexes perform a variety of functions critical for transcriptional regulation in animal cells.

Animals↗
PubMed1996

TAFII250 is a bipartite protein kinase that phosphorylates the base transcription factor RAP74.

Some TAF subunits of transcription factor TFIID play a pivotal role in transcriptional activation by mediating protein-protein interactions, whereas other TAFs direct promoter selectivity via protein-DNA recognition. Here, we report that purified recombinant TAFII250 is a protein serine kinase that selectively phosphotylates RAP74 but not other basal transcription factors or common phosphoacceptor proteins. The phosphorylation of RAP74 also occurs in the context of the complete TFIID complex. Deletion analysis revealed that TAFII250 contains two distinct kinase domains each capable of autophosphorylation. However, both the N- and C-terminal kinase domains of TAFII250 are required for efficient transphosphorylation of RAP74 on serine residues. These findings suggest that the targeted phosphorylation of RAP74 by TAFII250 may provide a mechanism for signaling between components within the initiation complex to regulate transcription.

Amino Acid Sequence↗
PubMed1996

Drosophila TFIIA-S is up-regulated and required during Ras-mediated photoreceptor determination.

Photoreceptor induction in the developing Drosophila eye is triggered by the activation of the Ras pathway. Subsequently, the Ras-mediated activation of site-specific transcription factors leads to the expression of putative "effector" genes. The coactivator function of the basal transcription factor TFIIA has been shown previously to enhance the trans-activation potential of site-specific transcription factors in vitro. Here, we show that the expression of the small subunit of TFIIA (dTFIIA-S) is specifically up-regulated in a transient manner during Ras-mediated photoreceptor induction. Furthermore, although null mutations in dTFIIA-S are cell lethal, a hypomorphic dTFIIA-S allele demonstrates an increased requirement for this factor during photoreceptor development. In addition, the cone cell to R7 photoreceptor transformation caused by ectopic activation of the Ras pathway during eye development is suppressed by the removal of one functional copy of the dTFIIA-S locus revealing the sensitivity of this process to reductions in dTFIIA-S activity. These results are the first in vivo evidence for the coactivator function in transcriptional enhancement proposed for TFIIA.

Animals↗