PubMed Health⌕ Search

Biomedical subjects

R Tjian

Publications and source records attributed to R Tjian.

At least 19 recordsLinked to original sources
PubMed2001

Requirement of tissue-selective TBP-associated factor TAFII105 in ovarian development.

Transcription factor TFIID, composed of TBP and TAFII subunits, is a central component of the RNA polymerase II machinery. Here, we report that the tissue-selective TAFII105 subunit of TFIID is essential for proper development and function of the mouse ovary. Female mice lacking TAFII105 are viable but infertile because of a defect in folliculogenesis correlating with restricted expression of TAFII105 in the granulosa cells of the ovarian follicle. Gene expression profiling has uncovered a defective inhibin-activin signaling pathway in TAFII105-deficient ovaries. Together, these studies suggest that TAFII105 mediates the transcription of a subset of genes required for proper folliculogenesis in the ovary and establishes TAFII105 as a cell type-specific component of the mammalian transcriptional machinery.

Animals↗
PubMed2001

Transcriptional regulation in Drosophila: the post-genome challenge.

Drosophila melanogaster has long been at the forefront of studies of transcriptional regulation in animals. Many fundamental ideas--such as cis control elements that act over long distances, the regulation of development by hierarchical cascades of transcription factors, dosage compensation, and position effect variegation--originated from studies of the fruit fly. The recent completion of the euchromatic DNA sequence of Drosophila is another breakthrough. The sequence data highlight important unanswered questions. For example, only one-fifth of the 124 Mb of Drosophila euchromatic DNA codes for protein. The function of the remaining 100 Mb of mostly unique DNA is largely unknown. Some proportion of this non-reading frame DNA must encode the functional recognition sites targeted by the approximately 700 sequence-specific DNA binding proteins that regulate transcription in Drosophila, but what proportion? Most or very little? Promoter sequences by definition contain all of the cis information that specifies how gene transcription is regulated. However, it has been difficult to decipher this information and predict the patterns of RNA expression. How do we break this "transcriptional code"? Mechanistic studies, using simple model promoters, indicate that transcription is controlled by the coordinate action of sequence-specific DNA binding proteins interacting with the general transcriptional machinery via intermediary adapters and chromatin remodeling activities. How can we integrate this biochemical information with data from genome-wide studies to describe the generation of highly complex patterns of transcription? Here, we discuss recent studies that may point the way ahead. We also highlight difficulties that the field faces in dissecting transcriptional control in the post-genome era.

Animals↗
PubMed2001

Transcription factor MIZ-1 is regulated via microtubule association.

A synthetic drug, T113242, activates low-density lipoprotein receptor (LDLR) transcription in the presence of sterols. T113242 also covalently binds to beta-tubulin and induces microtubule depolymerization. The myc-interacting zinc finger protein (MIZ-1) associates with microtubules, can bind directly to the LDLR promoter, and can activate LDLR transcription. MIZ-1 also binds to the promoter and activates transcription of other T113242-induced genes such as alpha(2) integrin. Soft X-ray, indirect immunofluorescence, and green fluorescent protein time-lapse microscopy reveal that MIZ-1 is largely cytoplasmic but accumulates in the nuclei of HepG2 cells upon treatment with T113242. Thus, MIZ-1 appears to be regulated by association with microtubules and may activate gene transcription in response to changes in the cytoskeleton.

Active Transport, Cell Nucleus↗
PubMed2001

Transcriptional coactivator complexes.

The last two decades have witnessed a tremendous expansion in our knowledge of the mechanisms employed by eukaryotic cells to control gene activity. A critical insight to transcriptional control mechanisms was provided by the discovery of coactivators, a diverse array of cellular factors that connect sequence-specific DNA binding activators to the general transcriptional machinery, or that help activators and the transcriptional apparatus to navigate through the constraints of chromatin. A number of coactivators have been isolated as large multifunctional complexes, and biochemical, genetic, molecular, and cellular strategies have all contributed to uncovering many of their components, activities, and modes of action. Coactivator functions can be broadly divide into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes. Strikingly, several distinct coactivator complexes nonetheless share many subunits and appear to be assembled in a modular fashion. Such structural and functional modularity could provide the cell with building blocks from which to construct a versatile array of coactivator complexes according to its needs. The extent of functional interplay between these different activities in gene-specific transcriptional regulation is only now becoming apparent, and will remain an active area of research for years to come.

Acetyltransferases↗
PubMed2000

A TRF1:BRF complex directs Drosophila RNA polymerase III transcription.

It has been generally accepted that the TATA binding protein (TBP) is a universal mediator of transcription by RNA polymerase I, II, and III. Here we report that the TBP-related factor TRF1 rather than TBP is responsible for RNA polymerase III transcription in Drosophila. Immunoprecipitation and in vitro transcription assays using immunodepleted extracts supplemented with recombinant proteins reveals that a TRF1:BRF complex is required to reconstitute transcription of tRNA, 5S and U6 RNA genes. In vivo, the majority of TRF1 is complexed with BRF and these two proteins colocalize at many polytene chromosome sites containing RNA pol III genes. These data suggest that in Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a major role in RNA pol III transcription.

Amino Acid Sequence↗
PubMed2000

Structure and function of a human TAFII250 double bromodomain module.

TFIID is a large multiprotein complex that initiates assembly of the transcription machinery. It is unclear how TFIID recognizes promoters in vivo when templates are nucleosome-bound. Here, it is shown that TAFII250, the largest subunit of TFIID, contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides. The 2.1 angstrom crystal structure of the double bromodomain reveals two side-by-side, four-helix bundles with a highly polarized surface charge distribution. Each bundle contains an Nepsilon-acetyllysine binding pocket at its center, which results in a structure ideally suited for recognition of diacetylated histone H4 tails. Thus, TFIID may be targeted to specific chromatin-bound promoters and may play a role in chromatin recognition.

Acetylation↗
PubMed2000

Promoter-selective properties of the TBP-related factor TRF1.

The TATA-binding protein (TBP)-related factor 1 (TRF1) is expressed in a tissue-restricted fashion during Drosophila embryogenesis and may serve as a promoter-specific recognition factor that can replace TBP in regulating transcription. However, bona fide target promoters that would preferentially respond to TRF1 have remained elusive. Polytene chromosome staining, chromatin immunoprecipitation, direct messenger RNA analysis, and transient cotransfection assays identified the Drosophila gene tudor as containing a TRF1-responsive promoter. Reconstituted in vitro transcription reactions and deoxyribonuclease I footprinting assays confirmed the ability of TRF1 to bind preferentially and direct transcription of the tudor gene from an alternate promoter. Thus, metazoans appear to have evolved gene-selective and tissue-specific components of the core transcription machinery to regulate gene expression.

Animals↗
PubMed2000

Different functional domains of TAFII250 modulate expression of distinct subsets of mammalian genes.

The TATA box-binding protein-associated factors (TAFs) are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. In hamster ts13 cells, a single amino acid change in TAF(II)250, which disrupts its acetyl-transferase activity at the restrictive temperature, alters the transcription of specific genes involved in cell cycle control. Likewise, disruption of the amino-terminal kinase domain of TAF(II)250 results in transcriptional defects in ts13 cells. However, it was not known whether the acetyl-transferase or kinase domains of TAF(II)250 modulate specific classes of genes and whether these two domains regulate distinct subsets of genes. Here we have used high-density gene-profiling to identify mammalian transcripts that require either the TAF(II)250 acetyl-transferase or protein kinase function for proper expression. We found that transcription of at least 18% of genes are differentially expressed at the restrictive temperature. The promoter region of one of these genes was subsequently characterized, and both upstream elements as well as the core promoter were shown to be TAF(II)250 responsive. We also found that expression of approximately 6% of genes in ts13 cells requires a functional TAF(II)250 amino-terminal kinase domain, but only approximately 1% of these hamster genes also require the TAF(II)250 acetyl-transferase activity. Our results suggest that the two TAF(II)250 enzymatic activities are important for regulating largely nonoverlapping sets of genes involved in a wide range of biological functions in vivo.

Acetyltransferases↗
PubMed2000

TAFs revisited: more data reveal new twists and confirm old ideas.

Synthesis of messenger RNA by RNA polymerase II requires the combined activities of more than 70 polypeptides. Coordinating the interaction of these proteins is the basal transcription factor TFIID, which recognizes the core promoter and supplies a scaffolding upon which the rest of the transcriptional machinery can assemble. A multisubunit complex, TFIID consists of the TATA-binding protein (TBP) and several TBP-associated factors (TAFs), whose primary sequences are well-conserved from yeast to humans. Data from reconstituted cell-free transcription systems and binary interaction assays suggest that the TAF subunits can function as promoter-recognition factors, as coactivators capable of transducing signals from enhancer-bound activators to the basal machinery, and even as enzymatic modifiers of other proteins. Whether TAFs function similarly in vivo, however, has been an open question. Initial characterization of yeast bearing mutations in particular TAFs seemingly indicated that, unlike the situation in vitro, TAFs played only a minor role in transcriptional regulation in vivo. However, reconsideration of this data in light of more recent results from yeast and other organisms reveals considerable convergence between the models derived from in vitro experiments and those derived from in vivo studies. In particular, there is an emerging consensus that TAFs represent one of several classes of coactivators that participate in transcriptional activation in vivo.

Animals↗
PubMed1999

Three-dimensional structure of the human TFIID-IIA-IIB complex.

The multisubunit transcription factor IID (TFIID) is an essential component of the eukaryotic RNA polymerase II machinery that works in concert with TFIIA (IIA) and TFIIB (IIB) to assemble initiation complexes at core eukaryotic promoters. Here the structures of human TFIID and the TFIID-IIA-IIB complex that were obtained by electron microscopy and image analysis to 35 angstrom resolution are presented. TFIID is a trilobed, horseshoe-shaped structure, with TFIIA and TFIIB bound on opposite lobes and flanking a central cavity. Antibody studies locate the TATA-binding protein (TBP) between TFIIA and TFIIB at the top of the cavity that most likely encompasses the TATA DNA binding region of the supramolecular complex.

Binding Sites↗
PubMed1999

Purification of transcription cofactor complex CRSP.

Transcription of protein coding genes in metazoans involves the concerted action of enhancer binding proteins and the RNA polymerase II apparatus. The cross talk between these two classes of transcription factors is mediated by an elaborate set of cofactor complexes. For the activation of transcription by the promoter specificity protein 1 (Sp1), TATA binding protein-associated factors in the TFIID complex originally were identified as necessary coactivators, but the identity of additional cofactors required for activated transcription was unknown. Recently, we have reported the isolation and properties of a cofactor complex, CRSP (cofactor required for Sp1), which functions in conjunction with the TATA binding protein-associated factors to promote efficient activation of transcription by Sp1. CRSP contains unique subunits as well as polypeptides that are shared with other cofactor complexes. Here, we report a detailed purification protocol for the isolation of CRSP from human HeLa cells. Our purification strategy takes advantage of the ability of CRSP to bind Ni2+-nitrilotriacetic acid-agarose resin as well as other conventional chromatographic resins. We also describe a streamlined purification protocol that allows a more rapid and efficient means to isolate active CRSP.

Chromatography, Affinity↗
PubMed1999

Composite co-activator ARC mediates chromatin-directed transcriptional activation.

Gene activation in eukaryotes is regulated by complex mechanisms in which the recruitment and assembly of the transcriptional machinery is directed by gene- and cell-type-specific DNA-binding proteins. When DNA is packaged into chromatin, the regulation of gene activation requires new classes of chromatin-targeting activity. In humans, a multisubunit cofactor functions in a chromatin-selective manner to potentiate synergistic gene activation by the transcriptional activators SREBP-1a and Sp1. Here we show that this activator-recruited cofactor (ARC) interacts directly with several different activators, including SREBP-1a, VP16 and the p65 subunit of NF-kappaB, and strongly enhances transcription directed by these activators in vitro with chromatin-assembled DNA templates. The ARC complex consists of 16 or more subunits; some of these are novel gene products, whereas others are present in other multisubunit cofactors, such as CRSP, NAT and mammalian Mediator. Detailed analysis indicates that the ARC complex is probably identical to the nuclear hormone-receptor cofactor DRIP. Thus, ARC/DRIP is a large composite co-activator that belongs to a family of related cofactors and is targeted by different classes of activator to mediate transcriptional stimulation.

Amino Acid Sequence↗
PubMed1999

TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family.

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the "basal" transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in Drosophila. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans, Drosophila, Caenorhabditis elegans, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2's primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on Drosophila chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.

Amino Acid Sequence↗
PubMed1999

The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1.

Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation. Transcriptional activation by enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has remained unknown. Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r) approximately 700K) that contains nine subunits with M(r) values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1. CRSP p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS. Immunodepletion studies confirm that these subunits have an essential cofactor function. The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.

Antibodies↗
PubMed1998

TAFII mutations disrupt Dorsal activation in the Drosophila embryo.

In this study, we present evidence that the Dorsal activator interacts with limiting amounts of the TFIID complex in the Drosophila embryo. In vitro transcription reactions and protein binding assays implicate the TAFII110 and TAFII60 subunits of the TFIID complex in contributing to Dorsal-mediated activation. Mutations in TAFII110 and TAFII60 result in altered patterns of snail and twist transcription in embryos derived from dl/+ females. These results suggest that TAFIIs contribute to the activation of transcription in vivo and support the hypothesis that subunits of TFIID may serve as targets of enhancer binding proteins.

Animals↗
PubMed1998

Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro.

The promoter selectivity factor Sp1 often cooperates with other enhancer-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vitro the synergy observed in vivo between Sp1 and the sterol-regulated factor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter. Using a highly purified human transcription system, we found that chromatin, TAFs, and a novel SREBP-binding coactivator activity, which includes CBP, are all required to mediate full synergistic activation by Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activation by SREBP-1a and Sp1 in a dominant-negative fashion that is both chromatin- and activator-specific. Whereas recombinant CBP alone is not sufficient to mediate activation, a human cellular fraction containing CBP can support high levels of chromatin-dependent synergistic activation. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that selectively binds to the SREBP-1a activation domain and is capable of mediating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promoters in the context of chromatin.

Animals↗