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Biomedical subjects

R G Roeder

Publications and source records attributed to R G Roeder.

At least 19 recordsLinked to original sources
PubMed1999

The human homologue of Drosophila TRF-proximal protein is associated with an RNA polymerase II-SRB complex.

Mammalian RNA polymerase II holoenzymes are large complexes that have been reported to contain, in addition to RNA polymerase II, homologues of several yeast SRBs, various general transcription factors, and other polypeptides. On the basis of its copurification with an SRB-containing RNA polymerase II complex by conventional chromatography procedures, we have identified a human homologue of Drosophila TRF-proximal protein, designated hTRFP, and isolated its cognate cDNA. Antibody specific for SRB7 can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally, antibody specific for hTRFP can immunoprecipitate RNA polymerase II and SRB7. These data indicate that hTRFP is an integral component of an RNA polymerase II-SRB complex. Whereas the precise function of hTRFP remains to be determined, the hTRFP-containing RNA polymerase II-SRB complex supports basal level transcription and, relative to RNA polymerase II alone, enhances transcriptional activation by Gal4-VP16 in the presence of cofactor PC4. Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.

Amino Acid Sequence↗
PubMed1999

Human TFIIIC relieves chromatin-mediated repression of RNA polymerase III transcription and contains an intrinsic histone acetyltransferase activity.

Human TFIIIC is a multisubunit factor that is essential for transcription by RNA polymerase III on tRNA and virus-associated RNA genes and initiates preinitiation complex assembly by direct recognition of promoter elements. We show that highly purified TFIIIC, at concentrations above those sufficient for transcription of naked DNA templates, effectively relieves nucleosome-mediated repression on an in vitro-reconstituted chromatin template. Highly purified TFIIIC alone can bind to the A and B boxes of a tRNA gene within a chromatin template and, further, displays a histone acetyltransferase activity that is intrinsic to at least one (and probably three) of its subunits. The possibility of a direct link between TFIIIC-dependent chromatin transcription and acetyltransferase activities is suggested by the partial loss of these activities, but not DNA transcription activity, following pretreatment of TFIIIC with p-hydroxymercuribenzoic acid.

Acetyltransferases↗
PubMed1998

Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation.

Transcriptional activation in human cell-free systems containing RNA polymerase II and general initiation factors requires the action of one or more additional coactivators. Here, we report the isolation of cDNAs encoding two novel human transcriptional coactivators (p52 and p75) that are derived from alternatively spliced products of a single gene and share a region of 325 residues, but show distinct coactivator properties. p52 and p75 both show strong interactions with the VP16 activation domain and several components of the general transcriptional machinery. p52, like the previously described PC4, is a potent broad-specificity coactivator, whereas p75 is less active for most activation domains. These results suggest that p52 is a general transcriptional coactivator that mediates functional interactions between upstream sequence-specific activators and the general transcription apparatus, possibly through a novel mechanism.

Adaptor Proteins, Signal Transducing↗
PubMed1998

A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID.

In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivo either with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex.

Acetyltransferases↗
PubMed1998

OCA-B integrates B cell antigen receptor-, CD40L- and IL 4-mediated signals for the germinal center pathway of B cell development.

Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is regulated synergistically by the B-cell antigen receptor, CD40L and interleukin signaling pathways. Consistent with the requirement for multiple T cell-dependent signals to induce OCA-B, we find that OCA-B protein is highly expressed in germinal center B cells. Accordingly, germinal center formation is blocked completely in the absence of OCA-B expression in B cells, whereas the helper functions of OCA-B-deficient T cells are indistinguishable from controls. The requirement for OCA-B expression in B cells is germinal center specific since the development of primary B cell follicles, the marginal zone and plasma cells are all intact. Thus, OCA-B is the first example of a transcriptional coactivator that is both synergistically induced by and required for integration of signals that mediate cell fate decisions.

Adoptive Transfer↗
PubMed1998

Analysis of the role of TFIIE in transcriptional regulation through structure-function studies of the TFIIEbeta subunit.

The general transcription factor TFIIE plays important roles at two distinct but sequential steps in transcription as follows: preinitiation complex formation and activation (open complex formation), and the transition from initiation to elongation. The large subunit of human TFIIE (TFIIEalpha) binds to and facilitates the enzymatic functions of TFIIH, but TFIIE also functions independently from TFIIH. To determine functional roles of the small subunit of human TFIIE (TFIIEbeta), deletion mutations were systematically introduced into putative structural motifs and characteristic sequences. Here we show that all of these structures that lie within the central 227-amino acid region of TFIIEbeta are necessary and sufficient for both basal and activated transcription. We further demonstrate that two C-terminal basic regions are essential for physical interaction with both TFIIEalpha and single-stranded DNA, as well as with other transcription factors including the Drosophila transcriptional regulator Krüppel. In addition, we analyzed the effects of the TFIIEbeta deletion mutations on TFIIH-dependent phosphorylation of the C-terminal domain of RNA polymerase II and on wild type TFIIEbeta-driven basal transcription. Both responsible regions also mapped within the essential 227-amino acid region. Our results suggest that TFIIE engages in communication with both transcription factors and promoter DNA via the TFIIEbeta subunit.

Animals↗
PubMed1998

The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion.

Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor alpha (TRalpha) from ligand-treated HeLa (alpha-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRalpha, and these interactions are mediated through the C terminus of TRalpha and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors [vitamin D receptor, retinoic acid receptor alpha, retinoid X receptor alpha, peroxisome proliferation-activated receptor (PPAR) alpha, PPARgamma and, to a lesser extent, estrogen receptor] in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor alpha, PPARalpha, and PPARgamma. Consistent with these results, TRAP220 moderately stimulates human TRalpha-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRalpha) and in cell free transcription systems (TRalpha and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRalpha during the function of the TRalpha-TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily.

3T3 Cells↗
PubMed1998

3' IgH enhancer elements shift synergistic interactions during B cell development.

IgH gene expression is tightly controlled over the course of B cell development, B cell activation, and the subsequent differentiation of these cells into Ig-secreting plasmacytes. There are several transcriptional enhancers that map within and downstream of the IgH locus, and some of these have been clearly implicated in the developmental regulation of IgH gene assembly and expression. While some of the individual enhancers from this locus have been studied extensively, the functional interactions possible among this group of enhancers have been largely unexplored. In the present study, we have measured the transcriptional activities of combinations of enhancers introduced into B-lineage cell lines at several different developmental stages. We detected a developmental progression in which the 3' enhancers are initially inactive, then become strongly active through synergistic interactions, and finally achieve a strong level of activity with little interdependency. The relative contributions of E mu (the intron enhancer) and of the 3' enhancers also change as a function of developmental stage. We discuss these results in light of parallel studies of developmental changes in transcription factor requirements.

Animals↗
PubMed1998

Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle.

PTF (PSE-binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter approximately 1.3 micron) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription 'factories' where Br-UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only approximately 30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.

Amanitins↗
PubMed1998

A cofactor, TIP30, specifically enhances HIV-1 Tat-activated transcription.

Replication of HIV-1 requires the viral Tat protein, which increases the extent of transcription elongation by RNA polymerase II after activation at the single viral long terminal repeat (LTR) promoter. This effect of Tat on transcription requires Tat interactions with a 5' region (TAR) in nascent transcripts as well as Tat-specific cofactors. The present study identifies a cellular protein, TIP30, that interacts with Tat and with an SRB-containing RNA polymerase II complex both in vivo and in vitro. Coexpression of TIP30 specifically enhances transactivation by Tat in transfected cells, and immunodepletion of TIP30 from nuclear extracts abolishes Tat-activated transcription without affecting Tat-independent transcription. These results implicate TIP30 as a specific coactivator that may enhance formation of a Tat-RNA polymerase II holoenzyme complex.

Acetyltransferases↗
PubMed1998

A dynamic model for PC4 coactivator function in RNA polymerase II transcription.

Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAFII250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state.

Animals↗
PubMed1998

DNA topoisomerase I and PC4 can interact with human TFIIIC to promote both accurate termination and transcription reinitiation by RNA polymerase III.

A human TFIIIC-containing complex (operationally designated holo TFIIIC) has been isolated by immunoaffinity methods and further resolved into two components that are both required for promoter-directed transcription of the VA1 gene. One component, designated TFIIIC, contains 5 polypeptides previously ascribed to TFIIIC2 and 4 additional polypeptides that correspond to TFIIIC1. Included within the other component are factors, namely DNA topoisomerase I and PC4, previously shown to serve as coactivators for transcription by RNA polymerase II. Topoisomerase I and PC4 both enhance TFIIIC interactions with down-stream promoter regions and promote multiple, but not single, round transcription by RNA polymerase III from preformed preinitiation complexes. Novel functions for holo TFIIIC in transcription elongation and accurate termination events that could be important for efficient reinitiation are also described.

Coenzymes↗
PubMed1998

Transcription activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs.

In contrast to previous findings in cell-free systems reconstituted with partially purified metazoan factors, we demonstrate dramatic activation of transcription in a TBP-dependent but TAFII-independent manner in HeLa nuclear extracts immunodepleted of TBP and major TAFIIs. Single-round transcription assays reveal that TAFII-independent activation is manifested at the level of productive preinitiation complex formation and that TAFIIs actually impair functional preinitiation complex assembly in a core promoter-specific manner. Furthermore, TAFIIs appear to elevate absolute levels of transcription under multiple-round transcription conditions, presumably by facilitating secondary initiation events. Finally, human coactivator activities related to those in yeast RNA polymerase II/mediator complexes appear to function in unfractionated HeLa nuclear extracts.

Cell Extracts↗
PubMed1998

Novel cofactors and TFIIA mediate functional core promoter selectivity by the human TAFII150-containing TFIID complex.

TATA-binding protein-associated factors (TAFIIs) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAFII150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAFIIs within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAFII150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAFII150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAFII-dependent TATA-less promoter. Therefore, TAFII-promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAFII-mediated synergism between TATA and initiator elements (TIC-1) and TAFII-dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC- and TAFII-dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAFIIs through core promoter elements and TAFII-dependent activator function.

ARNTL Transcription Factors↗
PubMed1998

Involvement of TFIID and USA components in transcriptional activation of the human immunodeficiency virus promoter by NF-kappaB and Sp1.

The purified Rel/NF-kappaB (p50/p65) complex and Sp1 markedly activate transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a highly purified HeLa reconstituted transcription system. Transcriptional activation by NF-kappaB and Sp1 requires both TFIID and the USA fraction. The USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator activity, both by repressing the basal level of transcription and by potentiating activator function to yield large increases in the levels of transcription induction. Under limiting concentrations, PC2 and PC4 also show synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65 subunit of NF-kappaB is a potent activator when assayed as a Gal fusion in the reconstituted transcription system and interacts both with TATA-binding protein (TBP) and with several human TBP-associated factors (TAFs) that include TAFII250. The p65 activation domain mediates transcription activation in the presence of partially reconstituted TFIID species that include a minimal complex containing only TBP and TAFII250. These studies also show that, like USA components, TAFs can serve both to repress TBP-mediated transcription and, following activator interactions, to reverse the repression and effect a net increase in activity. Taken together, these data underscore the importance of both TAFs and specific USA-derived coactivators for optimal activation of the HIV-1 promoter, as well as certain parallels in their overall mechanisms of action.

DNA-Binding Proteins↗
PubMed1998

Coactivation by OCA-B: definition of critical regions and synergism with general cofactors.

Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements showed that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B was found to interact directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators.

Amino Acid Sequence↗
PubMed1997

Cloning of an inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1.

The transcription factor TFII-I has been shown to bind independently to two distinct promoter elements, a pyrimidine-rich initiator (Inr) and a recognition site (E-box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII-I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E-box elements. The primary structure of TFII-I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix-loop/span-helix homology. These unique structural features suggest that TFII-I may have the capacity for multiple protein-protein and, potentially, multiple protein-DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII-I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E-box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.

Adenoviridae↗