Biomedical subjects

R G Roeder

Publications and source records attributed to R G Roeder.

At least 73 records · Page 4Linked to original sources

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↗

PubMed1997

Interaction between cell cycle regulator, E2F-1, and NF-kappaB mediates repression of HIV-1 gene transcription.

The NF-kappaB/Rel family of transcription factors is one of the main targets of cytokines and other agents that induce HIV-1 gene expression. Some of these extracellular stimuli arrest cells in the G1 phase of the mitotic division cycle and modulate the activity of the tumor suppressor protein Rb and its partner E2F-1. Earlier studies indicated that E2F-1, a transcription factor that stimulates expression of S-phase-specific genes, is able to repress transcription directed by the human immunodeficiency virus (HIV-1) type-1 promoter in a variety of cells, including those of glial and lymphocytic origin. Here, we demonstrate that E2F-1 may regulate the activity of the HIV-1 long terminal repeat through its ability to bind sequences in the NF-kappaB enhancer region and to interact with the NF-kappaB subunit, p50. Gel retardation and methylation interference assays show that E2F-1 is able to bind specifically to a site embedded within the two NF-kappaB elements. Gel retardation/immunoblot analysis using purified E2F-1 and p50 homodimers reveals the presence of complexes containing both proteins. Affinity chromatography and co-immunoprecipitation assays provide evidence for direct interaction of E2F-1 and p50 in the absence of their DNA target sequences. In vitro transcription assay demonstrates that E2F-1 represses NF-kappaB mediated transcription in a cell-free system. Functional studies in Jurkat T lymphocytic cells point to the importance of both the E2F and NF-kappaB binding sites in E2F-1 mediated repression of HIV-1 promoter, in vivo. The results of this study suggest that NF-kappaB activity may be regulated by its interaction with the cell cycle regulatory protein, E2F-1.

Binding Sites↗

PubMed1997

The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors.

Octamer binding transcription factors (Oct factors) play important roles in activation of transcription of various genes but, in some cases, require cofactors that interact with the DNA binding (POU) domain. In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein. MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). Further analyses showed that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner. MAT1-containing TFIIH was also shown to interact with POU domains of Oct-1 and Oct-2. MAT1 is shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2. Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK. However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific, suggesting that, in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo. These results suggest (i) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (ii) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (iii) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions.

Amino Acid Sequence↗

PubMed1997

Functional significance of the TATA element major groove in transcription initiation by RNA polymerase II.

The binding of TFIID to the TATA element initiates assembly of a preinitiation complex and thus represents one of the most important steps for transcriptional regulation. The fact that the TATA binding protein (TBP), a subunit of TFIID, exclusively contacts the minor groove of the TATA element led us to ask whether the major groove of the TATA element plays any role in transcription initiation or its regulation. Our results show that modifications of the major groove of the TATA element in the adenovirus major late promoter have no effect on TFIID binding affinity or on transcription in a cell-free system reconstituted with purified factors. However, major groove modifications do decrease the levels of both basal and activator-mediated transcription in unfractionated nuclear extracts, indicating that the intact structure of the major groove of the TATA element is functionally important for transcription initiation in a more physiological context.

Adenoviridae↗

PubMed1997

Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition.

Transcription by RNA polymerase III (Pol III) requires multiple general initiation factors that, in isolated form, assemble onto the promoter in an ordered fashion. Here, it is shown that all components required for transcription of the VA1 and tRNA genes, including TFIIIB, TFIIIC, and RNA Pol III, can be coimmunopurified from a HeLa cell line that constantly expresses a FLAG epitope-tagged subunit of human RNA Pol III. This finding of an RNA Pol III "holoenzyme" suggests similarities between transcription initiation by RNA Pol II and RNA Pol III and has led to the identification of a novel general initiation factor (TDF, translation dependent factor) that is present within the holoenzyme. TDF is selectively inactivated during protein synthesis inhibition by cycloheximide and at a late stage of adenovirus infection, thus accounting for the loss of RNA Pol III-mediated transcription of the tRNA and VA RNA genes under these conditions. On the basis of these observations, possible mechanisms for the global regulation of transcription by RNA Pol III and for disassembly of RNA Pol III initiation complexes are proposed.

Coenzymes↗

PubMed1997

Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.

The tumor suppressor p53 exerts antiproliferation effects through its ability to function as a sequence-specific DNA-binding transcription factor. Here, we demonstrate that p53 can be modified by acetylation both in vivo and in vitro. Remarkably, the site of p53 that is acetylated by its coactivator, p300, resides in a C-terminal domain known to be critical for the regulation of p53 DNA binding. Furthermore, the acetylation of p53 can dramatically stimulate its sequence-specific DNA-binding activity, possibly as a result of an acetylation-induced conformational change. These observations clearly indicate a novel pathway for p53 activation and, importantly, provide an example of an acetylation-mediated change in the function of a nonhistone regulatory protein. These results have significant implications regarding the molecular mechanisms of various acetyltransferase-containing transcriptional coactivators whose primary targets have been presumed to be histones.

Acetylation↗

PubMed1997

Considerations of transcriptional control mechanisms: do TFIID-core promoter complexes recapitulate nucleosome-like functions?

The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities. Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins. These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter. Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID. We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions. Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

Animals↗

PubMed1997

An RNA polymerase III-defective mutation in TATA-binding protein disrupts its interaction with a transcription factor IIIB subunit in drosophila cells.

A subunit of the Drosophila RNA polymerase III transcription factor IIIB (TFIIIB) complex has been identified using antibodies directed against the analogous human protein, hIIIB90. This protein has an apparent molecular mass of 105 kDa and has been designated dTAFIII105. Drosophila S-2 cell extracts that were immunodepleted of dTAFIII105 were substantially reduced in their capacity to support tRNA gene transcription. A protein (far Western) blot analysis revealed that dTAFIII105, present in a TFIIIB fraction, directly interacts with TATA-binding protein (TBP). Coimmunoprecipitation assays demonstrated that this protein associates with TBP in S-2 cell extracts. Our previous studies have identified a mutation at position 332 within Drosophila TBP that changes a highly conserved arginine residue to a histidine residue, which renders it specifically defective in its ability to support RNA polymerase III transcription in S-2 cells (Trivedi, A., Vilalta, A., Gopalan, S., and Johnson, D. L. (1996) Mol. Cell. Biol. 16, 6909-6916). We further demonstrate that extracts prepared from a stable cell line expressing epitope-tagged wild-type TBP exhibit an increase in tRNA gene transcription, whereas extracts derived from cells expressing the mutant TBP protein do not. Coimmunoprecipitation assays and far Western blot analysis demonstrate that this mutation in TBP abolishes its ability to stably interact with dTAFIII105. Thus, we have identified both a Drosophila protein that is directly associated with TBP in the TFIIIB complex, dTAFIII105, and an amino acid residue within the highly conserved carboxyl-terminal region of TBP that is critical for dTAFIII105-TBP interactions.

Animals↗

PubMed1997

Synergistic activation of transcription by CBP and p53.

The tumour suppressor p53 is a transcriptional regulator whose ability to inhibit cell growth is dependent upon its transactivation function. Here we demonstrate that the transcription factor CBP, which is also implicated in cell proliferation and differentiation, acts as a p53 coactivator and potentiates its transcriptional activity. The amino-terminal activation domain of p53 interacts with the carboxy-terminal portion of the CBP protein both in vitro and in vivo. In transfected SaoS-2 cells, CBP potentiates activation of the mdm-2 gene by p53 and, reciprocally, p53 potentiates activation of a Gal4-responsive target gene by a Gal4(1-147)-CBP(1678-2441) fusion protein. A double point mutation that destroys the transactivation function of p53 also abolishes its binding to CBP and its synergistic function with CBP. The ability of p53 to interact physically and functionally with a coactivator (CBP) that has histone acetyltransferase activity and with components (TAFs) of the general transcription machinery indicates that it may have different functions in a multistep activation pathway.

Adenovirus E1A Proteins↗

PubMed1997

RNA polymerase III transcription repressed by Rb through its interactions with TFIIIB and TFIIIC2.

The retinoblastoma susceptibility gene product (Rb) generally represses RNA polymerase III (Pol III)-directed transcription. This implies that Rb interacts with essential transcription factors. Mutations in either the A or B subdomains in the Rb pocket interfere with Rb-mediated repression of Pol III-directed transcription, which indicates that both subdomains are directly involved in this activity. Addition of either purified TFIIIB or purified TFIIIC2 partially relieves Rb-mediated repression and restores activity to nuclear extracts that had been depleted of essential factors by binding to Rb. Pull down and coimmunoprecipitation experiments as well as functional assays indicate that Rb interacts with both TFIIIB and TFIIIC2 and that the A subdomain is primarily required for binding TFIIIB and the B subdomain for binding TFIIIC2. While Rb interacts with both factors, the A subdomain is more important than the B subdomain in directing Rb-mediated repression, and TFIIIB is the principal target of that activity.

Amino Acid Sequence↗

PubMed1997

CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues.

Both the TATA and CCAAT boxes are widespread promoter elements and their binding proteins, TBP and NF-Y, are extremely conserved in evolution. NF-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YB and NF-YC contain a putative histone-like motif, a domain also present in TBP-associated factors (TAFIIs) and in the subunits of the transcriptional repressor NC2. Immunopurification of holo-TFIID with anti-TBP and anti-TAFII100 antibodies indicates that a fraction of NF-YB associates with TFIID in the absence of NF-YA. Sedimentation velocity centrifugation experiments confirm that two pools of NF-YB, and most likely NF-YC, exist: one associated with NF-YA and binding to the CCAAT box; another involved in high molecular weight complexes. We started to dissect NF-Y-TFIID interactions by showing that: (i) NF-YB and NF-YC interact with TBP in solution, both separately and once bound to each other; (ii) short stretches of both NF-YB and NF-YC located within the evolutionary conserved domains, adjacent to the putative histone fold motifs, are necessary for TBP binding; (iii) TBP single amino acid mutants in the HS2 helix, previously shown to be defective in NC2 binding, are also unable to bind NF-YB and NF-YC.

Amino Acid Sequence↗

PubMed1997

Three human RNA polymerase III-specific subunits form a subcomplex with a selective function in specific transcription initiation.

Transcription by RNA polymerase III involves recruitment of the polymerase by template-bound accessory factors, followed by initiation, elongation, and termination steps. An immunopurification approach has been used to demonstrate that human RNA Pol III is composed of 16 subunits, some of which are apparently modified in HeLa cells. Partial denaturing conditions and sucrose gradient sedimentation at high salt result in the dissociation of a subcomplex that includes hRPC32, hRPC39, and hRPC62. Cognate cDNAs were isolated and shown to encode three subunits that are specific to RNA Pol III and homologous to three yeast subunits. The human RNA Pol III core lacking the subcomplex functions in transcription elongation and termination following nonspecific initiation on a tailed template, but fails to show promoter-dependent transcription initiation in conjunction with accessory factors. The capability for specific transcription initiation can be restored either by the natural subcomplex or by a stable subcomplex composed of recombinant hRPC32, hRPC39, and hRPC62 polypeptides. One component (hRPC39) of this subcomplex interacts physically with both hTBP and hTFIIIB90, two subunits of human RNA Pol III transcription initiation factor IIIB. These data strongly suggest that the hRPC32-hRPC39-hRPC62 subcomplex directs RNA Pol III binding to the TFIIIB-DNA complex via the interactions between TFIIIB and hRPC39.

Amino Acid Sequence↗

PubMed1997

Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro.

Mammalian cells contain activities that amplify the effects of activators on class II gene transcription in vitro. The molecular identity of several of these cofactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity. PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID. This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD+, probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.

Animals↗

PubMed1997

Critical role of the second stirrup region of the TATA-binding protein for transcriptional activation both in yeast and human.

We previously identified three TATA-binding protein (TBP) point mutations (L114K, L189K, and K211L) that have severe effects on transcriptional activation by acidic activators, but no effect on basal transcription, in a yeast-derived TBP-dependent in vitro transcription system (Kim, T. K., Hashimoto, S., Kelleher, R. J., III, Flanagan, P. M., Kornberg, R. D., Horikoshi, M., and Roeder, R. G. (1994) Nature 369, 252-255). These activation defects were also demonstrated in vivo in yeast cells (Lee, M., and Struhl, K. (1995) Mol. Cell. Biol. 15, 5461-5469). Here, the transcriptional activities of these and other TBP mutations were examined in human by both in vitro and in vivo assays. Mutations L189K and E188K, which lie in the second stirrup region of TBP, show defective activation by acidic activators both in yeast and human. Somewhat surprisingly, mutations L114K and K211L have almost no demonstrable effect on activation by acidic activators in human, in contrast to their severe effects on defective activator responses in yeast. The implications of these results for TBP structure and function are discussed.

DNA-Binding Proteins↗

PubMed1997

Specific interactions and potential functions of human TAFII100.

Human transcription initiation factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). To investigate the structural organization and function of TFIID, we have cloned and expressed a DNA encoding the third largest human TFIID subunit, hTAFII100. Immunoprecipitation studies demonstrate that hTAFII100 is an integral subunit that is associated with all transcriptionally-competent forms of TFIID. They further suggest that at least part of the N-terminal region lies on the surface of TFIID, while a C-terminal region containing conserved WD-40 repeats appears inaccessible. Both in vivo and in vitro assays indicate that hTAFII100 interacts strongly with the histone H4-related hTAFII80 and the histone H3-related hTAFII31, as well as a stable complex comprised of both hTAFII80 and hTAFII31. Apparently weaker interactions of hTAFII100 with TBP, hTAFII250, hTAFII28, and hTAFII20, but not hTAFII55, also have been observed. These results suggest a role for hTAFII100 in stabilizing interactions of TAFs, especially the histone-like TAFs, in TFIID. In addition, functional studies show that anti-hTAFII100 antibodies selectively inhibit basal transcription from a TATA-less initiator-containing promoter, relative to a TATA-containing promoter, suggesting a possible core promoter-specific function for hTAFII100.

Amino Acid Sequence↗

PubMed1997

Drosophila TAF(II)230 and the transcriptional activator VP16 bind competitively to the TATA box-binding domain of the TATA box-binding protein.

The transcription initiation factor TFIID, consisting of the TATA box-binding protein (TBP) and many TBP-associated factors (TAFs), plays a central role in both basal and activated transcription. An intriguing finding is that the 80-residue N-terminal region of Drosophila TAF(II)230 [dTAF(II)230-(2-81)] can bind directly to TBP and inhibit its function. Here, studies with mutated forms of TBP demonstrate that dTAF(II)230-(2-81) binds to the concave surface of TBP, which is important for TATA box binding. Previously, it was reported that a point mutation (L114K) on this concave surface destroys the ability of TBP to bind VP16 and to mediate VP16-dependent activation in vitro, but has no effect on basal transcription. Importantly, the same TBP mutation eliminates TBP binding to dTAF(II)230-(2-81). Consistent with these effects of the L114K mutation, dTAF(II)230-(2-81) and the VP16 activation domain compete for binding to wild-type TBP. These results indicate that transcriptional regulation may involve, in part, competitive interactions between transcriptional activators and TAFs on the TBP surface.

Amino Acid Sequence↗

PubMed1997

The ts13 mutation in the TAF(II)250 subunit (CCG1) of TFIID directly affects transcription of D-type cyclin genes in cells arrested in G1 at the nonpermissive temperature.

The general transcription initiation factor TFIID contains the TATA-binding protein (TBP) and TBP-associated factors (TAFs) implicated in the function of gene-specific activators. Previous studies have indicated that a hamster cell line (ts13) with a point mutation in the TAF(II)250/CCG1 (TAF(II)250) gene shows temperature-sensitive expression of a subset of genes and arrests in late G1 at 39.5 degrees C. Here, we report the identification of cell cycle-specific (G1-specific) genes that appear to be regulated directly through TAF(II)250 both in vivo and in vitro. Transcription rates of several cell cycle-regulatory genes were determined by run-on assays in nuclei from ts13 cells grown at permissive (33 degrees C) and nonpermissive (39.5 degrees C) temperatures. Temperature-dependent differences in transcription rates were observed for cyclin A, D1, and D3 genes. In transient-transfection assays, the human cyclin D1 promoter fused to a luciferase reporter showed a temperature-dependent reduction in activity in ts13 cells but not in parental BHK cells. In in vitro assays, upstream sequence-dependent transcription from the human cyclin D1 promoter was significantly reduced in ts13 nuclear extracts preincubated at 30 degrees C but not in similarly treated BHK nuclear extracts, and transcription in the ts13 extract was restored by addition of an affinity-purified human TFIID. Preincubation of the ts13 nuclear extracts did not affect the function of several GAL4-activation domain fusion proteins (GAL4-VP16, GAL4-p65, and GAL4-p53) on either the adenovirus major late or cyclin D1 core promoter bearing GAL4 sites, further indicating that the effect of the TAF(II)250 mutation is both core promoter and activator specific.

Animals↗

PubMed1996

Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain.

The protein Tat is encoded by the HIV-1 genome and is essential for viral replication because of its activation of viral transcription. Tat enhances the ability of RNA polymerase II (Pol II) to move long distances down the DNA through a poorly understood mechanism that involves its binding the to the 5' end of the nascent HIV-1 transcript. It has been suggested that the stimulation of transcript elongation by conventional DNA-binding activators may involve phosphorylation of the carboxy-terminal domain (CTD) of Pol II by the transcription factor TFIIH through the associated CAK kinase. Here we show that Tat-enhanced HIV-1 transcription in vitro requires both TFIIH and the CTD of Pol II. In addition, Tat, through its activation domain, both interacts with a functional TFIIH-containing complex and stimulates phosphorylation of a CTD-containing substrate by the TFIIH kinase. Under conditions that jointly restrict transcriptional elongation and TFIIH-mediated CTD phosphorylation, Tat stimulates both these activities. Furthermore, RNA synthesis is required for Tat to stimulate phosphorylation of the CTD when it is part of an initiation complex, as expected from Tat's interaction with viral transcripts. Thus, stimulation of Pol II elongation by Tat may involve direct effects on TFIIH-mediated CTD phosphorylation.

Binding Sites↗