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R G Roeder

Publications and source records attributed to R G Roeder.

At least 37 records · Page 2Linked to original sources
PubMed2000

TIP30 has an intrinsic kinase activity required for up-regulation of a subset of apoptotic genes.

CC3 is a metastasis suppressor that inhibits metastasis of the variant small cell lung carcinoma (v-SCLC) by predisposing cells to apoptosis. The same protein was also reported as a cellular cofactor, TIP30, which stimulates HIV-1 Tat-activated transcription by interacting with both Tat and RNA polymerase II. We report here that TIP30/CC3 is a novel serine/threonine kinase. It phosphorylates the heptapeptide repeats of the C-terminal domain (CTD) of the largest RNA polymerase II subunit in a Tat-dependent manner. Amino acid substitutions in the putative ATP binding motif that abolish the TIP30 kinase activity also inhibit the ability of TIP30 to enhance Tat-activated transcription or to sensitize NIH 3T3 and v-SCLC cells to apoptosis. Furthermore, ectopic expression of TIP30/CC3 in v-SCLC cells induces expression of a number of genes that include the apoptosis-related genes Bad and Siva, as well as metastasis suppressor NM23-H2. These data demonstrate a molecular mechanism for TIP30/CC3 function and suggest a novel pathway for regulating apoptosis.

Acetyltransferases↗
PubMed2000

Isolation and characterization of monoclonal antibodies directed against subunits of human RNA polymerases I, II, and III.

Human nuclei contain three different RNA polymerases: polymerases I, II, and III. Each polymerase is a multi-subunit enzyme with 12-17 subunits. The localization of these subunits is limited by the paucity of antibodies suitable for immunofluorescence. We now describe eight different monoclonal antibodies that react specifically with RPB6 (also known as RPA20, RPB14.4, or RPC20), RPB8 (RPA18, RPB17, or RPC18), RPC32, or RPC39 and which are suitable for such studies. Each antibody detects one specific band in immunoblots of nuclear extracts; each also immunoprecipitates large complexes containing many other subunits. When used for immunofluorescence, antibodies against the subunits shared by all three polymerases (i.e., RPB6, RPB8) gave a few bright foci in nucleoli and nucleoplasm, as well as many fainter nucleoplasmic foci; all the bright foci were generally distinct from speckles containing Sm antigen. Antibodies against the two subunits found only in polymerase III (i.e., RPC32, RPC39) gave a few bright and many faint nucleoplasmic foci, but no nucleolar foci. Growth in two transcriptional inhibitors-5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole and actinomycin D-led to the redistribution of each subunit in a characteristic manner.

Animals↗
PubMed2000

Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells.

A novel multiprotein complex has recently been identified as a coactivator for transcriptional control of protein-encoding genes by RNA polymerase II in higher eukaryotic cells. This complex is evolutionarily related to the Mediator complex from yeast and, on the basis of its structural and functional characteristics, promises to be a key target of diverse regulatory circuits.

Animals↗
PubMed2000

Activator-dependent transcription from chromatin in vitro involving targeted histone acetylation by p300.

The transcriptional coactivator p300 shows physical and functional interactions with a diverse group of activators and contains an intrinsic acetyltransferase activity whose exact coactivator functions in the acetylation of nucleosomal histones versus other factors are poorly documented. Here, we show that p300 mediates acetyl-CoA-dependent transcription by GAL4-VP16 from a nucleosomal array template, that this involves p300 targeting by GAL4-VP16 and promoter-proximal histone acetylation prior to transcription, and that the affinities of different activators for p300 roughly correlate with corresponding levels of p300-dependent transcription. These results indicate that activators recruit p300 to nucleosomal templates by direct interactions and that bound p300 stimulates transcription, at least in part, by localized histone acetylation.

Acetyl Coenzyme A↗
PubMed2000

Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action.

The TRAP220 component of the TRAP/SMCC complex, a mammalian homologof the yeast Mediator that shows diverse coactivation functions, interacts directly with nuclear receptors. Ablation of the murine Trap220 gene revealed that null mutants die during an early gestational stage with heart failure and exhibit impaired neuronal development with extensive apoptosis. Primary embryonic fibroblasts derived from null mutants show an impaired cell cycle regulation and a prominent decrease of thyroid hormone receptor function that is restored by ectopic TRAP220 but no defect in activation by Gal4-RARalpha/RXRalpha, p53, or VP16. Moreover, haploinsufficient animals show growth retardation, pituitary hypothyroidism, and widely impaired transcription in certain organs. These results indicate that TRAP220 is essential for a wide range of physiological processes but also that it has gene- and activator-selective functions.

Animals↗
PubMed2000

The USA-derived transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts synergistically with other PCs.

PC2, the high-molecular weight constituent of the potent USA transcriptional coactivator fraction, was identified as a Mediator-like complex. Its composition resembles that of the TRAP/SMCC complex, but PC2 is distinguished by the prominent absence of the SRB10 and SRB11 kinase/cyclin pair, as well as several additional polypeptides. Furthermore, affinity-purified PC2, which lacks independent activity, acts in synergy with USA-derived coactivators PC3/topoisomerase I and PC4 to mediate the effects of a variety of activators (including VP16, the synthetic activator GAL4-AH, and the orphan nuclear receptor HNF4) and thus recapitulates partial USA coactivator function.

HeLa Cells↗
PubMed2000

HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF.

Histone acetyltransferases (HATs) play important roles in the regulation of gene expression. In this report, we describe the design, synthesis, and application of peptide CoA conjugates as selective HAT inhibitors for the transcriptional coactivators p300 and PCAF. Two inhibitors (Lys-CoA for p300 and H3-CoA-20 for PCAF) were found to be potent (IC(50) approximately = 0.5 microM) and selective (approximately 200-fold) in blocking p300 and PCAF HAT activities. These inhibitors were used to probe enzymatic and transcriptional features of HAT function in several assay systems. These compounds should be broadly useful as biological tools for evaluating the roles of HATs in transcriptional studies and may serve as lead agents for the development of novel antineoplastic therapeutics.

Acetyltransferases↗
PubMed1999

Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells.

The TATA-binding protein (TBP)-related factor TRF1, has been described in Drosophila and a related protein, TRF2, has been found in a variety of higher eukaryotes. We report that human (h)TRF2 is encoded by two mRNAs with common protein coding but distinct 5' nontranslated regions. One mRNA is expressed ubiquitously (hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted expression pattern and is extremely abundant in testis. In addition, we show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2. Neither recombinant human (rh)TRF2 nor the native flag.hTRF2-TFIIA complex is able to replace TBP or TFIID in basal or activated transcription from various RNA polymerase II promoters. Instead, rhTRF2, but not the flag.hTRF2-TFIIA complex, moderately inhibits basal or activated transcription in the presence of rhTBP or flag.TFIID. This effect is either completely (TBP-mediated transcription) or partially (TFIID-mediated transcription) counteracted by addition of free TFIIA. Neither rhTRF2 nor flag. hTRF2-TFIIA has any effect on the repression of TFIID-mediated transcription by negative cofactor-2 (NC2) and neither substitutes for TBP in RNA polymerase III-mediated transcription.

Amino Acid Sequence↗
PubMed1999

A novel RNA polymerase II-containing complex potentiates Tat-enhanced HIV-1 transcription.

The HIV-1-encoded Tat protein controls transcription elongation by increasing processivity of RNA polymerase II (Pol II). Here, we have identified a Tat stimulatory activity (Tat-SF) as a novel RNA Pol II-containing complex. Remarkably, Tat-SF contains the previously identified Tat cofactors Tat-SF1, P-TEFb and hSPT5/Tat-CT1, in addition to RNA Pol II and other unidentified polypeptides, but none of the SRB/MED proteins or other factors found associated with the previously described RNA Pol II holoenzyme complex. Tat-SF supports basal, Sp1-activated and Tat-activated transcription in a reconstituted system, and a Tat-SF-derived fraction lacking RNA Pol II can complement non-responsive RNA Pol II complexes for Tat-enhanced HIV-1 transcription, indicating that Tat-SF contains factors that are critical for Tat function. Both Tat-SF and RNA Pol II holoenzyme are present in HeLa nuclear extracts and each can be recruited to the HIV-1 promoter. Our results indicate that Tat-SF is a Tat cofactor-containing RNA Pol II complex whose recruitment to the promoter provides elongation factors important for Tat-enhanced HIV-1 transcription following TAR RNA synthesis.

Cell Nucleus↗
PubMed1999

Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III.

Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved 'physiological' buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br-UTP, before the resulting Br-RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that approximately 10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only approximately 2000 sites (diameter approximately 40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or 'factories', suggesting that different regions of the nucleus specialize in the transcription of different types of gene.

Binding Sites↗
PubMed1999

Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID.

Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes.

Animals↗
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

A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation.

A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified. This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex. The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD. These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes.

Cloning, Molecular↗
PubMed1999

Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators.

The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.

Amino Acid Sequence↗
PubMed1999

The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity.

Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III. Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC. hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits. Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription. hTFIIIC90 shows interactions with the hTFIIIC220, hTFIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III. These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC. We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.

Acetylation↗
PubMed1999

TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism.

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

Animals↗
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↗