Positive and negative TAF(II) functions that suggest a dynamic TFIID structure and elicit synergy with traps in activator-induced transcription.

Human transcription factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAF(II)s). To elucidate the structural organization and function of TFIID, we expressed and characterized the product of a cloned cDNA encoding human TAF(II)135 (hTAF(II)135). Comparative far Western blots have shown that hTAF(II)135 interacts strongly with hTAF(II)20, moderately with hTAF(II)150, and weakly with hTAF(II)43 and hTAF(II)250. Consistent with these observations and with sequence relationships of hTAF(II)20 and hTAF(II)135 to histones H2B and H2A, respectively, TFIID preparations that contain higher levels of hTAF(II)135 also contain higher levels of hTAF(II)20, and the interaction between hTAF(II)20 and hTAF(II)135 is critical for human TFIID assembly in vitro. From a functional standpoint, hTAF(II)135 has been found to interact strongly and directly with hTFIIA and (within a complex that also contains hTBP and hTAF(II)250) to specifically cooperate with TFIIA to relieve TAF(II)250-mediated repression of TBP binding and function on core promoters. Finally, we report a functional synergism between TAF(II)s and the TRAP/Mediator complex in activated transcription, manifested as hTAF(II)-mediated inhibition of basal transcription and a consequent TRAP requirement for both a high absolute level of activated transcription and a high and more physiological activated/basal transcription ratio. These results suggest a dynamic TFIID structure in which the switch from a basal hTAF(II)-enhanced repression state to an activator-mediated activated state on a promoter may be mediated in part through activator or coactivator interactions with hTAF(II)135.

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.

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.

Genomics and transcription analysis of human TFIID.

TFIID, a multisubunit protein comprised of TBP (TATA box-binding protein) and TAF(II)s (TBP-associated factors), has a central role in transcription initiation at class II promoters. TAF(II)s role as mediators of regulatory transcription factors, such as pRb and p53, and their involvement in signal transduction pathways suggest that some may participate in the control of cell proliferation and differentiation: therefore, they could be considered potential protooncogenes or antioncogenes. With the aim of starting to analyse these potential roles, we have determined the genomic position of nine human TAF(II) genes (TAF[II]250, TAF[II]135, TAF[II]100, TAF[II]80, TAF[II]55, TAF[II]43, TAF[II]31, TAF[II]28, TAF[II]20/15) and of two previously unknown sequences related to TAF(II)250 and TAF(II)31, respectively. Except for those encoding TAF(II)250 and TAF(II)31, these genes are present in a single copy and, with the exclusion of those for TAF(II)43 and TAF(II)28 (both at 6p21), are localized in different segments of the genome. Indeed, six of them map to a chromosomal region commonly altered in specific neoplasias, which defines them as candidates for involvement in oncogenesis. Our experiments also demonstrate that TAF(II) transcripts are synthesized ubiquitously, mostly at low levels similar to those of TBP. Interestingly, the amount of the major mRNA species detected by TAF(II)20/15 cDNA is higher, which suggests that the polypeptide it encodes may also perform functions independently of TFIID. TAF(II) isoforms, indicated by additional bands on Northern blots, may play a role in modulation of TFIID function. These data will be useful for analysing variations of TAF(II) mRNA phenotype during cell proliferation, differentiation and development, both normal and pathological.

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.

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.

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.

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.

Mitotic regulation of TFIID: inhibition of activator-dependent transcription and changes in subcellular localization.

Mitosis in higher eukaryotes is accompanied by a general inhibition of transcription. To begin to understand the mechanisms underlying this inhibition we have examined the behavior of the general transcription factor TFIID during mitosis. Immunocytochemistry and subcellular fractionation studies indicate that the majority of TFIID is displaced from the disassembling prophase nucleus to the mitotic cytoplasm around the time of nuclear envelope breakdown. However, a subpopulation of TFIID remains associated tightly with the condensed mitotic chromosomes. Metabolic labeling of mitotic cells revealed that several subunits of TFIID undergo mitosis-specific phosphorylation, but in spite of these changes, the TFIID complex remains intact. Functional analysis of purified TFIID from mitotic cells shows that phosphorylated forms are unable to direct activator-dependent transcription, but that this activity is restored upon dephosphorylation. These results demonstrate that TFIID regulation by phosphorylation is likely to have an important role in mitotic inhibition of RNA polymerase II transcription. In addition, they suggest a mechanism for regulating gene expression through the selective disruption of polymerase II promoter structures during mitosis.

Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors.

Human transcription initiation factor TFIID is composed of the TATA-binding polypeptide (TBP) and at least 13 TBP-associated factors (TAFs) that collectively or individually are involved in activator-dependent transcription. To investigate protein-protein interactions involved in TFIID assembly and in TAF-mediated activator functions, we have cloned and expressed cDNAs encoding human TAFII80 and TAFII31. Coimmunoprecipitation assays showed that TAFII80 interacted with TAFII250, TAFII31, TAFII20, and TBP, but not with TAFII55. Similar assays showed that TAFII80 interacted with TFIIE alpha and with TFIIF alpha (RAP74) but not with TFIIB, TFIIE beta, or TFIIF beta (RAP30). Further studies with TAFII80 mutations revealed three distinct interaction domains which fall within regions conserved in human TAFII80, Drosophila TAFII60, and yeast TAFII60. The N terminus of TAFII80 (residues 1-100) interacts with both TAFII31 and TAFII20, while two C-terminal regions are involved, respectively, in interactions with TAFII250 and TFIIF alpha (RAP74) (residues 203-276) and with TBP and TFIIE alpha (residues 377-505). The interactions between TAFII80 and general factors TFIIE alpha and TFIIF alpha (RAP74) could be important for recruitment of GTFs during activator-dependent transcription. Because TAFs 80, 31, and 20 show sequence similarities to histones H4, H3, and H2B, as well as some parallel interactions, this subset of TAFs may form a related core structure within TFIID.

Transcription of a quail gene expressed in embryonic retinal cells is shut off sharply at hatching.

The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.

Down regulation by p60v-src of genes specifically expressed and developmentally regulated in postmitotic quail neuroretina cells.

The avian neuroretina (NR) is composed of photoreceptors and different neurons that are derived from proliferating precursor cells. Neuronal differentiation takes place after terminal mitosis. We have previously shown that differentiating NR cells can be induced to proliferate by infection with Rous sarcoma virus (RSV) and that cell multiplication requires expression of a functional v-src gene. We speculated that the quiescence of NR cells could be determined by specific genes. Cell proliferation could then result from the negative regulation of these genes by the v-src protein. By differential hybridization of a cDNA library, we isolated eight clones corresponding to genes expressed in postmitotic NR cells from 13-day-old quail embryos, transcriptional levels of which are significantly reduced in NR cells induced to proliferate by tsNY68, an RSV mutant with temperature-sensitive mitogenic activity. Partial sequencing analysis indicated that one RNA encoded the calmodulin gene, whereas the other seven showed no similarity to known sequences. By using v-src mutants that induce NR cell proliferation in the absence of transformation, we showed that transcription of six genes was negatively regulated by the v-src protein and that of four genes was correlated with NR cell quiescence. We also report that a subset of genes are specifically transcribed in neural cells and developmentally regulated in the NR. These results indicate that the v-src protein regulates expression of genes likely to play a role in the control of neural cell growth or differentiation.

Evidence for two distinct adenylate cyclase catalysts in rat brain.

The Lubrol-soluble adenylate cyclase activity of brain synaptosomal membranes appeared, upon gel filtration or sucrose gradient centrifugation, as two overlapping peaks. Fractions corresponding to the peak of the largest Stokes radius (Biogel pool 1) or highest s value (gradient pool 1) contained an adenylate cyclase activity which could be detected whatever the enzyme assay conditions. In contrast, in fractions from the second peak (Biogel pool 2 or gradient pool 2), forskolin was needed to reveal adenylate cyclase activity. The enzyme activity of each Biogel pool was retained by forskolin-agarose and eluted by forskolin with a 34-83% yield. A polypeptide of 155 kDa made up 80% of the forskolin-agarose eluate 1, whereas it was almost absent from eluate 2. Since data from various groups point to the 155 kDa polypeptide as a brain adenylate cyclase catalyst, still another distinct catalyst of lower molecular mass is likely to be present in brain.