HIV Tat

The CIITA coactivator is essential for transcriptional activation of MHC class II genes and mediates enhanced MHC class I transcription. We now report that CIITA contains an intrinsic acetyltransferase (AT) activity that maps to a region within the N-terminal segment of CIITA, between amino acids 94 and 132. The AT activity is regulated by the C-terminal GTP-binding domain and is stimulated by GTP. CIITA-mediated transactivation depends on the AT activity. Further, we report that, although constitutive MHC class I transcription depends on TAF(II)250, CIITA activates the promoter in the absence of functional TAF(II)250.

The protooncogene MYC regulates a variety of cellular processes, including proliferation and metabolism. Maintaining MYC at homeostatic levels is critical to normal cell function; overexpression drives many cancers. MYC stability is regulated through phosphorylation: phosphorylation at Thr58 signals degradation while Ser62 phosphorylation leads to its stabilization and functional activation. The bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator with intrinsic kinase and histone acetyltransferase (HAT) activities that activates transcription of key protooncogenes, includingMYC. We report that BRD4 phosphorylates MYC at Thr58, leading to MYC ubiquitination and degradation, thereby regulating MYC target genes. Importantly, BRD4 degradation, but not inhibition, results in increased levels of MYC protein. Conversely, MYC inhibits BRD4’s HAT activity, suggesting that MYC regulates its own transcription by limiting BRD4-mediated chromatin remodeling of its locus. The MYC stabilizing kinase, ERK1, regulates MYC levels directly and indirectly by inhibiting BRD4 kinase activity. These findings demonstrate that BRD4 negatively regulates MYC levels, which is counteracted by ERK1 activation.

A novel regulatory element which contributes to the regulation of quantitative, tissue-specific differences in gene expression has been found between -771 and -676 bp upstream of the major histocompatibility complex (MHC) class I gene, PD1. Molecular dissection of this element reveals the presence of two overlapping functional activities: an enhancer and a silencer. Distinct nuclear factors bind to the overlapping enhancer and silencer DNA sequence elements within the regulatory domain. The levels of factors binding the silencer DNA sequence in different cell types are inversely related to levels of class I expression; in contrast, factors binding the enhancer DNA sequence can be detected in all cells. In cultured cell lines, inhibition of protein synthesis leads to the rapid loss of silencer complexes, with a concomitant increase in both enhancer complexes and MHC class I RNA. From these data, we conclude that a labile silencer factor competes with a constitutively expressed, stable enhancer factor for overlapping DNA-binding sites; the relative abundance of the silencer factor contributes to establishing steady-state levels of MHC class I gene expression.The immune responses of all vertebrate species are mediated by molecules encoded within the major histocompatibility complex (MHC) (19). The MHC class I molecules, which serve as the targets of cellular immune responses and allograft rejection, are receptors for peptide antigens (3). These molecules are transmembrane glycoproteins consisting of a polymorphic heavy chain of 42 to 45 kDa and a nonpolymorphic light chain (P2-microglobulin) of 12 kDa.Consistent with their pivotal role in immune surveillance, the class I molecules are expressed on nearly all somatic tissues (28). However, their level of expression varies markedly among the tissues. The highest levels of cell surface expression of class I antigens occur in the lymphoid tissues: lymph node, peripheral blood lymphocytes, and spleen. Markedly lower levels are found on other somatic tissues, such as kidney and liver; there is little detectable cell surface class I antigen expression on germ line tissue or brain (28). In general, these differences reflect tissue-specific regulation of gene expression.Analysis of the 5' flanking regions of a number of class I genes has revealed the presence of a series of DNA elements capable of participating in the regulation of class I gene expression (Fig. 1A). Among these is a major enhancer, enhancer A, located at bp -180 to -170, which overlaps an interferon response element (15,20,27). A functional enhancer A is necessary for optimal expression of the downstream class I gene (13,15,18). A variety of trans-acting factors which bind to enhancer A have been identified, and their genes have been cloned (17, 21). These factors act as inducers of class I expression. Thus, KbF1 and RIIBP, both of which bind enhancer A, are associated with high levels of expression. Factors which bind specifically to the interferon response element and are induced by interferon have also b...

Transcription consists of a series of highly regulated steps: assembly of a preinitiation complex (PIC) at the promoter nucleated by TFIID, followed by initiation, elongation, and termination. The present study has focused on the role of the TFIID component, TAF7, in regulating transcription initiation. In TFIID, TAF7 binds to TAF1 and inhibits its intrinsic acetyl transferase activity. We now report that although TAF7 remains bound to TAF1 and associated with TFIID during the formation of the PIC, TAF7 dissociates from the PIC upon transcription initiation. Entry of polymerase II into the assembling PIC is associated with TAF1 and TAF7 phosphorylation, coincident with TAF7 release. We propose that the TFIID composition is dynamic and that TAF7 functions as a check-point regulator suppressing premature transcription initiation until PIC assembly is complete.MHC class I ͉ regulation ͉ preinitiation complex ͉ TFIID I n eukaryotic cells, expression of most protein-encoding genes depends on the RNA polymerase II (Pol II)-dependent transcription machinery. The RNA Pol II machinery assembled on the promoter is composed of distinct complexes that are responsible for effecting the sequential steps of transcription initiation and elongation (1-5). The first step in transcription is the recognition of the core promoter by the TFIID complex and the assembly of a preinitiation complex (PIC) through an ordered recruitment of the general transcription factors (GTFs) TFIIB and TFIIA, followed by the RNA Pol II holoenzyme, the mediator and the remaining GTFs, TFIIF, TFIIE, and TFIIH. Once the PIC has been assembled, transcription initiation ensues: local melting of the promoter DNA, formation of the first phosphodiester bond, followed by the synthesis of a short nascent RNA at which point the polymerase pauses. The initiation of transcription is accompanied by the phosphorylation of serine 5 in the C-terminal domain (CTD) heptad repeat of RNA Pol II (CTD) by the kinase subunit of TFIIH, CDK7 (6-9).Despite the extensive understanding of the general requirements of transcription, many details remain unresolved and there is considerable variation among different promoters and cell types. Promoter recognition is mediated by members of either the TFIID complex family (TFIID, TFIID-like) or the SAGA complex family (e.g., TFTC, PCAF, SAGA) (10-12). In yeast, 90% of gene expression is TFIID-dependent transcription; the remaining 10% is largely dependent on the SAGA complex (13). The TFIID complexes are composed of either the TATAbinding protein (TBP) or a TBP related protein (TRF1, TRF2) and several TBP-associated factors (TAFs) (14-17). The SAGA family complexes do not contain TBP or TBP-related proteins; rather, they contain a GCN5-related histone acetyl transferase (AT) subunit, several adapter and Spt proteins and a subset of TAFs. The composition of the TAFs present in these different complexes varies depending on the structure of the promoter and the cell cycle and tissue-specific gene expression requirement. For example, in yeas...

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.MHC class I genes ͉ regulation ͉ transcription initiation I n eukaryotic cells, expression of protein-encoding genes depends on the ordered recruitment of the general transcription factors (GTF) TFIID, TFIIB, and TFIIA to the promoter, followed by association of Pol II, the mediator, and the remaining GTFs, TFIIF, TFIIE, and TFIIH, to form a preinitiation complex (PIC) (1). Once PIC assembly is complete, transcription initiation ensues; Pol II with the elongation complex dissociate from the PIC (2, 3). A required step during transcription initiation is the phosphorylation of serine 5 in the carboxy terminal domain (CTD) heptad repeat of Pol II by the kinase subunit of TFIIH, CDK7 (4, 5), after which Pol II pauses to ensure proper pre-mRNA capping (6-9). The transition from pausing to elongation is facilitated by the P-TEFb elongation complex, which also mediates efficient elongation (10). P-TEFb consists of two subunits, cyclin T1 and the kinase CDK9, which phosphorylates serine 2 of the CTD, required for productive elongation and the recruitment of complexes involved in mRNA processing (splicing and polyadenylation) (10-15).Although the general mechanics of transcription have been characterized, relatively little is known about how the transitions from PIC assembly to initiation/pausing to elongation are regulated. Promoter recognition is largely mediated by TFIID, which is composed of the TATA binding protein (TBP) and over a dozen TBP associated factors (TAFs) (16,17,18). The largest TFIID component, TAF1, has both acetyltransferase (AT) and kinase activities (19,20). We demonstrated that TAF1 and its intrinsic acetyltransferase activity are essential for transcription of an MHC class I gene (21). Importantly, MHC class I transcription is inhibited both in vitro and in vivo by the viral transactivator, HIV Tat, which binds to the TAF1 AT domain, inhibiting its enzymatic activity (22, 23). TAF7, a cellular 55-kDa TFIID component (24,25), also binds to TAF1 inhibiting its AT activity and repressing MHC class I transcription (26) Significantly, we have demonstrated that TAF7 remains boun...

The general transcription factor, TFIID, consists of the TATAbinding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. One of the TAFs, TAFII250, has acetyltransferase (AT) activity that is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAFII250, a yeast two-hybrid library was screened with a TAFII250 segment (amino acids 848-1279) that spanned part of its AT domain and it's the domain that binds to the protein, RAP74. The TFIID component, TAFII55, was isolated and found to interact predominantly with the RAP74-binding domain. TAFII55 binding to TAFII250 inhibits its AT activity. Importantly, the addition of recombinant TAFII55 to in vitro transcription assays inhibits TAFII250-dependent MHC class I transcription. Thus, TAFII55 is capable of regulating TAFII250 function by modulating its AT activity.T ranscription mediated by RNA polymerase II (RNAP) requires the assembly of a preinitiation complex at the promoter. Assembly is initiated by the association of the general transcription factor (GTF), TFIID, with the promoter. Subsequently, the GTFs TFIIB, TFIIE, TFIIF, and TFIIH enter the complex; interactions between the GTFs and RNAP result in transcription initiation and elongation (1). Recruitment of RNAP to the promoter results in the phosphorylation of its carboxyl terminal domain (CTD) by TFIIH, which is required for initiation. Reinitiation depends on the prior dephosphorylation of the CTD by a phosphatase that is activated by TFIIF (2). More recently, it has been demonstrated that the function of the TFIID complex itself is regulated. TFIID consists of the TATA-binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that, together, participate in the assembly of the transcription preinitiation complex. Binding of TBP to its site on DNA is inhibited by the interaction of TBP with N-terminal polypeptide of the TFIID component, TAF II 250 (3). Thus, binding of TFIID to DNA seems to be regulated by allosteric changes in TAF II 250 conformation that expose the TBP-binding site.In addition to preinitiation complex assembly, transcription depends on a cascade of enzymatic activities. Included among these is the acetyltransferase (AT) activity of TAF II 250 (4, 5). As we have shown, TAF II 250 AT is essential for MHC class I promoter activity (4). In a temperature-sensitive TAF II 250 mutant-cell line, the class I promoter is active at the permissive temperature, but inactive at the restrictive temperature where TAF II 250 loses its AT activity. Furthermore, the HIV transactivator (Tat) represses transcription from the MHC class I promoter by binding to the AT domain of TAF II 250 and inhibiting its AT activity (4). These findings suggested that the AT activity of TAF II 250 might be a normal cellular target for regulation of transcription.In the pres...

Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the role of the core promoter in mediating regulation has been largely undefined. We report here that the class I core promoter consists of distinct elements that are differentially utilized in basal and activated transcription pathways. These pathways recruit distinct transcription factor complexes to the core promoter elements and target distinct transcription initiation sites. Class I transcription initiates at four major sites within the core promoter and is clustered in two distinct regions: "upstream" (؊14 and ؊18) and "downstream" (؉12 and ؉1). Basal transcription initiates predominantly from the upstream start site region and is completely dependent upon the general transcription factor TAF1 (TAF II 250). Activated transcription initiates predominantly from the downstream region and is TAF1 (TAF II 250) independent. USF1 augments transcription initiating through the upstream start sites and is dependent on TAF1 (TAF II 250), a finding consistent with its role in regulating basal class I transcription. In contrast, transcription activated by the interferon mediator CIITA is independent of TAF1 (TAF II 250) and focuses initiation on the downstream start sites. Thus, basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. We propose that transcription initiation at the core promoter is a dynamic process in which the mechanisms of core promoter function differ depending on the cellular environment.