Regulation of MHC class I gene transcription is mediated by the coordinate activities of the basal promoter and upstream regulatory elements, to achieve tissue-specific levels of expression which are further dynamically modulated in response to extracellular signals. The recent research focus of the laboratory has been to define the critical sequence organization of the basal promoter, the transcription initiation complexes that are required for transcription of the basal promoter, and upstream elements that modulate basal promoter activity. The class I basal promoter consists of three elements: a TATAA box, an initiator (Inr), and a novel S-box element. The relative usage of TATAA and Inr elements varies among different cell types, but neither element is absolutely required in any cell type tested. Two sites, 3' of the TATAA box and 5' of the Inr, do appear to be necessary for basal promoter activity. The function of these elements in vivo is being examined in a series of transgenic mouse constructs. Transcription of the basal class I promoter -whether through the TATAA or Inr elements - depends on a functional TAFII250, a component of the TFIID complex which 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. TAFII250 is required for the transcription of a subset of genes, including those invovled in cell cycle and MHC class I. The tsBN462 cell line contains a temperature sensitive mutation of TAFII250, and differentially arrests transcription of many, but not all genes, at the restrictive temperature. We have found that the core promoter of MHC class I gene requires TAFII250. This dependence can be overcome by select upstream regulatory elements but not by core promoter elements. Thus, the coactivator CIITA rescues the basal promoter from the TAFII250 requirement, whereas introduction of a canonical TATAA box does not. Similarly, the SV40 core promoter requires TAFII250, but the presence of the 72bp enhancer overcomes this requirement. Further, the SV40 72 bp enhancer when placed upstream of the core class I promoter renders it independents of TAFII250. These data suggest that the assembly of transcription initiation complexes is dynamic and can be modulated by specific transcription factors. In support of this hypothesis, we find that cell type specific enhanceosomes contribute to the regulation of class I transcription. In particular, the interferon inducible regulator of MHC class II expression, CIITA, is a potent co-activator of class I expression. Coactivation by CIITA requires both the interferon response element and the CRE; in contrast, neither CBP, PCAF nor p300 affect class I transcription.TAFII250 is known to have acetyl transferase (AT) activity. We have shown AT activity 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 (848-1279 aa) that spanned both the AT domain (848-1120) and the RAP74 binding domain (1120-1279). The TFIID component, TAFII55, was isolated and found to interact with the RAP74-binding domain. Surprisingly, TAFII55 binding to TAFII250 inhibits its AT activity. Furthermore, addition of recombinant TAFII55 to in vitro transcription assays inhibits TAFII250-dependent transcription. Thus, TAFII55 regulates TAFII250 function by modulating its AT activity.
