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 roles of the core promoter and downstream elements in mediating regulation have been largely undefined. 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. Basal transcription is completely dependent upon the general transcription factor TAF1 whereas activated transcription is TAF1 independent. We have also found that the downstream region of the MHC class I promoter region, between +1 and +32 bp, contains three novel regulatory elements. One of the elements functions to increase transcription. The other two elements, DPE-L1 and DPE-L2 have sequence homology with previously characterized DPE elements, but are mechanistically and functionally distinct from other described DPE elements. Under constitutive, TAF1-dependent conditions, the two DPE-L's act in concert to up-regulate promoter activity However, under activated TAF1-independent conditions, only one of the element functions independently as an enhancer. Thus, the downstream regulatory elements associated with the class I promoter function to fine-tune and integrate both intracellular and extracellular signaling pathways to ensure the appropriate level of MHC class I transcription to maintain immune homeostasis. To understand these mechanisms in more detail, we have focused on the functions of the core promoter of the MHC class I gene in vivo. The minimal class I promoter has been localized to a segment between -65 bp and +14 bp. Contained within this segment are a canonical CCAAT box, a TATAA-like element, an Sp1 binding site (Sp1BS) and an Initiator (Inr). In past studies, we have reported that no single element is necessary for expression of a reporter in transient transfection assays. More recently, we examined the dependence of transcription on each of the core promoter elements in the context of the native gene in transgenic mice. Remarkably, all of the promoters with mutant elements supported transcription. Each element contributed uniquely to tissue-specificity, hormonal responses or both. The CCAAT box modulated constitutive expression in non-lymphoid tissues whereas the TATAA-like element controlled transcription in lymphoid tissues. The Sp1BS element and Inr negatively regulated constitutive transcription; the Inr regulated tissue specific patterns. Pol II binding, histone H3K4me3 patterns closely correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the WT, TATAA-like and CCAAT mutant promoters were activated by gamma-interferon, the Inr and Sp1 mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each contributes to fine tuning tissue-specific expression, extracellular signaling, promoter activity and chromatin structure. Transcription initiation is not a continuous process, but rather occurs in bursts which differ in amplitude and frequency. Although the roles of enhancers on the bursting characteristics of promoters in yeast and cultured mammalian cells have been described, the contributions of individual core promoter elements to bursting amplitude and frequency have not been assessed. Having demonstrated that the core promoter elements that constitute the MHC class I promoter all contribute to transcription initiation, we have examined their contribution to bursting characteristics in primary splenic B cells during both basal and activated transcription by single molecule RNA FISH. We have demonstrated that each core promoter element modulates different components of transcriptional bursting. Remarkably, the activities of the core promoter elements differ during basal or activated transcription. Thus, in primary B cells, SP1BS governs transcriptional burst size under basal, constitutive conditions but burst frequency in response to gamma-interferon induction. Conversely, while the Inr regulates burst frequency in the absence of gamma-interferon, it contributes to increased burst size in response to gamma-interferon induction. The TATAA-like element regulates burst size and frequency under both conditions. Paradoxically, in the presence of gamma-interferon, the burst frequency of the mutated TATAA-like element is reduced while size is increased, such that the overall effect was a modest increase in mean mRNA/cell, but an increase in noise. Finally, the CCAAT element does not contribute to bursting during constitutive transcription. However, in response to gamma-interferon activation, the CCAAT box modulates both burst size and frequency. The complex alterations in bursting of the core promoter element mutations in both constitutive and activated transcription indicate that core promoter architecture determines the specific bursting properties and enforces their roles in mediating proper response to signaling events. This combinatorial regulation of MHC class I transcriptional bursting patterns at the core promoter is also influenced by tissue-specific factors and, presumably, an interplay with upstream regulatory factors. In different cell types, transcriptional bursting of MHC class I is maintained. A comparison of the transcriptional bursting of an endogenous MHC class I gene, Kb, in B cells and T cells revealed distinct patterns in the two cell types. We hypothesize that these differences in tissue-specific bursting are intrinsic to the gene and its promoter architecture, but mediated through trans-acting factors. Although inheritance is clearly genetic, compelling evidence is accumulating in many species of non-genetic inheritance of traits such as longevity and metabolism. Transgenerational epigenetic inheritance has been attributed to chromatin modifications including histone modifications and changes in heterochromatin. We have discovered a novel role for the core promoter element, CCAAT, in maintaining transgenerational epigenetic inheritance. The CCAAT element within core promoter of the MHC class I gene, functions a 5' boundary element, forming a loop with the 3' boundary element that prevents heterochromatinization. The looping interactions are mediated by CTCF and cohesin and protect the promoter from inactivating epigenetic H3K9me3 and DNA methylation marks. Mutation of the CCAAT core promoter element results in loss of both the loop and transgenerational expression unless a new loop is reestablished with a distal CCAAT-like element.
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