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. To further characterize regulation of class I gene expression, we have undertaken to characterize core promoter elements in vivo. To understand these mechanisms in more detail, we have characterized elements within the core promoter of the MHC class I gene. The minimal class I core 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. However, we have now 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 mutant promoters 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. 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,E+4, 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 conditions, the two DPE-L's act in concert to up-regulate promoter activity, preferentially increasing the use of TSS located at the 5'end of the cluster of multiple start sites. These elements promote constitutive, TAF1-dependent transcription. 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. These novel downstream elements are functionally and mechanistically distinct from previously described downstream elements in mammalian promoters. Importantly, we have now identified a second promoter element within the core promoter region. This novel promoter element alone is capable of supporting transcription of a reporter gene in the presence or a heterologous enhancer. Furthermore, it is capable of supporting CIITA-activated transcription of a reporter gene. Most significantly, this novel promoter drives the expression of the native MHC class I gene both in stably transfected L cells and in transgenic mice. Thus, this promoter element, like the first, is capable of supporting both constitutive and activated transcription in vitro and in vivo. In recent studies, we have discovered non-coding sense and anti-sense transcripts derived from the 5prime extended promoters of MHC class I genes. The abundance of these transcripts is inversely correlated with sense transcription of the coding sequences in different tissues. The role of these transcripts in regulating MHC class I transcription is being investigated.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC009279-28
Application #
8763030
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2013
Total Cost
$490,344
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Mu, Jie; Tai, Xuguang; Iyer, Shankar S et al. (2014) Regulation of MHC class I expression by Foxp3 and its effect on regulatory T cell function. J Immunol 192:2892-903