|Ph.D Student||Cohen Helit|
|Subject||Characterization of DNA Sequence Elements that Regulate|
MHC Class I Gene Expression In-Vivo
|Department||Department of Biology||Supervisors||Professor Yoram Reiter|
|Dr. Dinah Singer|
|Full Thesis text|
Regulation of MHC class I genes expression level is critical to achieve proper immune surveillance. In this work, we identify DNA sequences downstream of the promoter, necessary for MHC class I regulation. We first demonstrate that in transgenic mice, the promoter of MHC class I gene, in the absence of downstream elements, is insufficient to direct expression of reporter genes. However, in the presence of the coding and 3' flanking sequences high levels of expression and proper tissue distribution are observed, pointing to the requirement for elements downstream of the promoter for MHC class I regulation. We identify two sets of elements downstream of the promoter that are necessary for appropriate regulation: Proper tissue-specific regulation requires the presence of the first two introns, possibly pointing to direct binding of tissue specific factors to sequence element(s) within the proximal introns. In addition, sequences beyond the polyA addition site are necessary for expression in vivo, but have no effect in transient transfection assays. Using transgenic mice and stably transfected cell lines, we demonstrate that a 3’ segment corresponding to the untranslated and 723bp intergenic regions functions as a barrier element, protecting the MHC class I gene from transcriptional silencing by propagation of repressive epigenetic modifications. Accordingly, truncation of the 3’ segment is correlated with gene silencing accompanied by repressive chromatin organization: increased nucleosomes density and decreased histone H3K9 acetylation and H3K4 methylation across the gene. Moreover, using chromatin immunoprecipitation, we show association of histone modifying enzymes with the 3' segment. Histone variant H2A.Z, which was associated both with barrier elements and activated promoters, is also associated with the MHC class I 3' segment, as well as the promoter. Taken together, these findings demonstrate the existence of a novel barrier element downstream of the MHC class I polyA addition site which functions to maintain transcription-permissive chromatin organization.