Molecular Mechanisms of Major histocompatibility complex (MHC) gene regulation

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My research program is focused on the multiple regulatory mechanisms that control major histocompatibility complex (MHC; HLA in human) class I (MHC-I) and class II (MHC-II) gene and protein expression. MHC molecules are antigen-presenting molecules displaying peptide antigens to T cells, and are thus of central importance for the adaptive immune response. MHC-dependent immune responses are intricately regulated. The MHC-II transactivator CIITA, a member of the NLR (nucleotide binding and leucine rich repeats) family of proteins, is the master regulator of MHC-II gene expression. We have found recently that protein turnover of CIITA is directly linked to its capacity to activate transcription. The first ten amino acids of CIITA isoform III act as a portable degron and transactivation sequence. The N-terminal end of CIITA isoform III is responsible for increased interaction with components of the transcription machinery. These experiments reveal a novel function of free N-terminal ends of proteins in degradation-dependent transcriptional activation. We have recently shown that NLRC5 is important for MHC-I expression. Intriguingly, NLRC5 is also an NLR protein, a family of proteins that are predominantly involved in innate immune responses. Thus far, CIITA and NLRC5 are the only NLR proteins that are transcriptional regulators. Using domain-swap experiments, we have shown that the NLRC5 N-terminal domain (NTD) behaves as a bona fide transcriptional activation domain, although its mechanism of transactivation is unknown. In collaboration with the group of Dr. T. Kufer, we propose to use our domain-swap constructs to analyze chromatin modifications at endogenous MHC-I and MHC-II promoters via ChIP (chromatin IP) experiments. We will use the NLRC5 NTD as a substrate to identify binding partners by affinity purification and mass spectrometry. Recently we have found that there is a crosstalk between IFN- induced HLA-II activation and estrogen receptor (ER) signaling. We propose to analyze the molecular mechanisms of this crosstalk. Bio-informatics analysis of ChIp-seq data revealed in vivo binding of ER at several distal sites around the CIITA gene, which in part overlap with distal regulatory elements of IFN- induced CIITA expression. At a number of sites, several potentially relevant transcription factors, such as Er, FoxA, Stat1, IRF1 and BRG1 bind in close proximity. We will carry out a ChIp-seq and RNA-seq analysis for ER, FoxA1 and IRF1 in breast cancer cells treated simultaneously with IFN- and E2. Functional tests of potential regulators will be carried out by shRNA-mediated knockdowns. Potential regulatory sites will be analyzed by somatic knockouts of potential endogenous distal regulatory sites. Taken together these experiments will inform us on the interactions of important signaling pathways in general and on the regulation of the CIITA gene in particular.