A comparative genomics and transgenic approach to regulation of IL-10 expression

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DESCRIPTION (provided by applicant): IL-10 is an immunoregulatory cytokine that plays a fundamental role in limiting inflammation. Very little is known about the mechanisms which control human IL-10 expression. Our broad goal is to define regulatory pathways and genomic requirements which control human IL-10 expression profiles. The specific hypothesis is that cell-type and receptor-specific human IL-10 expression patterns, are controlled by evolutionary conserved regulatory regions that encompass the il10 gene and extend into the flanking genes il19 and mapkapk2. We base this hypothesis on the observations that 1) the il10 gene is located in a syntenic cluster of 1110 homologues, 2) sequence alignments around the il10 gene cluster indicate numerous regions of homology between species, known as conserved non-coding sequences (CMS) which frequently harbor regulatory elements, 3) data from our laboratory and others show evidence of tissue-specific chromatin structure and expression profiles between il10 and its flanking genes 1119 and mapkapk2. To study genomic requirements of human IL-10 regulation, we have developed a transgenic mouse based on a bacterial artificial chromosome (BAC) which contains the human il10 gene as well as the flanking genes il19 and mapkapk2 (hIL10BAC mice). We believe the 175 kb BAC contains the genomic information to impart cell-specific expression of human IL-10. We have validated tissue-specific expression of human IL-10 in vivo and in vitro in hIL10BAC mice. The specific aims will: 1. Define tissue- and receptor-specific pathways which regulate human IL-10 transcription in Hil10BAC mice. We will characterize cell-specific human IL-10 induction patterns and identify mechanisms which underlie tissue-specific gene transcription by identifying; (i) regulatory boundaries between human i!10 and the flanking genes ill9 and mapkapk2, (ii) the presence/absence of human, tissue-specific intergenic transcripts and (iii) human IL-10 expression deficiencies in transcription factor null mice backcrossed to the hIL10BAC mice. 2. Determine epigenetic/genomic requirements for human IL-10 expression in hIL10BAC mice. We are targeting CMS sites in the human IL-10 BAC and using these sites as a guide to determine the extended chromatin structure surrounding the human il10 gene. 3. Determine if the hIL10BAC mouse is an appropriate in vivo model for analysis of genomic structure and biological function by; by crossing the hIL10BAC mice to the IL10-/- mouse (lL10BAC?/-) and attempting to rescue IL10-/- phenotypes focusing on LPS hypersensitivity models.