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

描述(申请人提供)：IL-10是一种免疫调节细胞因子，在限制炎症方面发挥基础作用。人们对控制人类IL-10表达的机制知之甚少。我们的主要目标是定义控制人类IL-10表达谱的调控途径和基因组要求。具体的假设是，细胞类型和受体特异性的人类IL-10表达模式，由围绕il10基因并延伸到侧翼基因il19和mapkapk2的进化保守调节区控制。我们基于以下观察结果：1)il10基因位于1110个同源基因的同线簇中；2)il10基因簇周围的序列比对表明物种之间存在大量的同源性区域，称为保守的非编码序列(CMS)，通常含有调控元件；3)来自我们实验室和其他实验室的数据表明，il10与其侧翼基因1119和mapkapk2之间存在组织特异性的染色质结构和表达谱。为了研究人类IL-10调控的基因组需求，我们开发了一种基于细菌人工染色体(BAC)的转基因小鼠(hIL10BAC小鼠)，该染色体含有人IL-10基因及其侧翼基因il19和mapkapk2。我们认为，175kb的BAC含有传递细胞特异性表达人IL-10的基因组信息。我们已经在hIL10BAC小鼠体内和体外验证了人IL-10的组织特异性表达。具体目标是：1.确定在Hil10BAC小鼠体内调节人IL-10转录的组织和受体特异性途径。我们将鉴定细胞特异性的人IL-10诱导模式，并通过鉴定：(I)人类I！10与侧翼基因ill9和mapkapk2之间的调控边界，(Ii)人类组织特异性基因间转录本的存在/不存在，以及(Iii)hIL10BAC小鼠回交转录因子缺失的人IL-10表达缺陷，来确定组织特异性基因转录的机制。2.确定hIL10BAC小鼠对人IL-10表达的表观遗传学/基因组需求。我们针对人类IL-10BAC中的CMS位点，并以这些位点为指南来确定人类IL10基因周围的延伸染色质结构。3.通过将hIL10BAC小鼠与IL10-/-小鼠(lL10BAC？/-)杂交，并试图挽救IL10-/-小鼠的内毒素超敏模型，以确定hIL10BAC小鼠是否是分析基因组结构和生物学功能的合适的体内模型。

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.