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

描述（由申请人提供）：IL-10是一种免疫调节细胞因子，在限制炎症中发挥重要作用。关于控制人IL-10表达的机制知之甚少。我们的主要目标是确定控制人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人IL-10表达模式由进化保守的调控区控制，所述调控区包含il 10基因并延伸到侧翼基因il 19和mapkapk 2中。我们基于以下观察结果提出了这一假设：1）il 10基因位于1110个同源物的同线簇中，2）il 10基因簇周围的序列比对表明物种之间存在许多同源区域，称为保守的非编码序列（CMS），其经常含有调控元件，3）来自我们实验室和其他实验室的数据显示了组织特异性染色质结构和IL 10及其侧翼基因1119和mapkapk 2之间的表达谱的证据。为了研究人IL-10调控的基因组需求，我们已经开发了一种基于细菌人工染色体（BAC）的转基因小鼠（hIL 10 BAC小鼠），所述BAC含有人IL-10基因以及侧翼基因IL-19和mapkapk 2。我们相信175 kb BAC含有赋予人IL-10细胞特异性表达的基因组信息。我们已经验证了人IL-10在hIL-10 BAC小鼠体内和体外的组织特异性表达。具体目标是：1.定义在Hil 10 BAC小鼠中调节人IL-10转录的组织和受体特异性途径。我们将描述细胞特异性的人IL-10诱导模式，并通过以下方式确定组织特异性基因转录的基础机制：（i）人IL-10和人IL-10之间的调控边界; 10和侧翼基因ill 9和mapkapk 2，（ii）存在/不存在人组织特异性基因间转录物和（iii）与hIL 10 BAC小鼠回交的转录因子缺失小鼠中人IL-10表达缺陷。2.确定hIL 10 BAC小鼠中人IL-10表达的表观遗传/基因组要求。我们靶向人IL-10 BAC中的CMS位点，并使用这些位点作为指导来确定人IL-10基因周围的延伸染色质结构。3.通过将hIL 10 BAC小鼠与IL 10-/-小鼠（IL 10 BAC？/-）杂交，确定hIL 10 BAC小鼠是否是用于分析基因组结构和生物学功能的适当体内模型并试图拯救IL 10-/-表型，集中于LPS超敏反应模型。

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.