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

调节 IL-10 表达的比较基因组学和转基因方法

• 批准号: 7657340
• 负责人: Jay H. Bream
• 金额: $ 40.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7657340
• 关键词: Acetylation; Allergic; Autoimmune Process; B-Lymphocytes; Backcrossings; Bacterial Artificial Chromosomes; Binding Sites; Biochemical; Biological Assay; Biological Process; Cell Lineage; Cells; Chromatin Structure; Chromosomes, Human, Pair 1; Colitis; DNA; DNA Binding; Data; Detection; Disease; Disease susceptibility; Dose; Electrophoretic Mobility Shift Assay; Elements; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Etiology; Evaluation; Functional RNA; Gene Cluster; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genetic Variation; Genomics; Goals; H19 gene; Homologous Gene; Human; Human Chromosomes; Hypersensitivity; IL10 gene; IL19 gene; In Vitro; Indium; Inflammation; Interleukin-10; Knockout Mice; Laboratories; Length; Link; Lymphoid; MAPKAPK2 gene; Malignant Neoplasms; Maps; Modeling; Modification; Molecular; Molecular Genetics; Molecular Profiling; Monitor; Mus; Myelogenous; Natural Killer Cells; Nucleic Acid Regulatory Sequences; Pathogenesis; Pathway interactions; Pattern; Phenotype; Play; Population; Predisposition; Principal Investigator; Production; Receptor Cell; Regulation; Regulator Genes; Regulatory Element; Regulatory Pathway; Research; Research Personnel; Role; Sequence Alignment; Sequence Analysis; Signal Pathway; Signal Transduction; Site; Structure; T-Lymphocyte; Testing; Time; Tissues; Transcript; Transcriptional Regulation; Transgenic Mice; Transgenic Model; Transgenic Organisms; Variant; Work; base; bisulfite; cell type; chromatin immunoprecipitation; chromatin remodeling; comparative; cytokine; design; human tissue; in vivo; in vivo Model; insight; macrophage; new therapeutic target; novel; programs; promoter; receptor; response; tool; transcription factor

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.

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。关于控制人类 IL-10 表达的机制知之甚少。我们的总体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。具体假设是，细胞类型和受体特异性的人 IL-10 表达模式由进化保守的调控区域控制，这些调控区域包含 il10 基因并延伸到侧翼基因 il19 和 mapkapk2。我们基于以下观察结果提出这一假设：1) il10 基因位于 1110 个同源物的同线簇中，2) il10 基因簇周围的序列比对表明物种之间存在许多同源区域，称为保守非编码序列 (CMS)经常含有调节元件，3）来自我们实验室和其他实验室的数据显示了 il10 及其侧翼基因 1119 和 之间组织特异性染色质结构和表达谱的证据地图卡APK2。为了研究人类 IL-10 调节的基因组要求，我们开发了一种基于细菌人工染色体 (BAC) 的转基因小鼠，其中包含人类 il10 基因以及侧翼基因 il19 和 mapkapk2（hIL10BAC 小鼠）。我们相信 175 kb BAC 包含赋予人 IL-10 细胞特异性表达的基因组信息。我们在 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. 定义在 Hil10BAC 小鼠中调节人 IL-10 转录的组织和受体特异性途径。我们将通过识别细胞特异性人类 IL-10 诱导模式来表征细胞特异性基因转录的机制，并确定组织特异性基因转录的机制； (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物的存在/不存在，以及 (iii) 与转录因子缺失小鼠回交的人类 IL-10 表达缺陷hIL10BAC 小鼠。 2. 确定 hIL10BAC 小鼠中人 IL-10 表达的表观遗传/基因组要求。我们以人 IL-10 BAC 中的 CMS 位点为目标，并使用这些位点作为指导来确定人 il10 基因周围的扩展染色质结构。 3. 确定 hIL10BAC 小鼠是否是用于分析基因组结构和生物功能的合适体内模型；通过将 hIL10BAC 小鼠与 IL10-/- 小鼠 (lL10BAC?/-) 杂交，并尝试拯救针对 LPS 超敏反应模型的 IL10-/- 表型。