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: 7900571
• 负责人: Jay H. Bream
• 金额: $ 39.82万
• 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/7900571
• 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 genomics; 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，在限制炎症中起着重要作用。我们对控制人类IL-10表达的机制知之甚少。我们的广泛目标是确定控制人类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，是由包含IL-10基因的进化保守调控区域控制的，该区域延伸到两侧的基因il19和mapkapk2。我们基于以下观察得出这一假设：1)il10基因位于1110同源物的同质集群中；2)il10基因集群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），这些区域经常包含调控元件；3)我们实验室和其他数据显示了il10及其侧基因1119和mapkapk2之间组织特异性染色质结构和表达谱的证据。为了研究人类IL-10调控的基因组要求，我们开发了一种基于细菌人工染色体（BAC）的转基因小鼠（hIL10BAC小鼠），该小鼠含有人类IL-10基因及其两侧基因il19和mapkapk2。我们认为175 kb的BAC包含了人类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hIL10BAC小鼠体内和体外的组织特异性表达。具体目标将：1.；确定在Hil10BAC小鼠中调节人IL-10转录的组织和受体特异性途径。我们将描述细胞特异性的人类IL-10诱导模式，并通过鉴定组织特异性基因转录的机制；（一）人与人之间的监管界限i！（ii）人类组织特异性基因间转录物的存在/缺失，以及（iii）转录因子缺失小鼠回交到hIL10BAC小鼠中的人类IL-10表达缺陷。2. 确定人IL-10在hIL10BAC小鼠中表达的表观遗传/基因组要求。我们的目标是人类IL-10 BAC中的CMS位点，并利用这些位点作为指导，确定人类IL-10基因周围的延伸染色质结构。3. 确定hIL10BAC小鼠是否适合用于基因组结构和生物学功能分析的体内模型；通过将hIL10BAC小鼠与IL10-/-小鼠(lL10BAC？/-)，并试图挽救IL10-/-表型，重点关注LPS超敏模型。