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: 8081828
• 负责人: Jay H. Bream
• 金额: $ 39.42万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8081828
• 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; interleukin-19; 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，在限制炎症中起着基本作用。关于控制人IL-10表达的机制知之甚少。我们的广泛目标是定义控制人IL-10表达曲线的调节途径和基因组要求。特定的假设是细胞类型和受体特异性的人IL-10表达模式，由涵盖IL10基因的进化保守调节区控制，并延伸到侧翼基因IL19和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侧面基因1119和MAPKAPK2。为了研究人类IL-10调节的基因组要求，我们基于细菌人工染色体（BAC）开发了一种转基因小鼠，该染色体（BAC）包含人类IL10基因以及侧翼基因IL19和MAPKAPK2（HIL10BAC小鼠）。我们认为175 kb的BAC包含基因组信息，以赋予人IL-10的细胞特异性表达。我们已经验证了人体内人IL-10的组织特异性表达和在HIL10BAC小鼠中的体外表达。具体目的将：1。定义组织和受体特异性途径，这些途径调节HIL10BAC小鼠的人IL-10转录。我们将表征细胞特异性的人IL-10诱导模式，并确定通过识别组织特异性基因转录基础的机制。 （i）人I！10与侧翼基因ILL9和MAPKAPK2之间的调节界限，（ii）人类，组织特异性基因间转录物的存在/不存在（III）人IL-10表达不足，在转录因子无效的NULL小鼠中反向HIL10BAC小鼠回到HIL10BAC小鼠中。 2。确定HIL10BAC小鼠中人IL-10表达的表观遗传/基因组需求。我们针对人IL-10 BAC中的CMS位点，并使用这些位点作为确定围绕人IL10基因的扩展染色质结构的指南。 3。确定HIL10BAC小鼠是否是用于分析基因组结构和生物学功能的合适的体内模型；通过将HIL10BAC小鼠跨到IL10 - / - 小鼠（LL10BAC？/ - ），并尝试挽救针对LPS超敏反应模型的IL10 - / - 表型。

项目成果

Cell type-specific regulation of IL-10 expression in inflammation and disease.

• DOI: 10.1007/s12026-009-8150-5
• 发表时间: 2010-07
• 期刊: Immunologic research
• 影响因子: 4.4
• 作者: Hedrich CM;Bream JH
• 通讯作者: Bream JH

Dynamic DNA methylation patterns across the mouse and human IL10 genes during CD4+ T cell activation; influence of IL-27.

• DOI: 10.1016/j.molimm.2010.09.009
• 发表时间: 2010-11
• 期刊: Molecular immunology
• 影响因子: 3.6
• 作者: Hedrich CM;Ramakrishnan A;Dabitao D;Wang F;Ranatunga D;Bream JH
• 通讯作者: Bream JH