Characterization of Enhancers in the Epidermal Differentiation Complex

表皮分化复合物中增强剂的表征

基本信息

批准号：
8307242
负责人：
Cristina de Guzman Strong
金额：
$ 23.7万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-16 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8307242
关键词：
1q21 Affect American Atopic Dermatitis Binding Bioinformatics Biological Assay Canis familiaris Cells Chromatin Chromosomes Complex Development Plans Didelphidae Disease EP300 gene Embryo Enhancers Epidermis Epigenetic Process Gene Cluster Gene Duplication Gene Expression Gene Expression Regulation Genes Genetic Genome Globin Goals Human Human Chromosomes Hypersensitivity In Vitro Inflammatory Institution Laboratories LacZ Genes Light Link Locus Control Region Luciferases Macaca mulatta Maps Mentors Mentorship Mus National Human Genome Research Institute Pan Genus Phase Positioning Attribute Process Psoriasis Quality of life Rattus Regulation Regulatory Element Reporter Research Sequence Alignment Sequence Analysis Skin Staging Stress Testing Transcriptional Regulation Transgenic Mice Universities Washington base career development chromatin immunoprecipitation comparative cost density genome sequencing in vivo keratinocyte mouse model promoter skin disorder transcription factor

项目摘要

Atopic dermatitis (AD) and psoriasis are two inflammatory skin diseases that affect over 22.5 million Americans collectively and are burdening the U.S. economy with over $7.5 billion dollars in management and quality-of-life issue costs. These diseases share linkage to the epidermal differentiation complex (EDC) on chromosome 1q21. The EDC spans 1.6Mb and contains a majority of genes that are specifically expressed upon terminal differentiation of the epidermis and subsequent skin barrier formation. The proximity and density of these genes on 1q21 suggest coordinate regulation via cis-regulatory elements. To date, only a few regulatory elements have been identified in the EDC. My goal is to identify enhancers that control gene expression in the EDC. Using comparative multi-species sequence analysis, we hypothesize that evolutionarily conserved noncoding sequences (CNS) function as enhancers to coordinate transcriptional regulation of genes within the EDC. To test this hypothesis, we propose the following 2 aims: In Specific Aim 1, we will test the function of the CNS to direct in vitro enhancer activity using luciferase reporter assays in mouse epidermal cells. In Specific Aim 2A, we will determine colocalization of the CNS EDC to open chromatin using DNasel hypersensitivity assays in mouse keratinocytes and barrier deficient mouse models. These aims will be completed during the mentored phase under Dr. Julie Segre at the National Human Genome Research Institute (NHGRI) in Bethesda, MD. The following aims will be pursued during the independent phase under the bridge mentorship of Dr. Anne Bowcock from Washington University: In Specific Aim 2B, we will also determine if these CNS EDC map to regions bound by p300 and monomethylated H3K4 enriched in enhancer sequences using chromatin immunoprecipitation (ChIP). In Specific Aim 3, will assess transcription factor binding to the CNS EDC using bioinformatics and ChIP. In Specific Aim 4, we will test select CNS EDC in vivo as epidermal locus control regions using CNS EDC- hsp68-lacZ transgenic mice. This proposal functions as Cristina de Guzman Strong's career development plan in obtaining an independent research position at an academic institution. Results from the research plan will shed light on the genetic and epigenetic mechanisms underlying epidermal differentiation and barrier formation as well contribute to our understanding of atopic dermatitis and psoriasis.

特应性皮炎（AD）和牛皮癣是两种炎症性皮肤病，影响超过2250万美国人集体以超过75亿美元的管理和生活质量的问题成本。这些疾病与表皮分化络合物（EDC）共享染色体1Q21。 EDC跨度为1.6MB，并包含大多数基因表皮的末端分化和随后的皮肤屏障形成。接近和这些基因在1q21上的密度表明通过顺式调节元件进行调节。迄今为止，只有一个在EDC中很少发现监管元素。我的目标是识别控制基因的增强子在EDC中的表达。使用比较多物种序列分析，我们假设进化保守的非编码序列（CNS）的功能作为增强子来协调转录 EDC内基因的调节。为了检验这一假设，我们提出以下两个目的：以特定目的 1，我们将使用荧光素酶报告基因测定法测试中枢神经系统的功能，以指导体外增强剂活性小鼠表皮细胞。在特定的目标2a中，我们将确定CNS EDC的共定位以打开在小鼠角质形成细胞和屏障缺陷小鼠模型中使用DNASEL超敏反应测定的染色质。这些目标将在朱莉·塞格尔（Julie Segre）博士的指导阶段完成马里兰州贝塞斯达的基因组研究学院（NHGRI）。以下目标将在华盛顿大学的安妮·鲍科克（Anne Bowcock）桥梁指导下的独立阶段：印第安纳州具体目标2B，我们还将确定这些CNS EDC是否映射到受P300和限制的区域和使用染色质免疫沉淀（CHIP）在增强子序列中富集的单甲基化H3K4。在特定的目标3将使用生物信息学和芯片评估转录因子与CNS EDC的结合。在特定的目标4，我们将使用CNS EDC-测试体内选择的CNS EDC作为表皮基因座控制区域 Hsp68-lacz转基因小鼠。该提案是克里斯蒂娜·德·古兹曼·斯特朗（Cristina de Guzman Strong）的职业发展计划在学术机构获得独立的研究职位。研究结果计划将阐明表皮分化和表观遗传机制和表观遗传机制障碍的形成也有助于我们对特应性皮炎和牛皮癣的理解。