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Direct dissection of genomic features determining transcription factor binding, and systematic characterization of long noncoding RNAs, which are associated with an increased risk of aggressive periodontitis

直接剖析决定转录因子结合的基因组特征，以及长非编码 RNA 的系统表征，这些特征与侵袭性牙周炎的风险增加相关

基本信息

批准号：
396785342
负责人：
Professor Dr. Arne Schäfer
金额：
--
依托单位：
Abteilung für Parodontologie und Synoptische Zahnmedizin
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/396785342?language=en
关键词：
Direct dissection genomic features determining

项目摘要

Periodontitis (PD) is among the most common inflammatory diseases worldwide with a heritability of 50%. It affects human populations at prevalence rates of 11% for the severe forms. The oral inflammation can affect large areas of the periodontal tissues and can thus pose a substantial stress for the immune system. Detailed knowledge of the precise genetic molecular biological mechanisms that drive the individual steps in the pathogenesis of PD is currently missing. To identify major susceptibility genes of PD, we performed a large genome-wide association study (GWAS) with aggressive periodontitis (AgP), the most severe and early-onset disease phenotype of PD. By integrating our imputed GWAS data together with variants in strong linkage disequilibrium to the most suggestive GWAS lead-polymorphisms with data of the ENCODE (Encyclopedia of DNA Elements) consortium on cell type specific genomic features, we identified putative causative variants (PCVs) that are highly suggestive to have a regulatory role in gene expression. To establish their causality and function, we formulated the following aims of research. (1) Dissection of the functional impacts of the effect alleles of the nominated PCVs on protein-DNA binding by EMSA (electrophoretic mobility shift assays). (2) To provide evidence for the direct effects of the PCVs on gene expression, we will design chromosomally integrated reporter assays in immortalized gingival and lymphoblast cell lines. (3) Following overexpression of the identified transcription factors, we will validate the predicted target genes of the PCVs by quantitative expression profiling and protein blotting. In addition, we will characterize the three most suggestive AgP-associated long noncoding RNAs (lncRNAs) by CRISPR-dCas9 activation and RACE-PCR (rapid amplification of cDNA-ends) in primary cells of blood and the gingiva, followed by whole transcriptome mRNA sequencing. The genes that are regulated by the lncRNA will be validated by protein blotting. The generated data will aggregate the AgP-associated lncRNA into etiological relevant gene networks and will substantially contribute to identify and characterize key PCVs and their effects on signaling pathways that drive the disease risk of AgP.

牙周炎是世界上最常见的炎症性疾病之一，遗传率高达50%。它影响人口的患病率为11%的严重形式。口腔炎症可以影响大面积的牙周组织，因此可以对免疫系统造成很大的压力。目前缺乏对PD发病机制中各个步骤的精确遗传分子生物学机制的详细了解。为了确定PD的主要易感基因，我们对侵袭性牙周炎（AgP）进行了一项大型全基因组关联研究（GWAS），AgP是PD最严重和早发性疾病表型。通过将我们的估算GWAS数据与最具暗示性的GWAS前导多态性的强连锁不平衡变体与ENCODE（DNA元件百科全书）联盟关于细胞类型特异性基因组特征的数据整合在一起，我们鉴定了高度暗示在基因表达中具有调节作用的推定致病变体（PCV）。为了确定它们的因果关系和功能，我们制定了以下研究目标。(1)通过EMSA（电泳迁移率变动试验）分析指定PCV的效应等位基因对蛋白质-DNA结合的功能影响。(2)为了提供PCV对基因表达的直接影响的证据，我们将在永生化牙龈和淋巴母细胞系中设计染色体整合的报告基因测定。(3)在确定的转录因子过表达后，我们将通过定量表达谱和蛋白质印迹来验证PCV的预测靶基因。此外，我们将通过CRISPR-dCas 9激活和RACE-PCR（cDNA末端快速扩增）在血液和牙龈的原代细胞中表征三种最具暗示性的AgP相关长非编码RNA（lncRNA），然后进行全转录组mRNA测序。将通过蛋白质印迹法验证由lncRNA调控的基因。生成的数据将聚集AgP相关lncRNA到病因学相关基因网络中，并将大大有助于识别和表征关键PCV及其对驱动AgP疾病风险的信号通路的影响。