Genomic, Epigenetic and Functional Analyses of Vertebrate Regulatory Regions

脊椎动物调节区的基因组、表观遗传学和功能分析

• 批准号: 8948368
• 负责人: Laura L Elnitski
• 金额: $ 142.79万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8948368
• 关键词: Aberrant DNA Methylation; Address; Affect; Alleles; Area Analyses; Biological; Biological Assay; Biological Markers; Biology; CASP8 gene; Categories; Cattle; Cell Line; Characteristics; Chromatin; Code; Colorectal Cancer; Communities; Complex; CpG Islands; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Methylation; Data; Detection; Development; Diagnostic; Diagnostic tests; Disease; Elements; Endometrial Carcinoma; Endometrial Neoplasms; Endometrium; Enhancers; Environment; Epigenetic Process; Epithelial; Etiology; Evolution; Exhibits; Exons; Gene Conversion; Gene Expression; Gene Expression Regulation; Gene Mutation; Genes; Genome; Genomics; Glioblastoma; Goals; Grouping; Histones; Human; Human Genome; Hypermethylation; Hypersensitivity; Indium; Individual; Internet; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Oviducts; Measures; Mediating; Messenger RNA; Methylation; Modeling; Modification; Mus; Mutation; Mutation Spectra; National Human Genome Research Institute; Nature; Neoplasm Metastasis; Normal Cell; Nucleic Acid Regulatory Sequences; Nucleotides; Ovarian Endometrioid Adenocarcinoma; Ovarian Endometrioid Tumor; Pattern; Phenotype; Play; Positioning Attribute; Process; Production; Promoter Regions; Proteins; Publishing; RNA Splicing; Rattus; Recurrence; Regulation; Regulator Genes; Regulatory Element; Reporting; Reproducibility; Research; Research Project Grants; Role; Sampling; Serous; Signal Transduction; Solid; Source; System; Testing; The Cancer Genome Atlas; Therapeutic; Transcript; Transcriptional Activation; Tumor Pathology; Untranslated RNA; Update; Variant; Visit; Work; base; biological systems; bisulfite; cancer type; cis acting element; cystic fibrosis patients; disease-causing mutation; exon skipping; expression vector; genome wide methylation; human disease; insight; melanoma; mouse model; neoplastic cell; novel; novel strategies; ovarian neoplasm; promoter; response; tumor; vector; vertebrate genome; web site

The analysis of individual parts of the genome enables a more comprehensive understanding of how the parts fit together in the broader context of disease. The following projects are described with respect to continuation of previous years' projects and represent integrative analyses of independent genomic data types to address the genome as a complex regulatory system. The analysis areas include cis-acting, trans-acting and epigenetic regulators of the human genome. Addressing the evolution of the human genome through the emergence of new human-specific genes regulated by bidirectional promoters. Research from my group previously established the enrichment of bidirectional promoters in vertebrate genomes including human, mouse, rat, and cow (Yang et al, 2008), which indicates evolutionary selection to maintain their presence. Despite the cross-species similarities, we discovered that some bidirectional promoters correspond to positions of unidirectional promoters in other vertebrate species; leading to the hypothesis that species-specific bidirectional promoters greatly and uniquely target the detection of species-specific transcripts in any genome. We confirmed this hypothesis while participating in the Bovine Genome Consortium (Bovine Seq. Cons. et al. 2009) and identified a spliced, highly expressed, multi-exon (noncoding) transcript regulated by a bidirectional promoter that was exclusive to the bovine lineage (Piontkivska et al. 2009). To find human-specific transcripts, my group identified a set of 1,400 nonconserved, novel noncoding transcripts flanking bidirectional promoters (Gotea et al. 2013). Once identified, we developed for positive selection in these transcripts and elsewhere, as an indicator of beneficial function of noncoding sequences to the human genome. We also realized that positive selection can be inaccurately measured in the genome and developed a test for GC-biased gene conversion to distinguish adaptive forces from non-adaptive forces in the genome (Gotea et al. 2014). After integrating computational and experimental data, we found nucleotide substitutions that facilitate the emergence of new exons in the bidirectional transcripts of the genome. The gene list provides the basis for studying the role of novel transcripts that are unique to the human genome. Moreover, using this approach, novel transcripts can be identified for any species. Postscript: The model for the emergence of new noncoding genes through bidirectional promoters is consistent with recent reports showing that the majority of lincRNA genes have bidirectional promoters, encompassing many species-specific transcripts. Comparing genome-wide methylation patterns in subtypes of ovarian tumors and mouse models. I am testing the hypothesis that altered DNA methylation in promoter regions can distinguish genes that are relevant to ovarian tumor pathology. Given the sporadic nature of 90% of ovarian cancers, disruption of normal gene regulation is a likely contributor to disease etiology. Methylation patterns at 25,475 unique loci in 43 samples of ovarian, endometrial or metastatic tumors, along with normal fallopian tube and normal endometrium have been assessed. Data from this project showed that methylation patterns mirror histopathological subdivisions of ovarian tumors and discriminated tumor types with finer granularity and greater reproducibility than published gene expression assays (Kolbe et al. 2012). The extensive differences we showed between tumor and normal samples are the first report of a methylator phenotype in ovarian endometrioid tumors, analogous to the methylator phenotype identified in colorectal cancer and glioblastoma. Ongoing studies will be to look for biomarkers for use in diagnostic tests. Profiling common epigenetic features in solid human epithelial tumors The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biollogical markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal controls. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter (Sanchez-Vega et al. 2013). We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, and unique to cell lines derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to PCR amplification of bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor diagnostics. Epigenetic modifications affecting the promoters of ZNF154, CASP8 and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer. Update of research projects on individual functional elements and community impact. Exon Skipping. My work to identify sequence mutations that cause exon skipping (Woolfe et al. 2010 applied statistical tests to determine which features showed statistically significant, predictive ability to discriminate neutral variants from disease-causing mutations. We implemented the results in a web server that evaluates variants of unknown function to predict those most likely to cause exon skipping, Skippy, (http://research.nhgri.nih.gov/skippy/), which continues to receive the most visits of all NHGRI webservers and downloads for private use. In the last year, the Skippy server had 39,381 total page views, 107 average page views per day and 12.21 average page views per visit. In an application of the Skippy toolset, my group showed that synonymous substitutions detected in cystic fibrosis patients cause exon skipping in CFTR. These variants are novel candidates for uncharacterized second allele mutations in CFTR (Scott et al, 2012 J. Cystic Fibrosis). This project extends into the collaborative project on the study of recurrent functional synonymous mutations in melanoma (Gartner et al. 2013). Negative regulatory elements. My group developed the first, systematic expression vector system to experimentally assay negative regulatory elements (Petrykowska et al. 2008 Gen. Res.). Despite the commonly held hypothesis that negative cis-acting elements are present in the human genome, examples have not been widely defined or characterized. My research to help identify negative elements has broader importance because mutations in these elements would be activating for disease and could play a role in a host of diseases. Annotations of NRE discovered by my group are posted on the UCSC Human Genome Browser test web site (EncodeNhgriNre). Since inception of the assay, I have provided the vectors as source materials to the community and continue to collaborate with other labs upon request. Furthermore, I have participated in the ENCODE Consortium analysis groups to experimentally assess the functional activity of putative negative regulatory elements predicted in genomic sequences (ENCODE Cons. et al. 2011 PLoS Bio. and ENCODE Cons. et al. 2012 Nature and Kellis et al, 2014 PNAS).

对基因组各个部分的分析可以更全面地了解这些部分如何在更广泛的疾病背景下组合在一起。以下项目是关于前几年项目的延续进行描述的，并代表独立基因组数据类型的综合分析，以将基因组视为一个复杂的调控系统。分析领域包括人类基因组的顺式作用、反式作用和表观遗传调节因子。 通过双向启动子调控的新人类特异性基因的出现来解决人类基因组的进化问题。 我的小组之前的研究证实了包括人类、小鼠、大鼠和牛在内的脊椎动物基因组中双向启动子的富集（Yang et al, 2008），这表明进化选择是为了维持它们的存在。尽管存在跨物种相似性，我们发现一些双向启动子对应于其他脊椎动物物种中单向启动子的位置；导致这样的假设：物种特异性双向启动子极大且独特地针对任何基因组中物种特异性转录本的检测。我们在参与牛基因组联盟时证实了这一假设（Bovine Seq. Cons. et al. 2009），并鉴定了一个剪接的、高表达的、多外显子（非编码）转录本，该转录本受牛谱系独有的双向启动子调节（Piontkivska et al. 2009）。为了寻找人类特异性转录本，我的团队鉴定了一组 1,400 个非保守的新型非编码转录本，它们位于双向启动子两侧（Gotea et al. 2013）。一旦确定，我们就在这些转录本和其他地方进行正向选择，作为非编码序列对人类基因组有益功能的指标。我们还意识到，基因组中的正选择可能无法准确测量，因此开发了一种 GC 偏向基因转换测试，以区分基因组中的适应性力量和非适应性力量（Gotea 等人，2014）。在整合计算和实验数据后，我们发现核苷酸取代促进了基因组双向转录本中新外显子的出现。基因列表为研究人类基因组特有的新转录本的作用提供了基础。此外，使用这种方法，可以鉴定任何物种的新转录本。 后记：通过双向启动子出现新非编码基因的模型与最近的报告一致，表明大多数 lincRNA 基因具有双向启动子，涵盖许多物种特异性转录本。 比较卵巢肿瘤亚型和小鼠模型的全基因组甲基化模式。 我正在测试这样的假设：启动子区域 DNA 甲基化的改变可以区分与卵巢肿瘤病理学相关的基因。鉴于 90% 的卵巢癌具有散发性，正常基因​​调控的破坏可能是导致疾病病因的一个因素。已经评估了 43 个卵巢、子宫内膜或转移性肿瘤样本以及正常输卵管和正常子宫内膜样本中 25,475 个独特位点的甲基化模式。该项目的数据表明，甲基化模式反映了卵巢肿瘤的组织病理学细分和可区分的肿瘤类型，其粒度比已发表的基因表达测定更细，重现性更高（Kolbe 等人，2012）。我们在肿瘤和正常样本之间显示的广泛差异是卵巢子宫内膜样肿瘤甲基化表型的首次报告，类似于结直肠癌和胶质母细胞瘤中鉴定的甲基化表型。正在进行的研究将寻找用于诊断测试的生物标志物。 分析人类实体上皮肿瘤的常见表观遗传特征 对癌症中异常 DNA 甲基化的研究是发现用于诊断的新型生物标志物的关键，并有助于描述疾病的重要机制。我们已经鉴定出 12 个位点在浆液性卵巢癌和子宫内膜样卵巢癌和子宫内膜癌中与正常对照相比存在差异甲基化。最强的信号显示肿瘤中 ZNF154 启动子内的 CpG 岛存在高甲基化（Sanchez-Vega et al. 2013）。我们发现，在 TCGA 的 16 种不同癌症类型中的 15 种实体人上皮肿瘤样本中，该位点的高甲基化现象反复出现。此外，ZNF154 高甲基化在多种 ENCODE 细胞系中引人注目地存在，并且是源自肿瘤细胞的细胞系所独有的。通过将我们的分析从 Illumina 27K Infinium 平台扩展到 450K 平台，再到亚硫酸氢盐处理的 DNA 的 PCR 扩增，我们证明高甲基化延伸到 ZNF154 CpG 岛的整个范围。我们还发现了与 CASP8 和 VHL 相关的两个基因组区域中反复出现的低甲基化。这三个基因在许多癌症类型中表现出甲基化和基因表达之间的显着负相关性，以及反映癌症与正常细胞系中不同染色质可及性的 DNaseI 超敏性和组蛋白标记模式。我们的研究结果强调 ZNF154 的高甲基化作为与肿瘤诊断相关的生物标志物。影响 ZNF154、CASP8 和 VHL 启动子的表观遗传修饰在多种肿瘤类型中是共有的，因此对于了解癌症的基因组图谱可能很重要。 关于个体功能要素和社区影响的研究项目的更新。 外显子跳跃。我的工作是识别导致外显子跳跃的序列突变（Woolfe et al. 2010）应用统计测试来确定哪些特征显示出统计学上显着的预测能力，以区分中性变异和致病突变。我们在网络服务器中实现了结果，该服务器评估未知功能的变异以预测那些最有可能导致外显子跳跃的变异，Skippy， (http://research.nhgri.nih.gov/skippy/)，该网站仍然是所有 NHGRI 网络服务器中访问量和私人使用下载量最多的网站。 去年，Skippy 服务器的总页面浏览量为 39,381 次，每天平均页面浏览量为 107 次，每次访问平均页面浏览量为 12.21 次。在 Skippy 工具集的应用中，我的小组表明，在 囊性纤维化患者会导致 CFTR 中的外显子跳跃。这些变体是 CFTR 中未表征的第二等位基因突变的新候选者（Scott 等人，2012 J. Cystic Fibrosis）。该项目延伸至黑色素瘤复发性功能同义突变研究的合作项目（Gartner 等人，2013 年）。 负调控因素。我的小组开发了第一个系统表达载体系统，用于通过实验测定负调控元件（Petrykowska 等人，2008 Gen. Res.）。尽管普遍认为人类基因组中存在负顺式作用元件，但例子尚未得到广泛定义或表征。我帮助识别负面元素的研究具有更广泛的重要性，因为这些元素的突变会激活疾病，并可能在许多疾病中发挥作用。我的小组发现的 NRE 注释已发布在 UCSC 人类基因组浏览器测试网站 (EncodeNhgriNre) 上。自检测开始以来，我已向社区提供载体作为源材料，并根据要求继续与其他实验室合作。此外，我还参加了 ENCODE 联盟分析小组，通过实验评估基因组序列中预测的假定负调控元件的功能活性（ENCODE Con​​s. 等人，2011 PLoS Bio. 和 ENCODE Con​​s. 等人，2012 Nature 和 Kellis 等人，2014 PNAS）。