Dissecting the transcriptional network governing differentiation of periderm

剖析控制周皮分化的转录网络

基本信息

批准号：
10521268
负责人：
Robert Aaron Cornell
金额：
$ 51.67万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10521268
关键词：
ATAC-seq Affect Algorithms Alleles Anatomy Area Binding Binding Sites Bioinformatics Biological Assay Biological Models CDH1 gene Candidate Disease Gene Cells ChIP-seq Code DNA Data Data Analyses Data Set Development Differentiated Gene Disease Elements Embryo Embryo Loss Embryonic Development Enhancers Epidermis Family Gene Expression Genes Genetic Genetic Transcription Genotype Health Heritability Heterozygote Human Individual Knowledge Link Mammalian Genetics Modeling Mus Nasal cavity Oral Outcome Outcome Study Palate Pathogenesis Pathogenicity Pathologic Patients Periderm Population Positioning Attribute Public Domains Regulatory Element Reporter Research Risk Risk Assessment Role Sampling Series Societies Sorting Structural Congenital Anomalies Structure Systems Biology Testing Time Tissue Differentiation Tissues Training Transgenic Organisms Untranslated RNA Variant Vertebrates Wild Type Mouse Zebrafish candidate identification clinically significant craniofacial differential expression disorder risk embryo tissue exome experimental study gene regulatory network genome sequencing genome wide association study improved in vivo loss of function loss of function mutation machine learning algorithm member model building model organism mutant novel oral cavity epithelium orofacial orofacial cleft paralogous gene promoter risk variant support vector machine tool transcription factor transcriptome sequencing whole genome

项目摘要

Our understanding of the pathogenic mechanisms for orofacial clefting (OFC) is limited by the fact that less than half of the heritable risk for this disorder has been assigned to specific genes. Towards identifying pathological sequence variants among the many irrelevant ones detected in exomes and whole genomes of patients with this disorder, an understanding of the gene regulatory networks (GRNs) that govern the development of relevant tissues, including the oral periderm, is essential. We propose a systems biology approach to analyzing the periderm GRN. Using this approach in the past enabled us to identify three novel OFC risk genes. We will utilize two model organisms, zebrafish and mouse, because the periderm differentiation GRN appears to be highly conserved. In zebrafish, the periderm differentiates very early in embryogenesis, greatly facilitating the execution and interpretation of genetic perturbation analyses. Mouse, on the other hand, has the advantage that its craniofacial anatomy is more similar to that of humans. In Aim 1, we will determine the zebrafish periderm differentiation GRN using a state-of-the-art network inference algorithm, NetProphet 2. This tool carries out both a coexpression analysis and a differential expression analysis. Input data sets will include RNA-seq expression profiles we will generate from loss-of-function (LOF) embryos for 4 key transcription factors (TF) known to participate in this GRN. We will also identify the direct gene linkages of these key TFs in the periderm GRN. Finally, we will test a novel candidate member of the periderm GRN, Tead, by carrying out LOF tests in zebrafish, thereby exploiting the strength of this model system. In Aim 2 we will deduce the murine oral periderm differentiation GRN, also using the NetProphet algorithm. Input datasets will include expression profiles of periderm isolated from the palate shelves of wild-type mouse embryos, and from heterozygous mutants of three key TFs: Irf6, Grhl3 and Tfap2a. For each of the mutant genotypes there is evidence of abnormal periderm differentiation. We will also identify murine periderm enhancer candidates by sorting GFP-positive and -negative cells from Krt17-gfp transgenic embryos, performing ATAC-seq on both populations, and H3K27Ac ChIP-seq on cells from palate shelves and the nasal cavity. As in Aim 1, we will also identify the direct gene linkages of the key TFs. We will train a machine learning algorithm on palate periderm enhancers, and use the resulting scoring function to prioritize OFC-associated SNPs near genes that are expressed in periderm for those that are likely to directly affect risk for OFC. Finally, we will perform allele- specific reporter assays on the top candidate SNPs from each of three loci. The expected outcome is a deeper understanding of the specific TFs and cis-regulatory elements that control differentiation of the periderm. This will have a broad impact because it will enable human geneticists to prioritize candidate risk variants that emerge from whole-exome and -genome sequencing analyses of OFC.

我们对口面裂（OFC）的致病机制的理解受到以下事实的限制该疾病的遗传风险的一半已分配给特定基因。识别识别许多与外来体和整个基因组中检测到的无关的病理序列变异患有这种疾病的患者，了解控制基因调节网络（GRN）相关组织的发展，包括口服periderm是必不可少的。我们提出了系统生物学分析Periderm GRN的方法。过去使用这种方法使我们能够识别三本小说 OFC风险基因。我们将利用两个模型生物，斑马鱼和鼠标，因为Periderm 分化GRN似乎是高度保守的。在斑马鱼中，佩里德尔在很早的早期就区分了胚胎发生，极大地促进了遗传扰动分析的执行和解释。鼠标，打开另一方面，其优势是其颅面解剖结构与人类更相似。在AIM 1中，我们将使用最先进的网络推理算法确定斑马鱼Periderm分化GRN， NetProphet 2。此工具同时进行了共表达分析和差异表达分析。输入数据集将包括我们将从功能丧失（LOF）胚胎中生成的RNA-seq表达曲线4 已知参与此GRN的关键转录因子（TF）。我们还将确定直接基因链接 Periderm GRN中的这些关键TF。最后，我们将测试Periderm Grn的新型候选人，通过在斑马鱼中进行LOF测试，从而利用了该模型系统的强度。在目标2中我们也将使用NetProphet算法推断出鼠口服periderm分化GRN。输入数据集将包括从野生型小鼠胚胎的口感架子中分离出的佩里德尔的表达曲线，以及来自三个关键TF的杂合突变体：IRF6，GRHL3和TFAP2A。对于每个突变基因型都有异常培训分化的证据。我们还将通过从KRT17-GFP转基因胚胎中对GFP阳性和阴性细胞进行排序，在这两种情况下都进行ATAC-SEQ 口感和鼻腔中的细胞上的人群和H3K27AC芯片序列。就像在AIM 1中一样，我们将还要确定密钥TF的直接基因链接。我们将训练一种机器学习算法的味觉 Periderm增强剂，并使用由此产生的评分函数来优先考虑与基因相关的SNP 对于可能直接影响OFC风险的人，用Periderm表示。最后，我们将执行等位基因 - 来自三个基因座的每个基因座的每个候选SNP上的特定记者测定法。预期的结果更深了解控制佩里德尔分化的特定TF和顺式调节元件。这将产生广泛的影响，因为它将使人类遗传学家能够优先考虑候选风险变体来自OFC的全外显体和 - 基因组测序分析。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Zebrafish models of orofacial clefts.

DOI：
10.1002/dvdy.24566
发表时间：
2017-11
期刊：
Developmental dynamics : an official publication of the American Association of Anatomists
影响因子：
0
作者：
Duncan KM;Mukherjee K;Cornell RA;Liao EC
通讯作者：
Liao EC

Genes conserved in bilaterians but jointly lost with Myc during nematode evolution are enriched in cell proliferation and cell migration functions.