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) 致病机制的理解受到以下事实的限制：这种疾病的遗传风险有一半以上归因于特定基因。走向识别在外显子组和全基因组中检测到的许多不相关序列中的病理序列变异患有这种疾病的患者，了解控制该疾病的基因调控网络（GRN）相关组织（包括口腔周皮）的发育至关重要。我们提出了系统生物学分析周皮 GRN 的方法。过去使用这种方法使我们能够识别出三种新颖的 OFC 风险基因。我们将利用两种模型生物，斑马鱼和小鼠，因为周皮分化GRN 似乎是高度保守的。斑马鱼的周皮分化很早胚胎发生，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，它的优点是其颅面解剖结构与人类更相似。在目标 1 中，我们将使用最先进的网络推理算法确定斑马鱼周皮分化 GRN， NetProphet 2。该工具可进行共表达分析和差异表达分析。输入数据集将包括我们将从 4 个功能丧失 (LOF) 胚胎中生成的 RNA-seq 表达谱已知参与此 GRN 的关键转录因子 (TF)。我们还将确定以下基因的直接联系：这些关键的 TF 位于周皮 GRN 中。最后，我们将测试 periderm GRN 的一个新候选成员， Tead 通过在斑马鱼中进行 LOF 测试，从而利用了该模型系统的优势。在目标 2 中，我们将同样使用 NetProphet 算法推导出小鼠口腔周皮分化 GRN。输入数据集将包括从野生型小鼠胚胎上颚架分离的周皮的表达谱，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，有周皮分化异常的证据。我们还将通过以下方式确定小鼠周皮增强剂候选者：从 Krt17-gfp 转基因胚胎中分选 GFP 阳性和阴性细胞，对两者进行 ATAC-seq 对上颚架和鼻腔细胞进行 H3K27Ac ChIP-seq。正如目标 1 一样，我们将还确定了关键转录因子的直接基因联系。我们将训练味觉机器学习算法周皮增强子，并使用所得的评分函数来优先考虑靠近基因的 OFC 相关 SNP 对于那些可能直接影响 OFC 风险的因素，在周皮中表达。最后，我们将执行等位基因对三个位点中每个位点的顶级候选 SNP 进行特定报告分析。预期的结果是更深层次的了解控制周皮分化的特定转录因子和顺式调控元件。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异来自 OFC 的全外显子组和基因组测序分析。

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）

Beyond MITF: Multiple transcription factors directly regulate the cellular phenotype in melanocytes and melanoma.

超越 MITF：多种转录因子直接调节黑色素细胞和黑色素瘤的细胞表型。

DOI：
发表时间：
2017
期刊：
影响因子：
4.3
作者：
Seberg, Hannah E;Van Otterloo, Eric;Cornell, Robert A
通讯作者：
Cornell, Robert A

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

两侧对称动物中保守但在线虫进化过程中与 Myc 共同丢失的基因在细胞增殖和细胞迁移功能方面得到丰富。

DOI：
发表时间：
2015-09
期刊：
影响因子：
2.4
作者：
Erives; Albert J
通讯作者：
Albert J

A single nucleotide polymorphism associated with isolated cleft lip and palate, thyroid cancer and hypothyroidism alters the activity of an oral epithelium and thyroid enhancer near FOXE1.

与唇腭裂、甲状腺癌和甲状腺功能减退相关的单核苷酸多态性会改变 FOXE1 附近的口腔上皮和甲状腺增强子的活性。

DOI：
10.1093/hmg/ddv047
发表时间：
2015-07-15
期刊：
Human molecular genetics
影响因子：
3.5
作者：
A. Lidral;Huan Liu;S. Bullard;Gregory Bonde;J. Machida;A. Visel;L. M. Uribe;Xiao Li;B. Amendt
通讯作者：
B. Amendt

Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis.

斑马鱼上颚形态发生过程中对 wnt9a 和 irf6 在延伸和整合机制中的不同要求。

DOI：
发表时间：
2013-01-01
期刊：
影响因子：
0
作者：
Dougherty, Ma;Kamel, George;Grimaldi, Michael;Gfrerer, Lisa;Shubinets, Valeriy;Ethier, Renee;Hickey, Graham;Cornell, Robert A;Liao, Eric C
通讯作者：
Liao, Eric C

Zebrafish models of orofacial clefts.

口面裂的斑马鱼模型。