Identification of SIX1-related genes as potential candidates for craniofacial birth defects

鉴定 SIX1 相关基因作为颅面出生缺陷的潜在候选基因

基本信息

批准号：
9807629
负责人：
Andre L P Tavares
金额：
$ 15.95万
依托单位：
GEORGE WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9807629
关键词：
Address Affect Alleles Applications Grants Area Binding Biological Assay Birth Branchial arch structure Cell Culture Techniques Cell Line Cephalic Congenital Abnormality Craniofacial Abnormalities Data Defect Deformity Disease Dysmorphology Ear Embryo Ensure Expression Profiling Face Fibrinogen First Pharyngeal Arch Future Gene Targeting Genes Genetic Genetic Transcription Genotype Goals Heterozygote Human Immunofluorescence Immunologic Immunofluorescence Microscopy In Situ Hybridization Incisor Individual Jaw Kidney Knockout Mice Laboratories Luciferases Mandible Maxilla Mesenchyme Mission Morphogenesis Mus Mutation National Institute of Dental and Craniofacial Research Neural Crest Cell Patients Pattern Phenotype Precipitation Proteins Reagent Regulation Regulator Genes Reporter Role Sampling Structure Syndrome Testing Time Tooth structure Transcription Coactivator Transcription Repressor/Corepressor Work base craniofacial craniofacial development craniofacial disorder cranium dosage gene interaction hearing impairment knock-down middle ear migration neural patterning novel overexpression transcription factor transcriptome sequencing vector

项目摘要

ABSTRACT Many congenital craniofacial disorders result from abnormal regulation of cranial neural crest cell (NCC) formation, migration or patterning within the mandibular portion of pharyngeal arch 1 (mandibular arch). The transcription factor SIX1 is critical for craniofacial development with mutations in SIX1 and its co-factor EYA1 identified as the underlying genetic causes of around 50% of Branchiootorenal spectrum disorder (BOS) cases. BOS is an autosomal dominant disorder, in which affected individuals that have one normal allele present variable degrees of craniofacial defects, hearing impairment, and renal abnormalities. Preliminary data from mice demonstrate that decreased levels of Six1 in heterozygotes (Six1+/-) affect the expression of four genes related to craniofacial development (Gbx2, Dlx3, Dlx2 and Hand2) more significantly than complete Six1 loss (Six1-/-); and that known (Eya1, Eya2) and putative (Pa2g4, Mcrs1, Sobp) co-factors are expressed in distinct regions of the mandibular arch with Six1. Thus, it is possible that normal dosage of Six1 regulates morphogenesis of multiple NCC-derived structures by inducing or repressing downstream genes, and that these transcriptional activities are the consequence of interactions with different levels of co-factors in distinct domains of the mandibular arch. In this R03 application I aim to identify: 1) SIX1-regulated genes within two specific mandibular arch domains; and 2) the functional interactions between SIX1 and putative co-factors PA2G4, MCRS1 and SOBP. These goals will be addressed in two specific aims. In Aim 1, genes that require proper Six1 dosage will be identified in the ventral plus intermediate domains of the mandibular arch. RNA-seq of dissected domains of the arch from Six1 heterozygotes and Six1-nulls will provide a list of genes that require normal levels of Six1. The top 10 most significantly affected genes from each genotype will be validated by quantitative real-time PCR and whole-mount and sectional RNAscope In Situ Hybridization (ISH). In Aim 2, the expression patterns of potential co-factors PA2G4, MCRS1 and SOBP will be characterized in relation to that of SIX1 in mouse embryos by dual probe whole-mount ISH and Immunofluorescence microscopy. Putative co- factors that overlap with SIX1 will be assayed in the NCC line O9-1 for whether they bind to SIX1, modulate SIX1 transcriptional activity and regulate the expression of SIX1-regulated genes from Aim 1. Results will provide a deeper understanding of SIX1 function and ultimately provide the key to understanding the underlying genetics of the remaining BOS cases or other craniofacial disorders. Validated SIX1-regulated genes and identified co-factors will be studied in future R01 applications in relation to BOS and other craniofacial disorders.

抽象的许多先天性颅面疾病是由颅神经嵴细胞（NCC）调节异常引起的咽弓 1（下颌弓）下颌部分内的形成、迁移或图案化。这转录因子 SIX1 对于颅面发育至关重要，SIX1 及其辅助因子 EYA1 发生突变被确定为约 50% 文昌鱼肾谱系障碍 (BOS) 病例的潜在遗传原因。 BOS 是一种常染色体显性遗传疾病，受影响的个体存在一个正常等位基因不同程度的颅面缺陷、听力障碍和肾脏异常。初步数据来自小鼠证明杂合子中 Six1 水平的降低 (Six1+/-) 会影响四个基因的表达与颅面发育（Gbx2、Dlx3、Dlx2 和 Hand2）的相关性比 Six1 完全丧失更显着（六1-/-）；已知的（Eya1、Eya2）和推定的（Pa2g4、Mcrs1、Sobp）辅助因子以不同的形式表达 Six1 的下颌弓区域。因此，Six1 的正常剂量可能会调节通过诱导或抑制下游基因来实现多个 NCC 衍生结构的形态发生，并且这些转录活性是不同水平的辅因子相互作用的结果下颌弓的区域。在此 R03 应用中，我的目标是确定：1) 两个内的 SIX1 调节基因特定的下颌弓区域； 2) SIX1 和假定的辅助因子之间的功能相互作用 PA2G4、MCRS1 和 SOBP。这些目标将通过两个具体目标来实现。在目标 1 中，需要的基因正确的 Six1 剂量将在下颌弓的腹侧和中间区域确定。 RNA测序来自 Six1 杂合子和 Six1 无效的拱门的解剖结构域将提供需要的基因列表 Six1 的正常水平。每个基因型中影响最显着的前 10 个基因将通过实时定量 PCR 以及整体和切片 RNAscope 原位杂交 (ISH)。在目标 2 中，潜在辅助因子 PA2G4、MCRS1 和 SOBP 的表达模式将与以下相关进行表征通过双探针整体原位杂交 (ISH) 和免疫荧光显微镜检测小鼠胚胎中的 SIX1。推定共同与 SIX1 重叠的因子将在 NCC 系 O9-1 中进行测定，以确定它们是否与 SIX1 结合、调节 SIX1 转录活性并调节目标 1 中 SIX1 调节基因的表达。结果将提供对 SIX1 功能更深入的理解，并最终提供理解 SIX1 功能的关键其余 BOS 病例或其他颅面疾病的潜在遗传学。经 SIX1 监管验证基因和已确定的辅助因子将在未来与 BOS 和其他相关的 R01 应用中进行研究颅面疾病。