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

• 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
• 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

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.

摘要 颅神经嵴细胞（NCC）的异常调节是导致许多先天性颅面疾病的重要原因 在咽弓1的下颌部分（下颌弓）内的形成、迁移或图案化。的 转录因子SIX 1对颅面发育至关重要，SIX 1及其辅因子EYA 1突变 被确定为约50%的鳃海绵肾谱系障碍（BOS）病例的潜在遗传原因。 BOS是一种常染色体显性遗传疾病，其中受影响的个体具有一个正常等位基因 不同程度的颅面缺陷、听力障碍和肾脏异常。的初步数据 小鼠证明，杂合子（Six 1 +/-）中Six 1水平的降低会影响四种基因的表达， 与颅面发育（Gbx 2，Dlx 3，Dlx 2和Hand 2）相关性比完全Six 1缺失更显著 （Six 1-/-）;已知的（Eya 1，Eya 2）和推定的（Pa 2g 4，Mcrs 1，Sobp）辅因子在不同的细胞中表达， 下颌弓的区域与Six 1。因此，正常剂量的Six 1可能调节 通过诱导或抑制下游基因的多种NCC衍生结构的形态发生， 这些转录活性是与不同水平的辅因子相互作用的结果， 在这个R 03应用程序中，我的目标是确定：1）SIX 1调节基因在两个 特定的下颌弓区域; 2）SIX 1和推定的辅助因子之间的功能相互作用 PA 2G 4、MCRS 1和SOBP。这些目标将在两个具体目标中实现。在目标1中，需要 将在下颌弓的腹侧加中间区域中鉴定适当的Six 1剂量。 从Six 1杂合子和Six 1-null中切割出的拱门结构域将提供一个基因列表， 正常水平的6。每个基因型的前10个受影响最显著的基因将通过以下方法进行验证： 实时定量PCR和整体和部分RNA原位杂交（ISH）。在目标2中， 潜在的辅因子PA 2G 4、MCRS 1和SOBP的表达模式将根据以下特征进行表征： 通过双探针整体包埋ISH和免疫荧光显微镜观察小鼠胚胎中SIX 1的表达。推定的共同- 与SIX 1重叠的因子将在NCC细胞系O 9 -1中进行测定，以确定它们是否与SIX 1结合，调节SIX 1的表达， SIX 1的转录活性和调节SIX 1调节基因的表达来自Aim 1。结果将 提供了对SIX 1功能的更深入理解，并最终提供了理解 其余BOS病例或其他颅面疾病的潜在遗传学。经验证的SIX 1调节 基因和确定的辅因子将在未来的R 01应用中研究与BOS和其他 颅面疾病