Six1 Co-factors in Craniofacial Development

鳃海绵肾谱系障碍（BOS）的特点是颅面缺陷，包括畸形 鳃弓（BA）、外耳、中耳和内耳的病变;一部分患者还患有肾缺陷。 两个致病基因与BOS诊断相关，但这些基因占不到一半， 患者病例：SIX 1转录因子和辅因子蛋白EYA 1，其结合SIX 1并修饰其表达。 转录活性因此，超过一半的BOS患者的致病基因尚未确定。我们 假设有其他关键辅因子蛋白与SIX 1结合以调节其活性， 这些辅因子的突变导致了BOS的未知原因。本研究的目的 该计划的目的是在易处理的模型系统中识别出功能改变的其他基因， 因此，这些基因最终可以被包括在人类遗传筛查。 利用果蝇相互作用组中Six 1的果蝇同源物数据，我们鉴定了11种新的推定辅助因子 在非洲爪蟾中发现，其中大部分在发育中的BA，耳朵和肾脏中表达，因此 可能与BOS有关。这些蛋白质在人类中高度保守，我们的初步数据显示， 其中Sobp、Zmym 2、Zmym 4、2G 4、Mcrs 1是胚胎发育所必需的 鳃弓（神经嵴[NC]）、中耳（NC）和内耳（耳基板）的前体。在目标1中， 我们将使用功能获得和功能丧失的方法来确定这些候选辅因子是否发挥作用 NC的形成或迁移，鳃弓软骨或内耳基因的表达和形成。在目标2中， 将评估这些基因产物与Six 1的生化相互作用，以及它们是否影响Six 1 转录功能在目标3中，我们将确定SIX 1中已知的BOS突变是否影响候选基因。 辅因子结合或功能，并绘制Six 1和Six 2的蛋白质-蛋白质相互作用结构域的区域。 每个辅因子介导结合和转录活性。我们以前的工作和建立的模型系统 独特的位置，我们来验证这些候选人是否是真正的Six 1辅因子，并阐明他们如何 有助于正常和畸形颅面发育。这些分析将提供重要的 无法从有限的可用患者材料中获得的信息。他们也有未来 可能解释BOS患者的表型变异性，并为纳入新的 BOS基因板中的致病基因。

Branchiootorenal spectrum disorders (BOS) are characterized by craniofacial defects that include malformation of branchial arches (BAs), external ear, middle ear, and inner ear; a subset of patients also has kidney defects. Two causative genes are associated with BOS diagnoses, but these genes account for fewer than half of patient cases: the SIX1 transcription factor and a co-factor protein, EYA1, which binds to SIX1 and modifies its transcriptional activity. Thus, the causative genes for over half of BOS patients are yet to be identified. We hypothesize that there are other key co-factor proteins that bind to SIX1 to regulate its activity, and that mutations in these co-factors contribute to the unknown causes of BOS. The goal of this research program is to identify, in tractable model systems, additional genes whose altered functions contribute to the craniofacial malformations of BOS so that these genes can ultimately be included in human genetic screening. Using the Drosophila interactome data for the fly homologue of Six1, we identified 11 novel putative co-factors in Xenopus and showed that most of these are expressed in the developing BAs, ear and kidney, and therefore are potentially relevant to BOS. These proteins are highly conserved in humans, and our preliminary data show that five of them (Sobp, Zmym2, Zmym4, 2G4, Mcrs1) are required for development of the embryonic precursors of the branchial arches (neural crest [NC]), middle ear (NC) and inner ear (otic placode). In Aim 1, we will use gain- and loss-of-function approaches to determine whether these candidate cofactors play a role NC formation or migration, branchial arch cartilages or inner ear gene expression and formation. In Aim 2, we will evaluate the biochemical interactions of these gene products with Six1 and whether they affect Six1 transcriptional function. In Aim 3, we will determine whether the known BOS mutations in SIX1 affect candidate co-factor binding or function, and map what regions of the protein-protein interaction domains of Six1 and of each co-factor mediate binding and transcriptional activity. Our previous work and established model systems uniquely position us to validate whether these candidates are bone fide Six1 co-factors, and elucidate how they contribute to normal and dysmorphic craniofacial development. These analyses will provide important information that cannot be obtained from the limited patient material available. They also have the future potential to explain the phenotypic variability in BOS patients and provide a rationale for including new causative genes in BOS gene panels.