Molecular and cellular mechanisms underlying Satb2-mediated variation in craniofacial disease

项目摘要 颅面畸形是人类出生缺陷中最常见和最令人衰弱的，影响1/500至 1/2000出生，取决于人口。颅颌面畸形的严重程度和分布 是一个被广泛观察到但却知之甚少的现象。这种变异引起复杂的临床问题， 例如在诊断、治疗和遗传咨询方面的困难， 颅面疾病特别是，人们普遍认为，类似的基因突变往往表达一种 疾病表型谱，但仍然不知道是什么机制导致这种变化。为 例如，具有SATB 2突变的人类患者表现出一系列颅面表型，包括小的 下颌（小颌畸形）和腭裂中的可变的倾斜度。同样，在小鼠中，Satb 2具有剂量效应 下巴的大小然而，引起这种变化的机制还没有很好地理解。Satb 2是一个矩阵附件 MAR区结合蛋白，已被证明可通过染色质调节成骨分化 组织，充当高阶转录因子。成骨细胞表达减少 分化基因被认为是Satb 2介导的颅面缺陷的基础。然而近日 已发表的数据表明Satb 2在骨生成中的作用更为复杂，包括调节前 成骨细胞增殖，也可能涉及DNA复制的作用。MAR与两种基因都有关联 Satb 2在成骨细胞的S期高表达，提示成骨细胞的凋亡可能与Satb 2的转录和DNA复制有关。 在DNA复制中的新作用。在该提议中要测试的具体假设是Satb 2在 MAR通过调节成骨细胞增殖相关基因的转录， 增殖和/或通过调节DNA复制。Satb 2水平的降低预计会降低 保真度并增加这些过程中的变化。这一假设将通过三个具体的 目标。具体目标1将确定Satb 2突变如何影响成骨祖细胞的基因表达。 具体目标2将确定细胞周期进展如何影响Satb 2在MAR上的定位。第3章将 确定Satb 2突变前成骨细胞的增殖如何受到核纤层蛋白A/C表达和氧化损伤的影响。 应激（先前发现Satb 2突变细胞中发生改变的因子）。实验将在两个 原代和永生化小鼠颅骨细胞。Satb 2突变细胞将与来自一个人的野生型细胞进行比较。 等基因背景本提案中的工作产生的数据将提供对以下新机制的深入了解： 成骨的satb 2调控。未来的研究计划包括进一步研究遗传， 发育和环境对受影响的患者疾病表型变化的贡献 SATB 2相关综合征（SAS）使用患者来源的iPSC。重要的是，这项工作也将大大 改善研究环境，为本科生提供令人兴奋的机会，进行高， 影响研究，为他们进入生物医学劳动力做好准备。

PROJECT SUMMARY Craniofacial anomalies are among the most common and debilitating human birth defects, affecting 1/500 to 1/2000 births depending on the population. Variation in the severity and penetrance of craniofacial anomalies is a widely observed, but poorly understood, phenomenon. This variation causes complex clinical problems, such as difficulties in the diagnosis, treatment, and genetic counseling of individuals affected by, or susceptible to, craniofacial disorders. In particular, it is widely recognized that similar genetic mutations often express a spectrum of disease phenotypes, but it is still unknown what mechanisms contribute to this variation. For example, human patients with mutations in SATB2 exhibit a range of craniofacial phenotypes, including small lower jaws (micrognathia) and variable penetrance in cleft palate. Similarly, in mice, Satb2 has a dosage-effect on jaw size. Yet, the mechanisms causing such variation are not well understood. Satb2 is a matrix attachment region (MAR)-binding protein that has been shown to regulate osteogenic differentiation through chromatin organization, acting as a high-order transcription factor. Reduction in the expression of osteogenic differentiation genes has been thought to underlie Satb2-mediated craniofacial defects. However, recently published data indicate a more complex role for Satb2 in osteogenesis that includes regulation of pre- osteoblast proliferation and may also involve a role in DNA replication. MARs are implicated in both gene transcription and DNA replication, and Satb2 is highly expressed in S-phase in osteoblasts, suggestive of a novel role in DNA replication. The specific hypothesis to be tested in this proposal is that Satb2 binding at MARs regulates osteogenic proliferation either through regulation of transcription of genes involved in proliferation and/or by regulation of DNA replication. Reductions in Satb2 levels are predicted to decrease the fidelity and increase variation in these processes. This hypothesis will be evaluated through three Specific Aims. Specific Aim 1 will determine how mutations in Satb2 affect gene expression in osteoblast progenitors. Specific Aim 2 will determine how cell cycle progression affects Satb2 localization on MARs. Specific Aim 3 will determine how proliferation in Satb2 mutant pre-osteoblasts is affected by Lamin A/C expression and oxidative stress (factors previously found to be altered in Satb2 mutant cells). Experiments will be performed in both primary and immortalized mouse calvarial cells. Satb2 mutant cells will be compared to wild-type cells from an isogenic background. Data generated from work in this proposal will provide insight into a novel mechanism for Satb2 regulation of osteogenesis. Future research plans include further investigation of genetic, developmental, and environmental contributions to variation in disease phenotypes in patients affected by the SATB2-associated syndrome (SAS) using patient-derived iPSCs. Importantly, this work will also substantially enhance the research environment and provide exciting opportunities for undergraduates to conduct high- impact research, preparing them to enter the biomedical workforce.