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

项目总结 颅面畸形是人类最常见和最令人衰弱的出生缺陷之一，影响到1/500到 1/2000出生率取决于人口。颅面畸形的严重程度和外显率的变化 这是一种被广泛观察到但却鲜为人知的现象。这种变异会导致复杂的临床问题， 例如，在诊断、治疗和遗传咨询方面有困难的患者或易感人群 致，颅面部疾患。特别是，人们广泛认识到，类似的基因突变通常表达一种 疾病表型谱，但目前仍不清楚是什么机制导致了这种变异。为 例如，SATB2基因突变的人类患者表现出一系列头面部表型，包括小 下颌骨(小颌下巴)和腭裂的不同外露。同样，在小鼠身上，Satb2也有剂量效应 在下巴大小上。然而，导致这种差异的机制还没有被很好地理解。Satb2是一种基质附件 区域(MAR)结合蛋白，已被证明通过染色质调节成骨分化 组织，发挥高位转录因子的作用。成骨相关基因表达的下调 分化基因被认为是Satb2介导的头面部缺陷的基础。不过，最近， 已发表的数据表明，Satb2在成骨过程中扮演着更复杂的角色，包括对Pre-2的调节。 成骨细胞的增殖也可能参与DNA复制。火星与这两种基因都有关联 转录和dna复制，而且sTb2在成骨细胞的S期高表达，提示 在DNA复制中的新角色。在这项提议中要检验的具体假设是，Satb2结合在 MARS通过调控相关基因的转录调控成骨细胞的增殖 增殖和/或通过调节DNA复制。Sat b2水平的降低预计将降低 在这些过程中，保真度和变化性都会增加。这一假设将通过三个具体的 目标。具体目标1将确定Satb2基因突变如何影响成骨细胞前体细胞的基因表达。 特定目标2将确定细胞周期进程如何影响Satb2在火星上的定位。具体目标3将 确定Lamin A/C表达和氧化对Satb2突变前成骨细胞增殖的影响 压力(以前在Satb2突变细胞中发现的改变的因素)。实验将在这两个地方进行 原代和永生化的小鼠颅骨细胞。Satb2突变细胞将与来自中国的野生型细胞进行比较 同源基因背景。本提案中的工作生成的数据将为以下方面提供新的机制 Satb2对成骨的调节作用。未来的研究计划包括进一步研究基因， 发育和环境因素对疾病表型变异的影响 使用患者来源的IPSCs的SATB2相关综合征(SAS)。重要的是，这项工作还将实质上 优化研究环境，为本科生提供进行高层次研究的机会 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.