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

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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.