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.
