Acquiring tissue diversity without a gradient: investigating Satb2 as a mandible-specific mediator of Gli3 transcription

About This Grant

Project Summary The concept of a morphogen gradient, where cell fate choices are determined along a concentration gradient of a diffusible signaling factor, has been a paradigm in developmental biology for decades. A textbook example of this paradigm is transduction of the Sonic Hedgehog (Shh) pathway, which is mediated by the glioma-associated oncogene (Gli) family of transcription factors (TFs). Classic studies in the developing limb and neural tube suggest that Gli3 activity is dependent upon a Shh morphogen gradient, with the gradient concentration dictating the robustness of the Gli transcriptional response, and subsequent cell type specification. In contrast, our previous work has shown that in the developing mandible, Gli3 activity does not require a Shh gradient. Instead, interactions with co-factors regulate transcriptional output. Elucidating how specific co-factor interactions can mediate distinct cell fate programs in the absence of a gradient could create a new paradigm for modeling tissue development and disease and is the overarching objective of this proposal. We previously reported that Gli3 binds cis-regulatory regions via low affinity binding motifs that require specific co-factors to initiate a gene regulatory network. Specifically, we described a synergistic transcriptional output when Gli3 and Hand2 utilized a low affinity divergent Gli binding motif (dGBM) in regions outside of the highest Shh concentration. Additional TF motifs were found in close proximity to these regions, suggesting that Gli3 and Hand2 may interact with other TFs at dGBMs to execute distinct gene regulatory networks. Our preliminary data provide evidence that Special AT-Rich Sequence-Binding Protein (Satb2) and Gli3 physically interact in association with chromatin in cranial neural crest cells (CNCCs) of the developing mandible (MNP). Satb2 is a compelling candidate to mediate Gli3 activity in the mandible because it is a bona-fide transcription factor and has been ascribed chromatin remodeler function. Additionally, mandibular phenotypes associated with loss of function of Satb2, Hand2, and Gli3 are similar. Based on these data, we hypothesize that Satb2 is a novel co-factor for Gli3 in mandibular CNCCs regulating genes within a skeletal GRN. We will test this hypothesis through two specific aims: In Specific Aim 1, we will determine if Satb2 is required for Gli-mediated transcription in the MNP. In Specific Aim 2, we will determine if Satb2 is required for binding of Gli3 at a sub-set of loci associated with genes essential for skeletal differentiation. This proposal will provide crucial preliminary data for new studies investigating 1) how signaling pathways (e.g., Shh/Gli3) confer graded responses without a gradient and 2) how pleiotropic genes (e.g., Gli3/Satb2) execute cell-type specific functions. These new studies have the potential to elucidate paradigm- shifting mechanisms regulating early development and patterning as well as contribute to novel precision medicine-based therapies. Future work: Our future work will explore if/how combinatorial interactions between Gli3, Satb2, and/or Hand2 activate lineage specific outputs and how specific disease causing SATB2 variants impact its ability to interact with Gli3 (and/or Hand2), predicting tissue specific phenotypes.