ARID1 proteins orchestrate cranial neural crest differentiation

(Abstract) Cranial neural crest cells (CNCCs) are a unique, multipotent, migratory population that give rise to both ectodermal/non-ectomesenchymal (neurons, glia) and mesodermal/ectomesenchymal (bone, cartilage) derivatives. The cellular and molecular mechanisms that allow for proper craniofacial development, including establishing multipotency, epithelial to mesenchymal transformation (EMT) and migration; however, remain controversial and heavily focus on transcription factor expression. BAF is one of the major protein complexes regulating chromatin accessibility. AT-rich interactive domain- containing proteins 1A and 1B (ARID1A, ARID1B) are the largest, mutually exclusive, subunits of BAF and mutations in these subunits result in Coffin-Siris Syndrome (CSS), a disorder characterized by craniofacial phenotypes. Our previous work revealed a dynamic requirement for ARID1A/B during CNCC development in which ARID1A was required for maintenance of pluripotency and then as iPSCs differentiated into neurosphere intermediates, was decommissioned, and replaced by ARID1B to initiate commitment to the neuroectodermal lineage via the repression of thousands of pluripotency enhancers and genes. Upon differentiation of neurospheres into CNCCs, ARID1B was downregulated and ARID1A was reactivated. We hypothesize that the return of ARID1A in CNCCs is essential for multipotency, EMT, and migration of CNCCs, and that as CNCCs differentiate, ARID1B is reactivated to ensure lineage commitment. Interestingly, there is potential integration of chromatin remodeling activity with key developmental signaling pathways whereby the induction of CNCCs is a Wnt-dependent process and phenotypes associated with loss of ARID1A/B are similar to mutations linked to the Hedgehog (Hh) pathway. Further, our preliminary data identified an enrichment for TCF/LEF and ZIC2/3 binding sites in ARID1A bound regions and proteomic studies highlighted physical interactions with the transcription factor, Gli3. Gli3 acts as a bimodal regulator (e.g., can function as both an activator and repressor) of the Hh signaling pathway and has not previously been ascribed chromatin remodeling capability. Thus, we hypothesize a novel mechanism of Gli regulation which utilizes Arid1 subunits. Herein, we propose three distinct Aims to test our hypotheses. Aim 1: will test if ARID1A regulates multipotency, induction, EMT, and migration of CNCCs, by cooperating with pluripotency factors (OCT4, SOX2, NANOG), Wnt signaling transcription factors (LEF1/TCF), and ZIC2. Aim 2: will determine if ARID1A/ARID1B mediated mechanisms (chromatin remodeling and/or RNA Pol II pausing) are necessary for CNCC differentiation. Aim 3: will determine if Gli3 cooperates with ARID1A and ARID1B to drive transcriptional activation and repression, respectively. The proposed studies will shed new light on cellular and molecular mechanisms required for CNCC induction, multipotency, and differentiation. Furthermore, they will be the first studies to reveal mechanisms by which the BAF complex contributes to transduction of signaling pathways (e.g., Hh and Wnt) essential for craniofacial development. Project Number: 1R01DE033986-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Samantha Brugmann | Institution: CINCINNATI CHILDRENS HOSP MED CTR, CINCINNATI, OH | Award Amount: $493,458 | Activity Code: R01 | Study Section: Skeletal Biology Development and Disease Study Section[SBDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11127195