Project Summary Aberrations in chromatin structure can be a causal mechanism of numerous human disease and developmental syndromes. R-loops are RNA containing chromatin structures that are deregulated in numerous developmental disorders and cancers. In most cases the underlying mechanisms and the functional significance of R-loop deregulation and whether they are causal to specific disease is not clear. Activity dependent neuroprotective protein (ADNP) is a homeodomain containing transcriptional repressor that is critical for neuronal differentiation and neurodevelopment. ADNP mutations cause ADNP syndrome, a condition characterized by intellectual disability and features of autism spectrum disorder. ADNP is upregulated during neurodevelopment. ADNP contains zinc finger motifs and a homeodomain, both of which can bind nucleic acids. ADNP syndrome mutations result in protein products that lack the homeodomain, underscoring the importance of this region to ADNP function. However, the precise contributions of the zinc fingers and homeodomain to ADNP localization and function in gene expression during neuronal differentiation is not well understood. Our preliminary data have uncovered a novel role for ADNP in the resolution of R-loops. Biochemical assays revealed that ADNP resolves R-loops – in a homeodomain dependent manner. ADNP also suppresses R-loops in vivo, as deletion of ADNP in mouse embryonic stem cells (mESCs) resulted in R-loop accumulation specifically at ADNP target sites, but not at sites not bound by ADNP. Last, ADNP-deficient mESCs and mESCs exclusively expressing an ADNP mutant lacking the homeodomain fail to differentiate into neural progenitor cells (NPCs), indicating an essential role for ADNP and its homeodomain in neuronal differentiation. These results suggest a potentially novel mechanism – R-loop resolution – for ADNP, and possibly other homeodomain-containing proteins, in gene regulation, which can link R-loop dysfunction to numerous disorders and diseases caused by mutations in homeodomain proteins. Based on our preliminary data, we hypothesize that ADNP drives neuronal differentiation by suppressing R-loops; as a corollary, we propose that resolving R-loops that accumulate upon ADNP loss may rescue gene expression programs to enable neuronal differentiation. We propose two aims to test our central hypothesis. In Aim 1, we will elucidate the molecular basis of R-loop resolution by ADNP by identifying roles of the ADNP protein domains and their interactions with nucleic acids. In Aim 2, we will elucidate how ADNP suppression of R-loops influences neuronal differentiation.