Molecular Functions of DPF1, DPF3, and PHF10 in BAF-Mediated Chromatin Remodeling - ABSTRACT All of DPF1, DPF3, and PHF10 are in the list of proteins open for study under this NOFO. DPF1, DPF3, and PHF10 are subunits of mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes, required for neuronal development, and associated with neurodevelopmental disorders and diseases, such as Nicolaides-Baraitser Syndrome (DPF1, DPF3, and PHF10), Baraitser-Winter Syndrome (DPF1, DPF3, and PHF10), Coffin-Siris Syndrome (DPF1 and PHF10), Autosomal Dominant Intellectual Developmental Disorder (DPF1 and PHF10), Autism Spectrum Disorder (DPF1, PHF10), and Hirschsprung Disease (DPF3). Mutations that impact these proteins also cause cancers. However, the molecular functions of these proteins are largely unknown. Genes encoding subunits of the BAF complexes are mutated in over 20% of human cancers. Mammalian BAF complexes exist as three major complexes -- cBAF, PBAF, and ncBAF, which differ based on their distinct subunit compositions. We have recently established the in vitro reconstitution system of cBAF, PBAF, and ncBAF, and we compared their biochemical activities. In addition, we reconstituted a variety of cBAF subcomplexes, defining roles for several subunits in high affinity nucleosome binding and nucleosome sliding activity. Remarkably, we found that the ARID1A subunit of cBAF, the most frequently mutated gene (~9 % of all types of cancer), is largely dispensable for nucleosome binding, nucleosome sliding, and ATPase activity, but ARID1A is required for cBAF to transfer histone octamers between DNA templates. Our study revealed the novel biochemical function of ARID1A in cBAF-mediated chromatin remodeling and suggests that histone octamer transfer activity is a key biochemical activity of BAF complexes for chromatin regulation. The overall objective of this proposed research is to determine biochemical roles for DPF1, DPF3, and PHF10 in BAF-mediated chromatin remodeling by exploiting a powerful combination of our in vitro reconstitution system and various biochemical and biophysical analyses. Given the limited time duration and budget of this NOFO, we will focus on investigating roles for these proteins in the biochemical activities of BAF complexes, especially in the histone octamer transfer activity of BAF complexes. This proposal has two specific aims. Aim1 will define roles for the DPF1 and DFP3 subunits in the biochemical activity of the cBAF complex. DPF1 and DPF3 are expressed specifically in brain and heart, whereas another isoform DPF2 is ubiquitously expressed in various tissues. DPF1, DPF2, and DPF3 are thought to be mutually exclusive subunits of cBAF. To define biochemical roles for DPF1 and DPF3 in BAF-medicated chromatin remodeling, we will reconstitute cBAF complexes containing either of DPF1, DPF2, or DPF3. We will also reconstitute a cBAF that lacks the DPF proteins (∆DPF complex). Then, we will measure various biochemical activities of these cBAF complexes, including nucleosome binding affinity, nucleosome sliding, and ATPase activity. We have recently developed the assays for monitoring the histone octamer transfer activity of BAF complexes. We will also measure the histone octamer transfer activity of these cBAF complexes. By comparing the biochemical activities of these complexes, we will determine biochemical roles for DPF1 and DPF3 in cBAF-mediated chromatin remodeling. We will also investigate impacts of histone modifications recognized by DPF1 and DPF3 on the BAF remodeling activity. Aim2 will determine a role for the PHF10 subunit in the biochemical activity of the PBAF complex. PHF10 is a PBAF-specific subunit. To define a role for PHF10 in the biochemical activity of PBAF, we will reconstitute a PHF10-containing PBAF complex (wild-type) and a PBAF complex that lacks PHF10 (∆PHF10 complex). We will measure various biochemical activities of these PBAF complexes, including nucleosome binding affinity, nucleosome sliding, and ATPase activity. In addition, we found that the not only cB
