Neurodevelopmental chromatin regulation by Polycomb Repressor Complex (PRC1). Polycomb repressor complexes 1 & 2 play diverse and fundamental roles in chromatin regulation, however their functions in the brain have been explored to a limited degree, despite linkages to neurologic disease. We found that the PRC1 product ubiquitinated histone H2A (H2A119ub) is remarkably dynamic during neurodevelopment, undergoing enrichment in the surprising context of euchromatin, especially at active enhancers. Currently we are exploring how H2AK119ub represses genes that are required during neurodevelopment but become obsolete in the mature brain. We are also performing Hi-ChIP for H2AK119ub to determine how this modification regulates enhancer function during neurodevelopment. 

Bap1 in neurodevelopmental chromatin regulation. To probe the neuronal functions of H2AK119ub, we generated brain-specific mutants in Bap1 (BRCA1-associated protein), which encodes a deubiquitinating enzyme that erases H2AK119ub. Patients with developmental delay due to BAP1 mutations were recently described, and mutant mice exhibit severe developmental delay with behavioral abnormalities and epilepsy. At the transcriptional level, we observe altered expression of many synaptic genes, and chromatin mapping using CUT&RUN demonstrated perturbation of H2AK119ub as well as several other essential regulatory histone modifications, including those associated with euchromatin. We are now applying a combination Hi-C and Hi-ChIP to understand how Bap1 loss impacts chromatin conformation and the formation of enhancer-promoter loops.

Regulation of heterochromatin by ubiquitin-mediated protein degradation. We previously identified key targets of the ubiquitin ligase Anaphase-Promoting Complex (APC) in differentiating neurons, in particular Ki-67 and the Chromosome Passenger Complex (CPC). Consisting of INCENP, Aurora B, Survivin and Borealin, the CPC regulates histone phosphorylation (H3S10ph) within constitutive heterochromatin in differentiating neurons. In collaboration with the Nick Brown lab at UNC-Chapel Hill, we are now using structural studies to understand how the APC recognizes and ubiquitinates the CPC, with implications for the degradation of protein complexes by the ubiquitin-proteasome system more broadly. 

Regulation of phase separation of neuronal heterochromatin by Ki-67. A major substrate of the APC in neurons which we identified in mouse models of the ANAPC7 neurodevelopmental syndrome (OMIM 619699, Ferguson-Bonni disorder), Ki-67 is a massive and highly disordered protein. During mitosis, Ki-67 organizes the chromosome periphery, whereas after mitotic exit Ki-67 contributes to the formation of heterochromatin. But how it accomplishes these feats is poorly understood. We have generated mouse models of Ki-67 loss in the brain and are currently exploring the impacts on neurodevelopment through a combination of microscopy, genomics and behavior assays. We are also collaborating with the Galia Debelouchina lab at UCSD to perform in vitro phase separation assays using minimally reconstituted chromatin assays. 

Role of Fig4 in neurodegeneration. Fig4, along with Vac14 and PIKfyve/Fab1, comprise the biosynthetic complex which regulates the lysosomal signaling lipids PI(3,5)P2 and PI5P. Mutations in Fig4 cause amyotrophic lateral sclerosis (ALS, LouGehrig’s disease) and a fatal form of Charcot-Marie-Tooth disease CMT4J. We carried out the first comprehensive interrogation of proteinopathy in the brain of Fig4 and Vac14 mutant mice, which revealed pathogenic connections to Alzheimer and Parkinson diseases, while also identifying molecular mediators of inflammatory cell death. We have now collaborated with Andrew Mendiola’s lab at UCSD to understand how microglia contribute to neuroinflammation and oxidative damage in the brain during neurodegeneration, and are employing cell-specific mouse models to understand the interaction between neurons, astroglia and microglia using a combination of proteomics, imaging and transcriptomic analyses.