High confidence ASD (hcASD) genes encode proteins that regulate synapse formation, neuronal activity and transcription regulation, including chromatin regulation. In addition to  Chd8, several less-studied chromatin regulators are also strongly associated with ASD risk, including pogo transposable element with ZNF domain (Pogz) and histone-lysine N-methyltransferase Suv420h1. POGZ and SUV420H1 are part of a gene-silencing pathway that produces heterochromatin, a compact chromatin state inaccessible to transcription factors that is distinguished by the H3K9me3 and H4K20me3 histone modifications. John Rubenstein hypothesizes that reduction of heterochromatin, resulting from either Pogz or Suv420h1 mutations, predisposes individuals to ASD by disrupting neuronal development and function.

Rubenstein proposes that studying ASD mutations in these genes has the potential to yield novel insights into the role of chromatin modifiers in ASD pathogenesis. Co-expression network analysis provides evidence that multiple ASD genes impact human brain function by disrupting midfetal development of the frontal cortex. Rubenstein’s team will focus on the roles of Pogz and Suv420h1 in the histogenesis of the mouse cortex and in gene regulation in maturing and mature cortical neurons. Pogz is particularly tractable for study, as established ASD rare variants are predicted to result in mutations in specific functional domains of this protein, thereby elucidating modules of the POGZ protein that contribute to ASD risk.

Specific Aim 1: Define the cortical phenotype of developing and adult Pogz mutant mice using RNA-seq transcriptomic analyses, histological analysis of cortical region and laminar patterning, and cortical neuron differentiation and the identification of developmental loci regulated by POGZ using ChIP-sequencing (ChIP-seq) in wild type versus mutant.

Specific Aim 2: Elucidate the effect of de novo ASD variants in POGZ using both missense mutations in POGZ zinc finger domain and truncating mutations in POGZ transposase domain.

Specific Aim 3: Define the cortical phenotype of conditional Suv420h1/h2 mouse mutants using RNA-seq transcriptomic analyses, histological analysis of cortical regional and laminar patterning, and cortical neuron differentiation and the identification of developmental loci regulated by Suv420h1/h2 using ChIP-seq in wild type versus mutant.

This study will provide novel information about the role of repressive chromatin in contributing to phenotypes that increase the risk for ASD. Although Pogz and Suv420h1 variants account for a small proportion of ASD cases, Rubenstein proposes that other ASD-associated chromatin modifiers could similarly be involved in dynamic epigenetic regulation during development. Therefore, these findings should be highly relevant to generally understanding how ASD is caused by alterations in the epigenetic state during cortical development. In addition, this study will identify cis-regulatory domains related to cortical development that, in the context of whole genome sequencing efforts, may identify noncoding mutations that predispose to ASD.