While autism (ASD) is a heterogeneous disorder, a significant fraction of ASD genetic risk is conferred by genes encoding for relatively selective functions, including synaptic proteins, and regulators of chromatin structure and gene expression. These observations suggest an overlap between ASD causal mechanisms; however, such points of convergence at the molecular and circuit level are largely unknown. Many ASD risk genes, particularly those encoding for ion channels and synaptic scaffolding proteins, tend to be longer (> 80 kb) than most expressed neuronal genes. Additionally, genes implicated in several syndromic forms of autism, such as MECP2 (Rett syndrome) and FMR1 (Fragile X syndrome (FXS)), preferentially regulate the expression of long genes, including other ASD risk genes. These results indicate that gene length could explain how mutations in gene regulatory mechanisms manifest with synaptic and circuit-level dysfunction in ASD. A deeper understanding of the molecular mechanisms that govern the expression of long neuronal genes could therefore inform new strategies for therapeutic intervention in ASD.

The transcription of long ASD-related genes is dependent on the DNA topoisomerases, TOP1 and TOP2B. Preliminary studies indicate that TOP2B activity within the genome is epigenetically regulated and stimulated in chromatin enriched in H3K36me3. Loss of SETD2, the enzyme that catalyzes H3K36 trimethylation, depletes TOP2B activity in the mouse brain. H3K36me3 suppresses spurious intragenic transcription, especially within long genes, and preliminary data suggest a similar role for TOP2B. Both TOP2B and SETD2 are ASD-risk genes and their interaction suggests a convergent mechanism that regulates the transcription of long ASD-related genes. The proposed research will investigate if TOP2B activity in SETD2 mutant mice activates cryptic transcription in long genes, which suppresses genic transcription by triggering collisions between RNA polymerases (RNAPII). Dr. Madabushi and team will compare gene expression patterns in excitatory cortical neurons lacking either SETD2 or TOP2B, and assess how TOP2B activity distributes in neurons without SETD2 expression. Preliminary data suggests that heterozygous SETD2 deletion (SETD2c Het) or Fmr1KO, results in hyperexcitability of developing neocortical circuits, in the form of long, persistent activity, or Up, states that is mediated by enhanced metabotropic glutamate receptor 5 (mGluR5), cannabinoid receptor 1 (CB1R) function and de novo protein synthesis. Therefore the team will investigate if reduced expression of long synaptic scaffolding proteins in Setd2, Fmr1, and Top2b mutants gives rise to the mislocalization of mGluR5, and causes an imbalance in mGluR5-eCB regulation of excitatory and inhibitory cortical circuits. This will be investigated by measuring Up states, synaptic transmission and intrinsic excitability in cortical slices from Setd2cHet, Top2bCKO and Top2bCHet mice and their regulation by mGluR5, CB1Rs. Results from these studies are expected to reveal whether the loss of Fmr1, Setd2 and Top2b converge onsynaptic mechanisms of circuit dysfunction and could provide a mechanistic understanding of gene length as a risk factor for the development of ASD.