Epigenetic Editing Regulates Alternative Splicing of Srsf11 and Cocaine Reward Behavior

Cocaine drives alternative splicing to a far greater extent than differential mRNA expression yet this mechanism of gene regulation is understudied in the context of drug abuse and addiction. During development, alternative isoform expression confers neuronal identity and is maintained throughout life. We posit that in adulthood, alternative exon expression maintained by stably-associated hPTMs, underlies chronic disease states, such as addiction. We and others have recently reported that alternative splicing is correlated with H3K36me3 enrichment in brain, a histone posttranslational modification (hPTM) which is also regulated by cocaine. H3K36me3 is catalyzed by the histone methyltransferase, SET2. We generated three within-sample, RNA/ChIP datasets from NAc of mice following either SET2 overexpression, 1-day or 28-days of abstinence following cocaine-self administration. In each dataset, we analyzed alternative isoform expression and splice junction enrichment of H3K36me3. To examine the direct causal relevance of H3K36me3 to alternative splicing, we expressed either full-length SET2 or locus-targeted dCas9-SET2 in NAc (control: catalytically dead SET2(R195)). Across three datasets, we identified exons that were both differentially expressed and enriched for H3K36me3 at the respective splice junction (FET P<0.05). This group included Serine and arginine rich splicing factor 11 (Srsf11). We selected this gene given that the Srsf11 target motif was enriched globally in spliced genes following cocaine- and SET2-treatment. Targeted enrichment of H3K36me3 by dCas9-SET2 to the relevant Srsf11 splice junction was confirmed by CUT&RUN-seq in N2a cells. This manipulation increased inclusion of the targeted skipped exon and increased cocaine conditioned place preference and self-administration behavior. Taken together, the combination of bioinformatic and epigenetic editing approaches provide conclusive evidence of the functional relevance of H3K36me3 enrichment at splice junctions to expression of specific isoforms.