Authors :
Presenting Author: Jose Ezekiel Espina, MS – University of Wisconsin-Madison
Olivia Hoffman, PhD – University of Wisconsin-Madison
Jennifer Koehler, BA – University of Wisconsin-Madison
Barry Schoenike, MS – University of Wisconsin-Madison
Avtar Roopra, PhD – University of Wisconsin-Madison
Rationale: The processes by which
status epilepticus (SE) initiates the cascade of molecular, cellular, and network alterations that lead to epilepsy are poorly understood. We previously discovered that the transcriptional repressor Enhancer of Zeste Homolog 2 (EZH2) is robustly induced in the hippocampus acutely post-SE. EZH2 is the catalytic sub-unit of Polycomb Repressive Complex 2 (PRC2) and functions through methylation of histone H3K27. Systemic inhibition of EZH2 activity or deletion of EZH2 in neurons exacerbates disease severity in the chronic phase, suggesting that EZH2 induction post-SE may exert a net protective effect to dampen disease progression. Based on these findings, we developed a potent disease-modifying therapy that enduringly suppresses seizures in mice. However, the molecular mechanisms underlying EZH2-mediated control of the protective and pathological pathways in epileptogenesis are still unknown.
Methods: To interrogate the mechanisms of EZH2 function post-SE, we performed bulk Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing against the PRC2 sub-units EZH2 and SUZ12, and H3K27me3 in tandem with bulk RNA-sequencing in hippocampi of naïve and 4d. post-SE mice induced by systemic kainate.
Results: In the naïve hippocampus, we found that H3K27me3 is enriched at silenced developmental genes such as Hox genes and Wnt signaling. However, only a fraction of PRC2 co-localized with H3K27me3. PRC2 was enriched primarily at transcribed genes involved in active processes such as RNA processing and nonsense-mediated decay, among other functions. Differential analysis showed that H3K27me3 is enriched post-SE at a subset of genes previously marked by H3K27me3 in naïve mice. A majority of H3K27me3-enriched genes were developmental genes – similar to naïve mice – with no change in RNA levels post-SE. This suggests H3K27me3 deposition may function in part to maintain gene silencing post-SE, which is consistent with canonical PRC2 function. We also identified a module of H3K27me3-enriched genes with decreased RNA levels post-SE. These genes represent broadly neuronal functions such as GABA biosynthesis, synaptic signaling, and resting membrane potential. In contrast, H3K27me3-enriched genes with increased RNA levels represent terms relating to gliogenesis, which is consistent with our previous finding that EZH2 deletion in neurons exacerbates astrogliosis post-SE. Finally, we found that the majority of repressed genes post-SE did not exhibit increased H3K27me3 deposition, suggesting that gene repression is not mediated by
de novo H3K27 methylation, but rather through indirect or other mechanisms.
Conclusions:
We have used multi-modal epigenomic and transcriptomic analysis to interrogate the mechanistic consequences of EZH2 induction post-SE. Future studies will focus on functional validation of identified pathways to determine their contribution to disease progression. This approach will provide mechanistic insights into the endogenous protective and pathological mechanisms in epileptogenesis that can be leveraged to develop more effective therapies for epilepsy.
Funding:
This work was supported by CURE, Lily’s Fund, and NIH grants R01NS108756 and R21NS093364.