Exploration of the Role of EZH2 and BRD4 in the Transcriptional Dysregulation of Epileptogenesis in Epilepsy

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults, is characterized by seizures originating in temporal structures and has high rates of drug refractoriness. Acquired TLE often develops following a brain insult e.g. traumatic brain injury, stroke or status epilepticus (SE), triggering widespread gene expression changes which have so far been poorly characterized. These changes may drive the various pathogenic processes involved in the development of the disease; a process known as epileptogenesis. A key process of the epileptogenic process is the gradual change in cellular properties including neurons, and glial cells. Epigenetic modifications and governance of gene expression are key regulators of cellular identity and function and have been shown to be altered by epileptic triggers. Epigenetics broadly refers to processes such as (i) DNA methylation, (ii) histone modifications and (iii) non-coding RNAs that control gene expression by influencing the accessibility of the chromatin to transcriptional machinery. All three major epigenetic processes have been found to be altered in both experimental and human epilepsies. EZH2 and BRD4 are two epigenetic regulating enzymes which are critical for cellular identity and function and have profound effects on gene expression patterns in the brain. Although both enzymes have been linked with seizure activity, little is known about how they may regulate the epileptogenic process, and their therapeutic potential as master regulators of the process. The current project sought to fill these knowledge gaps by exploring the effects of epileptogenic insults on the expression of key epigenetic enzymes. By profiling the binding profiles and gene regulatory networks governed by both EZH2 and BRD4 and assessing whether inhibition of BRD4 using small molecule inhibitors holds therapeutic promise as an anti-epileptogenic agent. To do this we used a clinically relevant mouse model of TLE and applied a combination of proteomic analyses, novel CUT&RUN and next generation sequencing approaches and pre-clinical analyses of small molecule inhibitors with long term EEG. Here, we identified altered expression of EZH2 and BRD4 protein levels and various other epigenetic enzymes in the hippocampus following SE suggesting possible widespread epigenetic reorganization of the genome initiated by epilepsy-inciting events. Additionally, we broadly mapped the protein-DNA binding events involving EZH2 and Brd4 using CUT&RUN, enabling us to further analyze the dynamic interactions between these epigenetic enzymes and DNA driving gene expression changes in epileptogenesis and chronic epilepsy. We then tested a small molecule BET inhibitor to evaluate therapeutic potential, which worsened seizure phenotype, potentially due to altered expression of genes associated with inflammation, microgliosis, and neurotransmission.