- Maureen McCoy
- Investigating regulation of pro-inflammatory gene
My research studies the transcriptional regulation of the pro-inflammatory gene NLRC4 which is involved in the response to bacterial infection and implicated in an acute autoimmune disease.
The NLRC4 inflammasome is critical for gut immune health. The NLRC4 inflammasome is activated when NAIP proteins sense specific components of bacterial infection. Ligand binding of the NAIP proteins causes the oligomerization of NLRC4, which processes the release of pro-inflammatory cytokines IL-1ß and IL-18. As the orchestrator of a potent proinflammatory response, NLRC4 misregulation can result in severe disease. Therefore, NLRC4 expression and activation must be highly controlled in vivo. Transcriptional control has been studied in inflammasomes such as NLRP3, where NF-κB must first prime NLRP3 expression to enable activation, but the transcriptional regulation of NLRC4 is relatively unknown. Two previous studies revealed a role for transcriptional regulators IRF8 and Brd4 in the transcription of the NAIP sensor proteins. IRF8 and Brd4 bind to the NAIP promoters, and knockout mice have greatly reduced NAIP expression, cytokine levels, bacterial clearance, and overall survival. IRF8 and Brd4 were not recruited to the NLRC4 promoter in these studies, yet the IRF8 and Brd4 knockouts had downregulated NLRC4 expression that may significantly contribute to the reduced inflammatory response. Interestingly, overlapping IRF8 and Brd4 binding peaks were observed in NLRC4 intron 5. Intronic regions, once considered unused DNA, are now known to contribute to transcription, and several studies have shown that introns can contain enhancers for their host gene. Therefore, we hypothesized that NLRC4 intron 5 acts as an enhancer for NLRC4 transcription and activation. Three potential methods of determining this regulatory mechanism have been proposed and are being investigated. First by determining the enhancer capabilities of the intronic region by cloning the NLRC4 promoter and intron regions into a PGL3 luciferase expression system. Next, by deleting the suspected enhancer region using CRISPR-Cas9 and examining changes to downstream inflammatory effects, and finally by using chromosome conformation capture (3C) to look for promoter and enhancer looping interactions.