Ca2+-sensitive Mef2c protein interactions and chromatin function in microglia-like cells

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder for which there are no disease-modifying therapies. Genetic studies have identified over 50 susceptibility loci for sporadic AD including the locus encoding the transcription factor MEF2C. Most of the genes implicated in AD-risk are exclusively or preferentially expressed in microglia. Furthermore, AD-risk variants are enriched in microglial open chromatin regions that contain DNA binding motifs for MEF2C. Therefore, genetic variants that disrupt MEF2C binding to DNA in microglia may alter cis-gene expression, contributing to AD-risk. Understanding how MEF2C functions in microglia may provide valuable insights into the genetic basis of AD-risk. To investigate the role of Mef2c in AD, mass spectrometry was used to identify proteins that co-purify with the endogenous protein in BV2 microglia-like cells. Two major Mef2c isoforms exist in BV2 cells that associate with 110 putative interactors including the transcriptional repressors, Hdac4, Hdac5, and Cabin1. Ionomycin treatment, that raises intracellular [Ca2+], caused the partial dissociation of these repressors from Mef2c and resulted in recruitment of the microglial amyloid-β response proteins, Yes1 and Smpdl3b to the Mef2c complex. However, no Mef2c-activating proteins were identified in the remodelled complex. Having demonstrated that ionomycin treatment remodels the Mef2c interactome, the effect of this treatment on chromatin accessibility in BV2 cells was investigated using ATAC-seq. This revealed that while the motifs for three transcription factors, Atf4, NFATC3 and p53, were enriched at Ca2+ -dependent differentially accessible sites, Mef2c sites were not similarly enriched. However, Mef2c motif-containing differentially accessible regions were associated with genes that control the microglial inflammatory response. This thesis investigated two mechanisms by which [Ca2+] levels potentially influence gene regulation; altered protein interactions and chromatin accessibility and further contributes to our understanding of the transcription factor Mef2c, Ca2+ signalling, and chromatin function in BV2 cells. In conclusion, Ca2+ dysregulation in AD may result in remodelling of the Mef2c interactome leading to abnormal Mef2c-mediated inflammatory responses in microglia