SAT-031 Understanding Gβγ Isoform Interactome Profiles in Fibrotic Gene Regulation

Abstract

Communication between cardiomyocytes and fibroblasts is strongly implicated in cardiac disorders, however the mechanisms of intercellular interplay by which cardiac hypertrophy and fibrosis are regulated remain unclear. In order to define new therapeutic targets, it is essential to identify links between fibroblast activation by the G protein-coupled type 1 receptor for angiotensin II and intracellular signaling leading to fibrotic gene transcription. There are 5 isoforms of Gβ and 12 isoforms of Gγ subunits of heterotrimeric G proteins in humans, yet we still don’t fully understand their distinct functions. Based on previous work from our lab, we showed that Gβγ dimers are found at over 700 promoters in HEK 293 cells some of which are likely mediated by an interaction between Gβγ subunits and RNA polymerase II (RNAP II). Our results suggest that Gβ1 acts as a regulator of gene expression and that its absence dysregulates the fibrotic response. (1) This project will explore the impact of different Gβγ subunits on transcription in HEK 293 cells and in rat neonatal cardiac fibroblasts (RNCFs) to understand their unique roles in regulating the fibrotic response. To do this we have adapted a proteomic screen to identify interacting partners of Gβγ isoforms at various stages in the transcription of individual genes. We will use a technique called caspex to biotinylate proteins in proximity of a DNA sequence of interest. The APEX2 peroxidase is fused to dCas9, allowing the targeting of specific DNA sequences by guide RNAs and labelling of nearby proteins by biotinylation. (2) We will initially use the screen to study specific gene loci under control conditions and following carbachol-stimulation of endogenous M3-mAChR in HEK 293 cells. Once labelled, these can be identified by mass spectrometry and confirmed by co-immunoprecipitation with FLAG-tagged Gβγ subunits. Following our experiments in HEK 293 cells, we will apply the screen in RNCFs and investigate the proteomes at specific gene loci regulated by Gβγ during the fibrotic response to Ang II with a view toward identifying how and when Gβγ subunits are recruited to target genes. Our results will establish a link between particular Gβγ isoforms and fibrotic gene regulation through the generation of Gβγ dimer-specific interactomes. (1) Khan et al., BioRxiv. (2018) doi: 415935 (2) Myers et al., Nature Methods. 15, 437-439 (2018)