SAT-031 Understanding Gβγ Isoform Interactome Profiles in Fibrotic Gene Regulation
Bibliographic record
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)
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".