The JAK-STAT signalling pathway is a classical model for the negative feedback loop, linking extracellular stimuli to alteration in gene expression. Binding of ligand (e.g. IL-6) to receptor (IL6Ra-gp130) on the cell surface initiates an intracellular signalling cascade of JAK proteins leading to activation of STAT transcription factors. STATs alter the expression of target genes, necessary for the cell’s response to stimulus. These genes include the Suppressor of Cytokine Signalling genes (e.g. Socs3), who’s protein product impedes the actions of JAKs, thereby preventing incessant signalling.
The functions of the JAK, STAT and Socs proteins have been well characterized through genetic knockout and structural studies, but it remains possible that other regulators exist. Using the GeCKO whole genome gRNA library, we performed a CRISPR/Cas9 screen for IL-6 unresponsiveness in the model murine cell line, M1. Our screen highlighted the essential roles of receptor and STAT genes for signal transduction and, for the first time, the H3K9 methyltransferase, Setdb1.
ChIP-seq for H3K9me3 in M1 cells showed the entire Socs3 locus is enriched for H3K9me3, but this enrichment is absent in Setdb1 knockout M1s. We hypothesized that H3K9me3 at the locus was indicative of heterochromatin-associated silencing, keeping expression of Socs3 off until needed, so as to prevent suppression of the pathway before IL-6 stimulation. Interestingly, ChIP-seq for H3K9me3 over a time course of IL-6 stimulation showed that the locus is cleared of the mark after just four minutes, an incredibly rapid response as H3K9me3 is typically associated with stable repression of repeat elements in the genome, not individual genes.
These data, alongside ATAC-seq and RNA-seq performed using the same material, reveal a complex interplay between chromatin accessibility and Socs3 expression over time, linking cell signalling to regulation of the epigenome in a highly dynamic spatio-temporal manner.