Activation of the JAK-STAT signaling pathway by interleukin 6 (Il6) is required for multiple cellular immune and inflammatory responses, including macrophage activation and the repair of damaged tissue. Binding of Il6 to Il6 receptor alpha (Il6ra) initiates signaling through the transmembrane gp130 protein leading to activation of the JAK-STAT signaling cascade and, ultimately, changes in expression of STAT target genes. To date the functions of the proteins within the pathway have been well characterized, but it remains possible that other regulators exist.

To screen for novel regulators of the JAK-STAT pathway, we used a Cas9-expressing M1 murine leukaemia cell line, infected with the GeCKO whole genome gRNA library. M1 cells form tight, clonal colonies of cells when plated in semi solid agar, which disperse and differentiate into macrophages when treated with Il6. Factors that perturb the JAK-STAT pathway result in unresponsiveness to Il6 treatment and cells fail to differentiate, thus providing a clear readout for screening experiments. Unresponsive clones can then be expanded and sequenced to identify gRNAs that may be driving the phenotype.

In our whole genome screen, one Il6 unresponsive M1 clone contained a gRNA targeting the H3K9 methyltransferase, Setdb1. Subsequent infection of M1-Cas9 cells with six gRNAs targeting Setdb1 resulted in clonal lines that were all hypo-responsive to Il6 stimulation, even at ten times the standard concentration. As Setdb1 is involved in gene silencing via tri-methylation of H3K9 tails, we have carried out RNA-Seq to identify differentially expressed genes when comparing the Setdb1 knockout lines to the M1-Cas9 line. In addition, we have performed ChIP-Seq to look for altered H3K9Me3 at genes in the knockout vs. M1-Cas9 cells. Combining these data sets, we aim to identify the Setdb1-regulated gene or gene network critical for this new level of control of the Il6 activated JAK-STAT signaling pathway.