Investigating regulatory mechanisms of the AP1 transcription factor in hypoxic (low oxygen) cell adaptation

We plan to test the hypothesis that components of the AP-1 transcription factor are activated in response to key hypoxic signalling pathways and reactive oxygen species (ROS) induced post translational modifications.

Specifically we will:
(1) Identify key hypoxic signalling pathways that regulate AP-1 protein activity in hypoxia using a CRISPR CAS9 knockout screen of kinase signalling molecules.
We will use a CRISPR CAS9 knockout screen of all known kinases and identify kinases and their signalling pathways that regulate AP-1 activity in hypoxia.
We will validate the results of the screen by testing the impact of CRISPR knockouts and therapeutically relevant inhibitors against identified kinases on cell survival (2D and 3D cell culture models) and transcriptional regulation (RNA-SEQ) in hypoxia.

(2) Investigate the impact of ROS induced-redox PTMs on AP1 subunits and HIF proteins.
We will identify PTMs on AP-1 subunits using CoIP and in colorectal cancer tissue using immunohistochemistry. The impact of Redox-PTMs on AP-1 function will be investigated at a activity level (Reporter assay, western blot), transcriptional level (RNA-SEQ, QPCR) and phenotypic level (3D cell culture models, immunofluorescence etc), using mutational studies."