Découverte de régulateurs de l'activité de HNF1B par CRISPR-interference

Hepatocyte nuclear factor-1β (HNF1B) is a bookmarking transcription factor expressed in the liver, pancreas, intestine, and kidneys. Hnf1b mutations are frequently the cause of monogenic syndromic CAKUT (Congenital Abnormalities of the Kidney and the Urogenital Tract) which often leads to renal failure. There is a knowledge gap about how HNF1B is regulated and what are the signaling pathways that lead to the regulation of its transcriptional activity. To identify genes that have an impact on HNF1B, we performed a CRISPR-interference screening. We took advantage of a biosensor for HNF1B activity: a plasmidic construct that contains a cassette encoding for a fluorescent protein under the control of multimerized HNF1B binding sites. By using a Cas9 protein fused to a repressor of transcription we could downregulate the expression of nearly 20.000 genes and select which gene perturbation led to changes in HNF1B activity. Among the hits, we identified a member of the AP-1 family of transcription factors: FOSL2. Silencing of FOSL2 induces a decrease in HNF1B mRNA levels, suggesting that it positively regulates HNF1B expression. Potentially, FOSL2 is a direct activator of HNF1B transcription as it was found to physically bind to an enhancer upstream of the HNF1B promoter in KEC cells. An increase in FOSL2 expression in HNF1B deficient mice suggests that these two proteins might be involved in a feedback regulatory loop, where HNF1B negatively regulates FOSL2 expression. Surprisingly, FOSL2 and HNF1B tend to co-localize on specific chromatin sites, especially on genes that regulate Epithelial-Mesenchymal Transition (EMT). FOSL2 was shown to drive EMT and matrix deposition which lead to kidney fibrosis. Interestingly, HNF1B was shown to inhibit EMT as HNF1B deficient mice show partial EMT. Downregulation of both FOSL2 and HNF1B in KEC cells could prevent the upregulation of mesenchymal marker ACTA-2 which is normally observed upon HNF1B downregulation. This suggests that HNF1B and FOSL2 might act as antagonists in the regulation of EMT, where HNF1B normally negatively regulates EMT gene expression in epithelial cells and FOSL2 activates EMT gene expression in the absence of HNF1B.