Evangeline Breeta Raja David Isac, Sara Sigurbjörnsdóttir, Ramile Dilshat and Eirikur Steingrimsson
Introduction: Melanoma cells evades drug treatment by changing their phenotype from proliferative to migrative cells and vice versa, a process known as phenotype switching. The Microphthalmia-associated transcription factor (MITF) is a main regulator of melanocyte development and plays an important role in the process of phenotype switching. Previously we have shown that MITF regulates the expression of E-Cadherin (CDH1) and N-Cadherin (CDH2), key markers of the epithelial-to-mesenchymal (EMT) process. Nevertheless, the role of CDH1 and CDH2 in phenotype switching or their correlation with MITF remains to be studied. Our main objective was to determine how MITF activates CDH1 expression in melanoma cells.
Methods: Using Cut-and-Run and ChiP-seq data we identified where MITF binds in the regulatory region of CDH1. These regions were cloned into the pGL3 promoter vector and luciferase assays were performed in melanoma cells. Furthermore, potential MITF-binding sites were mutated to identify which exact sites mediate the effects of MITF.
Results: The luciferase assay showed that a 1kb intronic CDH1 fragment (CDH1-B) leads to MITF-dependent activation of CDH1 expression. To further support these observations, all of the E-boxes found in the CDH1-B fragment were mutated such that MITF cannot bind. Transactivation assay showed that MITF activates CDH1 by binding to specific E-boxes (CAGCTG and CATGTG) within this regulatory region. To further validate these results, CRISPRi was performed targeting these prospective MITF binding sites.
Conclusion: Transactivation assays and CRISPRi experiments demonstrate that MITF directly activates CDH1 expression by binding to several E-boxes located in the particular intronic region.
