CRISPR/Cas9-mediated mutations in both a cAMP response element and an ETS-binding site suppress FLT1 gene expression.

ABSTRACT

The Fms-like tyrosine kinase-1 (FLT1) gene is expressed in various types of cells, including vascular endothelial cells and placental trophoblasts, and regulates angiogenesis, inflammation, and pregnancy. However, the basal transcriptional machinery of FLT1 is still not well understood. In this study, we first examined FLT1 promoter activity in three different types of cells, that is, trophoblast-derived cells, vascular endothelial-related cells, and HEK293 cells, using plasmid-based luciferase reporter assays, and showed that a cAMP-response element (CRE) and an ETS-binding site (EBS) are important for FLT1 expression in all cell types. To further examine the importance of these sites at the chromosomal level using HEK293 cells, we introduced CRISPR/Cas9-mediated mutations in these sites on the genomic DNA. HEK293 cells carrying these mutations clearly showed a significant decrease in endogenous FLT1 gene expression. These results suggest that CRE and EBS transcription regulatory elements are crucial for FLT1 gene expression in human tissues.