Protocol to identify DNA methylation-sensitive enhancer sites

Here, we present a protocol to identify DNA methylation-sensitive enhancer sites using the leptin-regulating enhancer RS1 in adipose tissue. We describe steps for validating RS1’s endogenous function with a dCas9-KRAB repressor system and quantifying RS1 CpG methylation by pyrosequencing. We then detail procedures for assessing methylation sensitivity by luciferase reporter assay. This protocol is adaptable to other enhancers and tissues. For complete details on the use and execution of this protocol, please refer to Taege et al. 1 1. Taege, N. ∙ Britsemmer, J.H. ∙ Israel, A. ... A Novel mechanism for depot-specific leptin gene expression regulation and its persistence after weight loss iScience. 2026; 29 , 115101 Full Text Full Text (PDF) Scopus (0) PubMed Google Scholar