Determination of Chromatin Accessibility in MidgutEnterocytes by 5mC Labeling.

Regulation of gene expression involves dynamic changes in chromatin organization, where in many cases open chromatin structure correlates with gene activation. Several methods enable monitoring changes in chromatin accessibility, including ATAC-seq, FAIRE-seq, MNase-seq and DNAse-seq methods, which involve Next-generation-sequencing (NGS). Focusing on the adult differentiated gut enterocytes (ECs) we used a sequencing-free method that enables visualizing and semi-quantifying large-scale changes in chromatin structure using methylation assay with the bacterial CpG Methyltransferase, M. Sssl, that determine chromatin accessibility. In brief, as CpG methylation is minimal in differentiated somatic cells, we used the bacterial M. SssI enzyme to methylate CpG dinucleotides depending on their chromatin accessibility. The methylated dinucleotides are detected using 5mCytosine monoclonal antibody and nuclei are visualized microscopically. Thus, the 5mC method enables to monitor large-scale chromatin changes in heterogenic cellular tissue focusing on the cell type of interest and without the need for cell purification or NGS.