In situ Quantification of Cytosine Modification Levels in Heterochromatic Domains of Cultured Mammalian Cells

Epigenetic modifications of DNA, and especially cytosine, play a crucial role in regulating basic cellular processes and thereby the overall cellular metabolism. Their levels change during organismic and cellular development, but especially also in pathogenic aberrations such as cancer. Levels of respective modifications are often addressed in bulk by specialized mass spectrometry techniques or by employing dedicated ChIP-seq protocols, with the latter giving information about the sequence context of the modification. However, to address modification levels on a single cell basis, high- or low-content microscopy techniques remain the preferred methodology. The protocol presented here describes a straightforward method to detect and quantify different DNA modifications in human cell lines, which can also be adapted to other cultured mammalian cell types. To this end, cells are immunostained against two different cytosine modifications in combination with DNA counterstaining. Image acquisition takes place on a confocal microscopy system. A semi-automated analysis pipeline helps to gather data in a fast and reliable fashion. The protocol is comparatively simple, fast, and cost effective. By employing methodologies that are often well established in most molecular biology laboratories, many researchers are able to apply the described protocol straight away in-house.