SAMPLE PREPARATION FOR DOWNSTREAM ANALYSIS OF EPIGENETIC MODIFICATIONS (DNA METHYLATION AND HISTONE MODIFICATIONS) IN THE REGULATION OF GENE EXPRESSION

Epigenetic changes are defined as inherited/non-inherited modifications that control alterations in gene expression without any structural changes in their nucleotide sequence. Epigenetic mechanisms include DNA methylation, methylation, phosphorylation, ubiquitination, acetylation and glycosylation of histones. These processes are associated with the adaptation of the structural and functional organization of chromatin to the local physiological and metabolic changes in response to environmental conditions. It is known by now that the number of diseases is associated with disruptions in the mechanisms of epigenetic regulation; however, epigenetic changes are dynamic and their potential reversibility makes them the promising targets for pharmaceutical interventions to correct alterations associated with epigenetic reprogramming.

Experimental approaches to the investigation of epigenetic mechanisms may seem complicated, since they all are time-consuming and require experience in performing complex experimental tasks. In our work, we present the protocols for the preparation of samples for the study of DNA methylation and histone modifications that are the result of integration and adaptation of the methodological approaches described in various scientific publications. All experimental procedures we tested on the cellular models of adipogenic differentiation of mesenchymal stem cells. These protocols will be useful for those who plan to study the mechanisms of epigenetic regulation of specific genes, or for full-genome mapping of certain epigenetic modifications; these protocols can also be adapted for immunoprecipitation of any regulatory DNA-protein complexes.

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