Balance between the open and locked structure of chromatin is orchestrated by posttranslational histone modifications. Acetylation of histone H3 on core lysine 56 (H3K56ac) increases conformational entropy in alpha-N helix part and destabilize protein structure. This mechanism is distinct to N-tail acetylation that decreases electrochemical attraction between negatively charged DNA and basic histones. Nucleosomes containing H3K56ac show increased chromatin breathing and facilitate gene expression. However, the most H3K56ac research is connected with yeasts. Mammalian cells have a very low level of H3K56ac in comparison with N-tail histone acetylation, because cells do not contain histone acetyltransferases with high specificity to H3K56. However, recent studies reveal important role of H3K56ac in regulation of chromatin structure of the most transcription active genes. In yeasts, H3K56ac serves as a marker for distinguish maternal and daughter DNA strand needed for proper DNA repair. We are focused on study of H3K56ac role during mammalian cell cycle and its regulation.