Investigation of histone modifications in interphase chromosomes obtained by premature chromosome condensation and mitotic chromosomes by immunofluorescence method

Epigenetic" changes, which regulate gene expression without altering the DNA sequence and can be inherited in cell divisions, are mediated by DNA methylation, posttranslational histone modifications and non-coding RNAs. Histone modifications can be detected on chromosomes by immunofluorescence methods using specific antibodies. Although there have been a limited number of studies on metaphase chromosomes using these methods, which combine cytogenetic techniques with epigenetic studies and can therefore be called "Cytoepigenetics", such studies have not yet been reported on interphase chromosomes obtained by the PCC method. PCC is a method of obtaining chromosomes in interphase using phosphatase inhibitors such as calyculin A. In this study conducted on isolated lymphocytes obtained from healthy adult volunteers, histone modifications H3K79me2 and H3K27me3 were demonstrated on both metaphase and interphase chromosomes obtained by PCC method using specific antibodies. To obtain interphase and metaphase chromosomes, chromosome spreads were prepared at the harvest stage of cultures using methods that do not contain acidic fixatives and preserve histone proteins. Immunofluorescence was applied to the prepared slides using antibodies specific to the selected histone modifications, and the slides were examined under a fluorescence microscope. The H3K79me2 and H3K27me3 modifications were shown to exhibit distinct band patterns on both interphase and metaphase chromosomes. It has been observed that the band patterns of H3K27me3 and H3K79me2 modifications are generally similar to each other and usually remain unchanged in both PCC and metaphase chromosomes. However, to understand whether PCC chromosomes preserve the histone modification pattern of interphase chromatin, studies targeting different modifications need to be conducted. Our study is the first to demonstrate histone modifications in interphase chromatin at the single cell level and on chromosomes and will contribute to the elucidation of the epigenetic basis of cell cycle and cell differentiation mechanisms.