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The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pericentromeric gamma-satellite DNA is organized in constitutive heterochromatin structures. It comprises a 234 bp sequence repeated several thousands times surrounding the centromeric sequence of all murine chromosomes. Potential binding sites for transcription factor Yin Yang 1 (YY1), a repressor or activator of several cellular and viral genes, are present in pericentromeric gamma-satellite DNA. Using gel retardation and chromatin immunoprecipitation, we demonstrate in this work that YY1 specifically interacts in vitro and in vivo with gamma-satellite DNA. Using immunoFISH and confocal microscopy we show that YY1 specifically co-localizes with pericentromeric gamma-satellite DNA clusters organized in constitutive heterochromatin in murine L929 and 3T3 fibroblasts cell lines. Immunoelectron microscopy experiments further confirmed YY1 localization in heterochromatic areas. Overall, our results demonstrate for the first time that a fraction of YY1 is directly associated with constitutive heterochromatin structures. This association appears physiologically relevant since the association of YY1 with pericentromeric gamma-satellite DNA observed in cycling 3T3 fibroblasts strongly diminished in quiescent (G0) 3T3 fibroblasts. We discuss the implications of these results in the context of heterochromatin formation as well as with regard to the YY1-induced repression of euchromatic genes.