Constitutive heterochromatin at satellite-based and satellite-less horse centromeres

My thesis work originates from a collaboration between the laboratory of Molecular Cytogenetics, directed by Professor Elena Raimondi, and the laboratory of Molecular and Cellular Biology, directed by Professor Elena Giulotto, to study the molecular organization of mammalian centromeres using the equid species as a model system. The centromere is the chromosomal locus that allows a proper genome inheritance during eukaryotic cell division. In the majority of vertebrates, centromeric DNA is composed of extended arrays of tandemly repeated DNA sequences named satellite DNA. This suggests that these repeated sequences may contribute to establishing the centromere function. Nevertheless, some centromeres are void of satellite DNA, but completely functional and stably maintained through generations. It has been demonstrated that centromere identity is established by the presence of epigenetic markers. The most important one is the CENP-A protein, a centromere specific variant of histone H3. However, additional molecular features contribute in defining centromere activity, including the composition of pericentromeric chromatin and post-translational modifications of CENP-A itself. Moreover, a centromere-specific ratio between typical euchromatic and typical heterochromatic histone modifications is crucial for centromere identity. The genus Equus is an excellent model system to analyze centromere biology due to the presence of centromere repositioning events that occurred in some chromosomes during evolution giving rise to evolutionarily new centromeres void of satellite DNA. The karyotype of the equids is peculiar because canonical satellite-based centromeres coexist with evolutionarily new, satellite-free centromeres. The purpose of this thesis work was to investigate the epigenetic landscape of horse centromeres, by means of high-resolution molecular cytogenetic approaches. We could directly compare some features characterizing the centrochromatin of satellite-based and satellite-free centromeres. The results demonstrated that satellite-free centromeres, as well as the satellite-based ones, are immersed in a heterochromatic environment, even if the first ones contain much smaller amounts of constitutive heterochromatin. The results suggest that, independently from sequence composition, heterochromatin specific post-translational histone modifications are needed to drive centromere function.