Universitą degli studi di Pavia
Contenuto della pagina
Cerutti research activity
Study of the centromere function and the complex chromatin architecture of mammalian centromeres by using the species of the genus Equus (horses, asses, zebras) as model system.
The research activity of my PhD will focus on the study of the centromere function and the complex chromatin architecture of mammalian centromeres by using the species of the genus Equus (horses, asses, zebras) as model system.
The centromere is the genetic locus required for the faithful chromosome segregation during cell division processes. As might be expected from the functional conservation among eukaryotes, centromere/kinetochore proteins generally share substantial homology, even between evolutionarily distant organisms. In contrast, centromeric DNA sequences are highly variable in size and complexity. This paradox suggests that the centromere is indeed an epigenetic structure, which does not depend strictly on primary DNA sequence. The functional and molecular analysis of mammalian centromeres has been so far particularly difficult due to the highly repetitive nature of centromeric DNA.
In recent years, we have demonstrated that the rapid evolution of the species of the genus Equus was marked by an exceptionally high frequency of centromere repositioning events, that is the shifting of the centromeric function along the chromosome without altering the DNA sequence; we also showed, at the cytogenetic level, that several Equus neocentromeres are devoid of extended blocks of tandemly repeated satellite sequences (Piras et al., 2010). Taking advantage of the complete sequence of the horse genome, we then demonstrated, at the molecular level, the complete absence of satellite repeats on horse chromosome 11 (ECA11), the first satellite-free neocentromere stably fixed in different horse populations ever reported (Wade et al., 2009). These observations allowed us to propose the satellite-less Equus centromeres as a new model system for studying the fine functional architecture of mammalian centromeres.
The first goal of our study was to characterize the presence and localization of centromeric proteins in four Equus species (horse, donkey, Burchell’s zebra, and Grevy’s zebra) using a series of antibodies and sera. Our results showed that antibodies against human CENP-A, CENP-B, CENP-C, CENP-F and CENP-H bind to all Equid centromeres. However, in Grevy’s zebra, CENP-B localizes not only to all centromeres, including the satellite-less ones, but also to the terminal sites of several chromosomes. Our findings suggest that, unlike reported by many past works, the centromere localization of CENP-B is not satellite DNA-related. We hypothesized the existence, at those telomeric sites, of ancestral centromeres that were inactivated in the course of evolution. In corroboration to this idea, we performed immuno-FISH experiments on Grevy’s zebra chromosomes and observed that CENP-B and satellite sequences co-localize at the chromosomic termini. Through the use of plasmid vectors expressing different horse-CENP-B domains fused to a reporter protein, we are currently investigating which domains of this protein are responsible for its centromere and telomere localization in Grevy’s zebra cells. We are also performing ChIP-seq experiments using a human serum containing a high titer of anti-CENP-B antibodies to study the sequences of CENP-B binding in Grevy’s zebra and horse. This last work is part of a wider project aiming to analyze in detail the DNA sequences bound by different CENP proteins in different Equus species. We are currently performing ChIP-seq experiments with sera specifically directed against CENP-A both in horse and in other Equus species in which we identified satellite-less centromeres that, as such, might be similar to ECA11 centromere (Carbone et al., 2006; Piras et al., 2009; Piras et al., 2010).
The research activity of my PhD will focus on the study of the centromere function and the complex chromatin architecture of mammalian centromeres by using the species of the genus Equus (horses, asses, zebras) as model system.
The centromere is the genetic locus required for the faithful chromosome segregation during cell division processes. As might be expected from the functional conservation among eukaryotes, centromere/kinetochore proteins generally share substantial homology, even between evolutionarily distant organisms. In contrast, centromeric DNA sequences are highly variable in size and complexity. This paradox suggests that the centromere is indeed an epigenetic structure, which does not depend strictly on primary DNA sequence. The functional and molecular analysis of mammalian centromeres has been so far particularly difficult due to the highly repetitive nature of centromeric DNA.
In recent years, we have demonstrated that the rapid evolution of the species of the genus Equus was marked by an exceptionally high frequency of centromere repositioning events, that is the shifting of the centromeric function along the chromosome without altering the DNA sequence; we also showed, at the cytogenetic level, that several Equus neocentromeres are devoid of extended blocks of tandemly repeated satellite sequences (Piras et al., 2010). Taking advantage of the complete sequence of the horse genome, we then demonstrated, at the molecular level, the complete absence of satellite repeats on horse chromosome 11 (ECA11), the first satellite-free neocentromere stably fixed in different horse populations ever reported (Wade et al., 2009). These observations allowed us to propose the satellite-less Equus centromeres as a new model system for studying the fine functional architecture of mammalian centromeres.
The first goal of our study was to characterize the presence and localization of centromeric proteins in four Equus species (horse, donkey, Burchell’s zebra, and Grevy’s zebra) using a series of antibodies and sera. Our results showed that antibodies against human CENP-A, CENP-B, CENP-C, CENP-F and CENP-H bind to all Equid centromeres. However, in Grevy’s zebra, CENP-B localizes not only to all centromeres, including the satellite-less ones, but also to the terminal sites of several chromosomes. Our findings suggest that, unlike reported by many past works, the centromere localization of CENP-B is not satellite DNA-related. We hypothesized the existence, at those telomeric sites, of ancestral centromeres that were inactivated in the course of evolution. In corroboration to this idea, we performed immuno-FISH experiments on Grevy’s zebra chromosomes and observed that CENP-B and satellite sequences co-localize at the chromosomic termini. Through the use of plasmid vectors expressing different horse-CENP-B domains fused to a reporter protein, we are currently investigating which domains of this protein are responsible for its centromere and telomere localization in Grevy’s zebra cells. We are also performing ChIP-seq experiments using a human serum containing a high titer of anti-CENP-B antibodies to study the sequences of CENP-B binding in Grevy’s zebra and horse. This last work is part of a wider project aiming to analyze in detail the DNA sequences bound by different CENP proteins in different Equus species. We are currently performing ChIP-seq experiments with sera specifically directed against CENP-A both in horse and in other Equus species in which we identified satellite-less centromeres that, as such, might be similar to ECA11 centromere (Carbone et al., 2006; Piras et al., 2009; Piras et al., 2010).
