Final Activity Report Summary - CHROMOSEG (Molecular mechanisms of chromosome segregation: regulation of CENP-A and cohesin at the centromere)
Chromosome segregation is a fundamental aspect of cell proliferation and appears to be altered in many cancers. The centromere is the central element of this process in which it plays a dual role as effector of chromosome movement and as regulator of mitotic progression. In most organisms centromeres are embedded in long stretches of repetitive DNA and are determined epigenetically by the presence of a histone H3 variant named CENP-A. This project aimed to dissect the molecular mechanisms of centromere specification and function in the context of chromosome dynamics, and in particular, of sister chromatid cohesion mediated by cohesin. Our main goal was to identify novel factors regulating the association of cohesin and CENP-A with centromeres using Xenopus laevis egg cell-free extracts and mammalian tissue culture cells as experimental systems.
We have generated and characterised a number of custom polyclonal antibodies against several proteins thought to be involved in the mechanism of CENP-A deposition and cohesin regulation. These antibodies are useful tools to dissect the pathways of chromosome assembly and segregation in both Xenopus extracts and human cells. Once published, these materials will be available to the scientific community. In case there is high demand for some of them, an effort will be made to make them commercially available through agreements with biotech companies.
We have developed an assay to measure CENP-A incorporation by immunofluorescence microscopy in chromosomes assembled in vitro in Xenopus extracts. In combination with immunodepletion experiments using the aforementioned antibodies we have started to dissect the requirements of this process. We expect to publish soon our first conclusions regarding timing of deposition, independence from DNA replication, dependence on a preceding mitotic event, etc.
We have characterised the role of HP1 proteins in regulation of cohesin in human cells. There has been wide discussion about this issue in the recent literature and controversial results from different experimental systems. We have found that HP1alpha, beta and gamma are not involved in recruitment or maintenance of cohesin at the centromeric region.
We have studies the mechanism by which Shugoshin protects centromeric cohesion. We have found that it binds and re-localises protein phosphatase PP2A from chromosome arms to centromeres at the onset of mitosis. In this way, it allows dissociation of cohesin from arms upon phosphorylation of cohesin SA subunit by Polo while preventing its release from centromeres. We have published a paper with our conclusions in Chromosoma. In addition, we expect to finish and publish soon a study of the involvement of Shugoshin in spindle assembly and checkpoint function. This study is the result of a collaboration with the group of Dr Funabiki (Rockefeller University, New York, United States) that has been partly co-financed by an EMBO short-term fellowship awarded last year to Teresa Rivera, the graduate student in my lab heading this project.
We have generated and characterised a number of custom polyclonal antibodies against several proteins thought to be involved in the mechanism of CENP-A deposition and cohesin regulation. These antibodies are useful tools to dissect the pathways of chromosome assembly and segregation in both Xenopus extracts and human cells. Once published, these materials will be available to the scientific community. In case there is high demand for some of them, an effort will be made to make them commercially available through agreements with biotech companies.
We have developed an assay to measure CENP-A incorporation by immunofluorescence microscopy in chromosomes assembled in vitro in Xenopus extracts. In combination with immunodepletion experiments using the aforementioned antibodies we have started to dissect the requirements of this process. We expect to publish soon our first conclusions regarding timing of deposition, independence from DNA replication, dependence on a preceding mitotic event, etc.
We have characterised the role of HP1 proteins in regulation of cohesin in human cells. There has been wide discussion about this issue in the recent literature and controversial results from different experimental systems. We have found that HP1alpha, beta and gamma are not involved in recruitment or maintenance of cohesin at the centromeric region.
We have studies the mechanism by which Shugoshin protects centromeric cohesion. We have found that it binds and re-localises protein phosphatase PP2A from chromosome arms to centromeres at the onset of mitosis. In this way, it allows dissociation of cohesin from arms upon phosphorylation of cohesin SA subunit by Polo while preventing its release from centromeres. We have published a paper with our conclusions in Chromosoma. In addition, we expect to finish and publish soon a study of the involvement of Shugoshin in spindle assembly and checkpoint function. This study is the result of a collaboration with the group of Dr Funabiki (Rockefeller University, New York, United States) that has been partly co-financed by an EMBO short-term fellowship awarded last year to Teresa Rivera, the graduate student in my lab heading this project.