Final Report Summary - EPICENTROMERE (Determining the Epigenetic Mechanism of Centromere Propagation)
The centromere is a specialised chromosomal locus responsible for nucleating the kinetochore during mitosis. Centromeres are maintained largely epigenetically, where centromeric DNA is neither necessary nor sufficient for centromere specification. Instead centromeres are maintained across mitotic divisions through self-propagating properties of centromeric chromatin. This chromatin domain is characterised by the specific incorporation of CENP-A, a histone H3 variant that is critical for epigenetic inheritance of centromere identity. Using a fluorescent pulse labeling method called SNAP-tagging we have previously demonstrated that CENP-A is an extremely stable resident at centromeres and that assembly of nascent CENP-A is restricted to a unique cell cycle window in late telophase implicating mitotic events in controlling the maturation of centromeric chromatin. This project aims at determining several aspects of CENP-A biology including, but not exclusively, establishing the molecular nature of cell cycle coupling of CENP-A assembly.
Here we summarize the specific aims and results that have been published at the end of the project.
Determining the role of mitosis in propagation of centromeric chromatin in dividing cells.
Early models of cell cycle control of CENP-A assembly implicated specific mitotic events in triggering CENP-A assembly in early G1 phase. Using pharmacogenetic studies in both human and chicken cell systems we have identified cyclin dependent kinase 1 (Cdk1) and Cdk2 as a master regulators of CENP-A assembly. Treatment of cells with Cdk inhibitors prior to mitosis induces rapid assembly of newly synthesised CENP-A at centromeres. We have shown the CENP-A assembly machinery to be present and poised for activity throughout the cell cycle. Inhibition of Cdk activity is sufficient to trigger the canonical pathway with no requirement for proteolysis other than the Cdk activator cyclin B. More recently, we have gained further insight into the mechanism of this process and have shown that a non-phosphorylatable mutant of the key CENP-A assembly factor, Mis18BP1 gains the capacity to target to centromeres prior to mitosis strongly suggesting that Cdk mediated phosphorylation of CENP-A assembly factors is controlling their centromere localization and in this way, CENP-A assembly. This work was published in Developmental Cell in 2012.
Determining the role of centromeric DNA transcription in CENP-A assembly.
In a collaborative effort with Dr Bill Earnshaw, from the wellcome trust centre for cell biology, Edinburgh, UK we have helped show that loss of transcription and transcription-associated chromatin modifications at artificial centromeres results in centromere dysfunction, most likely due to a failure to recruit new CENP-A into centromeric chromatin. This work was published in the EMBO Journal in 2011.
Determine the role of constitutive centromere proteins in CENP-A assembly.
CENP-N is a central centromere component that directly binds to CENP-A nucleosomes in vitro. We have identified this protein as an important player in the recruitment of nascent CENP-A to newly replicated centromeres. The requirement of CENP-N for CENP-A chromatin assembly while itself dependent on CENP-A nucleosomes places this protein at the heart of an epigenetic feedback loop that helps drive centromere propagation. This work was conducted in collaboration with Aaron Straight at Stanford University and published in 2009 in Nature cell biology.
Determine the pattern of assembly and local stability of histones
Using the SNAP methodology that we have successfully employed for CENP-A we have now analysed the turnover and dynamics of canonical histones and shown that CENP-A and histone H4 but not any other canonical histone analysed thus far is preferentially assembled and retained at the centromere in a manner that depends on key residues within CENP-A but is independent of local DNA sequence context. These results identify the stable epigenetic core of the centromere. This work is currently prepared for submission.
As part of the broader objectives of this aim we have also employed gene targeting methods for human somatic cells to better assess the cell biology of centromeres. We have further developed this and established an efficient Flow cytometry (FACS)-based strategy to rapidly isolate human somatic cell knockin or knockout clones. This work was published in public library of science (PLoS) one 2012.
In a related effort, we have in collaboration with Genevieve Almouzni, determined the role of specific histone chaperone in histone H3.1 and H3.3 assembly (Ray-Gallet et al., (2011) Molecular cell).
Here we summarize the specific aims and results that have been published at the end of the project.
Determining the role of mitosis in propagation of centromeric chromatin in dividing cells.
Early models of cell cycle control of CENP-A assembly implicated specific mitotic events in triggering CENP-A assembly in early G1 phase. Using pharmacogenetic studies in both human and chicken cell systems we have identified cyclin dependent kinase 1 (Cdk1) and Cdk2 as a master regulators of CENP-A assembly. Treatment of cells with Cdk inhibitors prior to mitosis induces rapid assembly of newly synthesised CENP-A at centromeres. We have shown the CENP-A assembly machinery to be present and poised for activity throughout the cell cycle. Inhibition of Cdk activity is sufficient to trigger the canonical pathway with no requirement for proteolysis other than the Cdk activator cyclin B. More recently, we have gained further insight into the mechanism of this process and have shown that a non-phosphorylatable mutant of the key CENP-A assembly factor, Mis18BP1 gains the capacity to target to centromeres prior to mitosis strongly suggesting that Cdk mediated phosphorylation of CENP-A assembly factors is controlling their centromere localization and in this way, CENP-A assembly. This work was published in Developmental Cell in 2012.
Determining the role of centromeric DNA transcription in CENP-A assembly.
In a collaborative effort with Dr Bill Earnshaw, from the wellcome trust centre for cell biology, Edinburgh, UK we have helped show that loss of transcription and transcription-associated chromatin modifications at artificial centromeres results in centromere dysfunction, most likely due to a failure to recruit new CENP-A into centromeric chromatin. This work was published in the EMBO Journal in 2011.
Determine the role of constitutive centromere proteins in CENP-A assembly.
CENP-N is a central centromere component that directly binds to CENP-A nucleosomes in vitro. We have identified this protein as an important player in the recruitment of nascent CENP-A to newly replicated centromeres. The requirement of CENP-N for CENP-A chromatin assembly while itself dependent on CENP-A nucleosomes places this protein at the heart of an epigenetic feedback loop that helps drive centromere propagation. This work was conducted in collaboration with Aaron Straight at Stanford University and published in 2009 in Nature cell biology.
Determine the pattern of assembly and local stability of histones
Using the SNAP methodology that we have successfully employed for CENP-A we have now analysed the turnover and dynamics of canonical histones and shown that CENP-A and histone H4 but not any other canonical histone analysed thus far is preferentially assembled and retained at the centromere in a manner that depends on key residues within CENP-A but is independent of local DNA sequence context. These results identify the stable epigenetic core of the centromere. This work is currently prepared for submission.
As part of the broader objectives of this aim we have also employed gene targeting methods for human somatic cells to better assess the cell biology of centromeres. We have further developed this and established an efficient Flow cytometry (FACS)-based strategy to rapidly isolate human somatic cell knockin or knockout clones. This work was published in public library of science (PLoS) one 2012.
In a related effort, we have in collaboration with Genevieve Almouzni, determined the role of specific histone chaperone in histone H3.1 and H3.3 assembly (Ray-Gallet et al., (2011) Molecular cell).
