Periodic Reporting for period 1 - CHROMSEG (Structural Basis for Centromere-Mediated Control of Error-free Chromosome Segregation)
Periodo di rendicontazione: 2023-03-01 al 2025-08-31
Accurate transfer of genetic information (which is in the form of chromosomes) during cell division requires attachment of sister-chromatids (duplicated chromosomes) to microtubules (a polymeric protein assembly) emanating from opposite poles of the microtubule-based apparatus, the mitotic spindle and accurate segregation of sister chromatids mediated by the mitotic spindle to the newly formed daughter cells. Two chromosomal sites regulate these processes: centromeres, the microtubule attachment sites defined by the enrichment of CENP-A nucleosomes (a variant version of a fundamental DNA packaging unit), and the inner centromere, a region between the sister-chromatids that recruits enzymatic activities (protein modifiers such as kinases and phosphatases and motor proteins). The inner centromere-associated enzymes selectively stabilise chromosome-microtubule attachments suitable for chromosome bi-orientation (microtubule attachment of sister chromatids to opposite spindle poles), control sister chromatid cohesion and achieve timely chromosome segregation. Errors in these processes can lead to aneuploidy, a numerical chromosomal aberration implicated in miscarriages, birth defects and cancers. Using an integrative structure-function approach (protein structure determination methods such as X-ray crystallography, cryo electron microscopy and Crosslinking/Mass Spectrometry and biochemical/biophysical methods with human cell-line based functional assays), we aim to obtain detailed mechanistic understanding of: (1) how the inner centromere is assembled, (2) how the inner centromere associated interaction network recruits regulators to achieve chromosome bi-orientation and accurate segregation, and (3) how centromere identity (defined by the presence of CENP-A nucleosomes) is maintained through multiple generations. This work builds on our recently obtained exciting structural/molecular knowledge that has led to unexpected insights and new questions, and will exploit our recently generated battery of molecular reagents. The outcome of our work will provide unprecedented details of centromere-mediated control of chromosome segregation and allow us to build a comprehensive mechanistic model for error-free chromosome segregation, a process that has fascinated researchers for more than a century.
We have made outstanding progress on the proposed research as summarised below:
WP1 Establishment of inner centromere signalling platform: One of the key protein assemblies that associates with the inner centromere is the Chromosomal Passenger Complex (consisting of four different proteins, Borealin, Survivin, INCENP and Aurora B (a protein kinase)). The CPC interacts with two other major regulators knows as Shugoshin 1 and HP1. Towards understanding how these proteins interact with each other to establish the inner centromere signalling platform, we first focused on determining the structure of CPC bound to nucleosome. We have been successful in determining a high-resolution cryo-EM structure of the core inner centromere component of CPC (consisting of Borealin, Survivin and INCENP) bound to nucleosome at 2.8 angstrom resolution. The molecular details of their interactions reveal that CPC-nucleosome binding stabilises the centromeric chromatin (nucleosomes forming the microtubule attachment sites). We are now working towards characterising the interaction of CPC, Shugoshin 1 (Sgo1) and HP1 with nucleosomes. Structural and biochemical characterisation these interactions are in progress.
WP2 Mechanisms of recruitment of enzymatic activities by the inner centromere interaction network: Protein phosphatase 2A (PP2A), which exists in different forms due to the presence of different regulatory protein subunits (B55 and B56) plays critical roles in regulating centromere functions such as maintaining centromere cohesion, error-correction (selective stabilisation of bi-polar attachments) and timely onset of anaphase (cell division stage when sister chromatids move towards the opposite poles of the mitotic spindle). However, the mechanistic basis of how different Shugoshin isoforms (Sgo1 and Sgo2) preferentially recruit PP2A to different sub-centromeric locations remains an open question. We have now reconstituted these protein complexes for further structural characterisation.
WP3. Mechanisms of centromere maintenance: As outlined above, the centromere is defined by the enrichment of a Histone H3 Variant, CENP-A, containing nucleosomes, which is critical for assembling the microtubule-capturing protein hub called the kinetochore. Importantly, the CENP-A levels undergo dilution during S-phase (a specific phase of the cell cycle, where the genetic information is duplicated for subsequent segregation to the newly formed daughter cells), as existing CENP-A is shared among the new and old DNA. To ensure stable maintenance of the centromere, the original CENP-A levels must be restored through active CENP-A deposition. By characterising the architecture of the CENP-A deposition machinery and its interaction with a protein modifying PLK1 kinase, we unravelled molecular details of how PLK1 allows CENP-A loading machinery to deposit CENP-A at the right time during the cell cycle, a question that remained unanswered for more than a decade.
During this grant period, through a collaborative effort, we have also characterised CENP-32 (an RNA modifying enzyme critical for the assembly of functional chromosome segregation apparatus, the mitotic spindle) using biochemical, structural, cell biological and clinical approaches. Our work showed that CENP-32 mutations found in patients with developmental defects affect CENP-32 enzymatic activity. We also showed that mutants compromising CENP-32 activity lead to spindle defects, causing chromosome segregation errors and altered cell proliferation.
WP1 Establishment of inner centromere signalling platform: One of the key protein assemblies that associates with the inner centromere is the Chromosomal Passenger Complex (consisting of four different proteins, Borealin, Survivin, INCENP and Aurora B (a protein kinase)). The CPC interacts with two other major regulators knows as Shugoshin 1 and HP1. Towards understanding how these proteins interact with each other to establish the inner centromere signalling platform, we first focused on determining the structure of CPC bound to nucleosome. We have been successful in determining a high-resolution cryo-EM structure of the core inner centromere component of CPC (consisting of Borealin, Survivin and INCENP) bound to nucleosome at 2.8 angstrom resolution. The molecular details of their interactions reveal that CPC-nucleosome binding stabilises the centromeric chromatin (nucleosomes forming the microtubule attachment sites). We are now working towards characterising the interaction of CPC, Shugoshin 1 (Sgo1) and HP1 with nucleosomes. Structural and biochemical characterisation these interactions are in progress.
WP2 Mechanisms of recruitment of enzymatic activities by the inner centromere interaction network: Protein phosphatase 2A (PP2A), which exists in different forms due to the presence of different regulatory protein subunits (B55 and B56) plays critical roles in regulating centromere functions such as maintaining centromere cohesion, error-correction (selective stabilisation of bi-polar attachments) and timely onset of anaphase (cell division stage when sister chromatids move towards the opposite poles of the mitotic spindle). However, the mechanistic basis of how different Shugoshin isoforms (Sgo1 and Sgo2) preferentially recruit PP2A to different sub-centromeric locations remains an open question. We have now reconstituted these protein complexes for further structural characterisation.
WP3. Mechanisms of centromere maintenance: As outlined above, the centromere is defined by the enrichment of a Histone H3 Variant, CENP-A, containing nucleosomes, which is critical for assembling the microtubule-capturing protein hub called the kinetochore. Importantly, the CENP-A levels undergo dilution during S-phase (a specific phase of the cell cycle, where the genetic information is duplicated for subsequent segregation to the newly formed daughter cells), as existing CENP-A is shared among the new and old DNA. To ensure stable maintenance of the centromere, the original CENP-A levels must be restored through active CENP-A deposition. By characterising the architecture of the CENP-A deposition machinery and its interaction with a protein modifying PLK1 kinase, we unravelled molecular details of how PLK1 allows CENP-A loading machinery to deposit CENP-A at the right time during the cell cycle, a question that remained unanswered for more than a decade.
During this grant period, through a collaborative effort, we have also characterised CENP-32 (an RNA modifying enzyme critical for the assembly of functional chromosome segregation apparatus, the mitotic spindle) using biochemical, structural, cell biological and clinical approaches. Our work showed that CENP-32 mutations found in patients with developmental defects affect CENP-32 enzymatic activity. We also showed that mutants compromising CENP-32 activity lead to spindle defects, causing chromosome segregation errors and altered cell proliferation.
Maintaining centromere identity, defined by the enrichment of CENP-A nucleosome, is paramount to achieving error-free chromosome segregation. Despite this, a mechanistic understanding of molecular processes ensuring centromere maintenance remains poorly understood. One critical aspect of centromere maintenance is to ensure the correct amount of CENP-A deposition at centromeres at the right time. Identifying and characterising molecular players acting as licensing factors is critical for understanding centromere maintenance. A decade ago, PLK1 was identified as a licensing factor with kinase activity that is crucial for timely CENP-A deposition at centromeres. However, the mechanistic basis for how PLK1 exerts its licensing role remained an open question. Our recently published study in the journal Science characterised PLK1 – CENP-A deposition machinery interactions and showed that PLK1 directly interacts with Mis18 and Mis18BP1 subunits of the Mis18 complex via a self-priming phosphorylation cascade. The phosphorylation cascade culminates with the activation of the Mis18 complex via a conformation switch involving Mis18, facilitating the efficient recruitment of the CENP-A chaperone HJURP for timely CENP-A deposition. Considering the vital requirement of centromere maintenance in ensuring stable inheritance of genetic information through generations, we think this work is a breakthrough discovery.