Master Regulation of Centromere Function by the Highly Conserved Mis18 Complex

The cells in our body duplicate their genomes every time they divide. In order to make sure that the process of cell division proceeds to completion without any glitches, the genomic DNA packaged in chromosomes following duplication in the parental cell must be equally and accurately segregated between two daughter cells. This means that every time a cell divides, the newly formed daughter cells will inherit the same amount of genetic material that the parental cell initially harboured. Specialised structures known as 'kinetochores' assemble on specific sites on chromosomes known as 'centromeres' to ensure that chromosome segregation into daughter cells is always accurate and error-free. Centromeres are crucial for genome stability, as kinetochores that assemble on them must attach themselves to thread-like structures known as microtubules that will then pull each duplicated chromosome towards one and only one daughter cell during cell division. Centromere dysfunction and consequent kinetochore defects can lead to chromosome mis-segregation, which has been frequently observed in a variety of cancers, and in genetic diseases such as Down's syndrome.

In most organisms, kinetochore-microtubule attachment happens at only one site on each individual chromosome. Each chromosome therefore must have only one centromere on it. While much of the chromosomal DNA in cells is tightly wrapped around conventional 'histone' proteins, centromeric DNA is unique in that it is wrapped around a specialised centromere-specific histone protein known as CENP-A. It is CENP-A, rather than the underlying DNA sequence itself, that specifies the site of centromere assembly on each chromosome. CENP-A is known to be essential for centromere function and kinetochore formation: mutations in this protein have been shown to adversely affect chromosome segregation. How CENP-A recognizes centromeric DNA and marks functional centromeres, has remained a mystery for long.

The assembly of CENP-A at centromeres is dependent on the highly conserved centromere protein Mis18, and the CENP-A specific chaperone protein HJURP. The sequence of events that regulates the timing of CENP-A loading is first initiated by Mis18, which recruits HJURP, that in turn then deposits CENP-A at centromeres. Mis18 thus master-regulates CENP-A assembly at centromeres, and is the most fundamental factor required for centromere function. Mis18 was first identified in fission yeast in 2004, as a gene which when mutated led to chromosome mis-segregation. Subsequent studies in human cells have shown that Mis18 function is highly conserved. Not surprisingly, Mis18 has been found to be mis-regulated in a variety of human cancers. How Mis18 initially recognizes centromeres to subsequently effect CENP-A assembly, however, remains unclear. The research proposed in this application is broadly aimed at elucidating how Mis18 and its associated proteins, together regulate CENP-A assembly and centromere function. The insights gained from our research will be key to building a more complete understanding of how cells faithfully segregate their DNA during cell division, through the course of evolution.

Centromeres are the specialised sites on eukaryotic chromosomes where kinetochores, that govern spindle microtubule attachment, assemble during cell division. A plethora of evidence suggests that DNA sequence is neither necessary nor sufficient for the establishment of a functional centromere. The centromere-specific histone H3 variant CENP-A (Centromere Protein A) however, specifically associates with and marks active centromeres. By replacing canonical histone H3 in centromeric chromatin, CENP-A serves as the epigenetic mark that establishes and maintains centromere identity. Through the experiments proposed here, my research group will uncover the molecular mechanisms that govern centromere specification and inheritance by regulation of the CENP-A chromatin state at centromeres. We will mainly focus on the highly conserved centromere protein Mis18, and the factors that it closely associates with, which together master-regulate CENP-A assembly.

The proposed research will use a systematic approach involving genetics, biochemistry, cell biology, genomic & proteomic techniques to dissect the regulation and function of the Mis18 complex, using the fission yeast Schizosaccharomyces pombe as a model organism. Informed by insights gained from our work in fission yeast, a simple unicellular eukaryote whose centromeric chromatin and kinetochore framework are remarkably similar to that of metazoans, we will perform cross-species complementation experiments as well as functional assays in human cells to confirm the evolutionary conservation of our findings. Conversely, we will use fission yeast as a transgenic model to study the evolutionary conservation of the Mis18 complex. Our findings will provide crucial insights into the highly conserved mechanisms that safeguard genome integrity through the regulation of centromere function and chromosome segregation across species.

Who will benefit from this research?
The research proposed in this application is basic in nature, and will have an immediate impact on the life sciences research community with interests in epigenetics and cell biology in an evolutionary context. In the long term, our findings have the potential to impact on the areas of healthcare (specifically the biopharmaceutical industry with interests in anti-cancer drug targets) and agriculture (specifically plant breeders). Through these avenues, our research will have a wider impact on the general public, the society & economy, given its potential to shed light on the fundamental mechanisms that regulate chromosome segregation during cell division.

How will they benefit from this research?
The Life Sciences research community:
The proposed research is inter-disciplinary in nature, and will be of interest to scientists interested in a range of areas including epigenetics, centromere & kinetochore biology, as well as biochemistry & structural biology. The specification of centromere identity is a fundamental, but poorly studied process in any organism. Our research will provide crucial insights into this process using fission yeast, a simple model organism. The proposed efforts to investigate the evolutionary conservation of our findings will make our work attractive to a wide range of stakeholders within the life sciences research community, including evolutionary biologists and cancer biologists.

The Biopharmaceutical Industry interested in anti-cancer drug targets:
Centromere integrity is crucial to the process of chromosome segregation. Not surprisingly, in a variety of cancers with increased levels of aneuploidy, centromere proteins including CENP-A and Mis18 have been shown to be mis-expressed. Our work aimed at dissecting the molecular-level regulation of Mis18 complex function will therefore be directly of interest to the biopharmaceutical industry interested in discovery and validation of anti-cancer drug targets, especially given the high level of structural conservation between fission yeast and human Mis18.

Plant Breeders:
Centromere-mediated genome elimination involving manipulation of CENP-A (also known as CenH3) is of immense interest to plant breeders, as this allows for ploidy to be reduced, thus facilitating breeding in crops. Studies on the regulation of CENP-A, such as ours, are therefore likely to be of significant interest to the plant breeding community. The cell cycle regulation of CENP-A deposition in plants is very similar to that in fission yeast: our findings are therefore extremely relevant to plant centromere biology, and have the potential to accelerate the innovation of novel methodologies for plant breeding through centromere-mediated genome elimination.

The general public, society & economy:
Centromere dysfunction can lead to a variety of cancers including breast cancer, lung adenocarcinoma, colorectal cancer and hepatocellular carcinoma. Aneuploidy, a consequence of centromere/kinetochore dysfunction, has been observed in one-third of human miscarriages and 0.3% of live human births, and is the leading cause of disorders such as Down's syndrome that result in trisomic individuals with a significantly reduced quality of life. These illnesses have huge implications for society and the economy. Our research will benefit the economy, the National Health Service and the public by accelerating the development of improved drugs. Our research will also facilitate improved crop production through its relevance to plant breeding, thus providing widespread benefits to society and the economy at large.