Investigating the interplay between SMC complexes and Topoisomerase II

DNA is the repository for all the genetic information of a cell. To encode all this information DNA molecules are extraordinarily long. For example, a human cell contains nearly 2m of DNA in a nucleus smaller than 20 microns in diameter. Not only must all this DNA be packed into the nucleus but its organisation must be constantly re-organised so that its packaging can promote normal gene expression during most of the cell cycle before being tightly packaged during mitosis in a manner that allows faithful segregation of all chromosomes to daughter cells. Failure to appropriately organise chromosomes during the different stages of the cell cycle leads to chromosome fragility, aberrant chromosome numbers and cell death. Features often associated with cancer, ageing and human growth disorders.

Two types of protein complexes appear to be essential to establish and maintain chromosomal organisation, SMC complexes and type II topoisomerases (Top2). The ancient family of SMC complexes are found in all the kingdoms of life, where they are required to appropriately structure chromosomes for genetic inheritance. Both the eukaryotic SMC complexes cohesin and condensin are thought to organise chromosomes by promoting DNA looping along chromosomes. Type II topoisomerases are also found in all cell types where they are thought to organize DNA by allowing one section of DNA to pass through another, promoting untangling of chromosomes and relaxing any DNA topological stress that builds up on the DNA. The eukaryotic type II topoisomerase Top2 is proposed to have both an enzymatic and structural role in chromosome structure. However, analysis to date of how SMC complexes and Top2 may work together to dynamically organise chromosome structure has produced seemingly contradictory results. Some of these studies indicate that SMC complexes and Top2 work in concert with each other to promote proper gene expression and chromosome segregation. However, others have found that they can also operate independently or even antagonistically in the maintenance of normal chromosome structure. These contrasting results indicate a complex and potentially context dependent interplay between Top2 and the different SMC complexes. Understanding the nature of this interplay is crucial for how these two ancient DNA manipulating machines work together to organise DNA and ensure normal biological function. Indeed, mutations in the genes encoding Top2 and SMC proteins are closely linked with cancer progression and the growth of animals and plants.

In this proposal, we will investigate the interplay and co-dependencies of SMC complexes and Top2 on chromosome structure. Recently we have used the Hi-C chromosome conformation technique to show that cohesin and condensin organise the structure of budding yeast chromosomes in distinct ways. Here, we will examine how chromosome structure is altered by Top2 and how such changes are regulated by the different SMC complexes cohesin and condensin. We will go onto to examine if Top2 dependent changes to chromosome structure require the enzymatic activity of Top2 or only its binding to chromosomes and if these are regulated by SMC complexes. Finally, we will examine if DNA supercoiling and its regulation by Top2, and potentially SMCs, organizes chromosomes.

Together this proposal aims to provide a comprehensive and coherent analysis of how the fundamental DNA structuring activities of Top2 and the SMC complexes cohesin and condensin interact to generate functional chromosome structure.

Two of the most conserved DNA metabolism enzymes are Type II topoisomerases and SMC complexes. Both are required for faithful genetic inheritance in archea, bacteria and eukarya. Although they have distinct biochemical roles on DNA, Type II topoisomerases (Top2) and SMC complexes have been found to have a mixed and complex set of genetic interactions. SMC complexes from both bacteria and eukarya have been found to promote the de-catenation of sister-chromatids by Top2. Top2 action has also been linked to TAD formation via SMC dependent chromosomal looping. However, Top2 has also been reported to act independently of SMC activity during chromosome re-organization and even to act antagonistically to SMC complexes in mitotic contexts.

Using Hi-C chromosome conformation analysis, we have found that Top2 is required for normal mitotic chromosome structure in budding yeast. However, the role of Top2 appears to be distinct in different genomic contexts. In some regions, its action is consistent with supporting SMC dependent chromosome organization. In other contexts, our data suggests that Top2 activity antagonizes SMC action. These observations indicate that this system will be an ideal model to understand the complex and evolutionarily conserved interplay of SMC complexes with Top2.

In this proposal, we will carry out a comprehensive analysis of the functional interplay of the SMC complexes cohesin and condensin with Top2 in budding yeast. These experiments will combine defined genetic manipulation of these factors with analysis of chromosome conformation (Hi-C), PALM and conventional live cell microscopy, genome wide analysis of Top2 strand passage activity and DNA topological stress in cells. Together these data will provide a clear and comprehensive analysis into how these two ancient and evolutionarily conserved DNA metabolic activities structure chromosomes to allow faithful genetic inheritance.

Who might benefit from this research?
This research will investigate the processes that dynamically re-organize the structure of chromosomes in cells. This is a fundamental yet still poorly understood area of cell biology and bio-physics. This work seeks to inform how two activities at the core of chromosome organisation, provided by SMC complexes and Top2 topoisomerases, work together to organise chromosomes.

The primary beneficiaries of this work in the short term will be the scientists working in the area of chromosome structure and related issues. In the medium term this work will benefit clinical geneticists researching how mutations in SMC complex factors or Top2 influence human development and disease.

The students of Sussex University and schools in our partnership program will also benefit from our research. The concepts of Hi-C and describing chromosome structure in terms of contact probability forces students to conceptualize chromosome structure as a highly dynamic process, a fact not illustrated by traditional microscopy of fixed cells.

The capacity of the University of Sussex and U.K. science generally, will benefit from having our research group actively and successfully generating Hi-C libraries. Finally, the post-doctoral research associate on the project will benefit from the extensive training in data driven techniques and project management of this multidisciplinary project.

How might they benefit from this research?
In the short term our genome wide data sets will provide a comprehensive analysis of how the essential activities of the SMC complexes cohesin and condensin interplay with the activity of topoisomerase II and how this is influenced by genomic context. The raw genome wide data sets along with single molecule and live cells imaging will underpin future modelling of the dynamics of chromosome though biophysical modelling. Our work will also provide paradigms for other researchers working in other systems as to how these two fundamental activities work together in cells.

In the medium term this work will benefit clinical geneticists by providing mechanistic insight into how mutations in SMC complex factors or Top2 influence human development and disease.

Through my workshop at the University in "modern methods in genetics" on Hi-C and also provision of undergraduate and summer projects we have engaged and trained students in the data rich analysis of Hi-C assays. My lab has a school partnership program where we are conducting genome instability experiments in schools using yeast colony sectoring. In addition to the impact of carrying out genome stability experiments with school age students this program allows us to discuss current themes of biological science with the school teachers and see if our genome wide experiments can be used to illustrate concepts on their curricula both for teaching sciences and coding.

Hi-C is increasingly becoming an essential technique in the field of genetics and molecular biology for analyzing nuclear structure. We have successfully established the experimental and processing facilities to analyze Hi-C datasets. This project will cement the future of Hi-C experimentation and processing at the GDSC. We freely provide training in this assay to other labs both internal and external . Internally we have worked with the in Neale Lab in the GDSC. To benefit wider UK science we have also provided training to the Marston lab based in Edinburgh. The PDRA supported by the grant will continue to provide this training service in the future to any academic group in the UK.

Biological research is increasingly becoming a data driven research area. Strategically the GDSC is focused on producing researchers with the skills to produce both the highest quality data and the best quality analysis of that data. In this proposal the PDRA will be trained in both these areas, internally and externally, gaining crucial skills for the U.K.