Periodic Reporting for period 1 - COSMOS (The Conformation Of S-phase chroMOSomes)
Período documentado: 2021-02-01 hasta 2023-01-31
The organization of DNA within a nucleus is essential for all processes that occur. It’s extremely important for DNA replication and mitotic DNA segregation to occur without excessive entanglements and subsequent DNA damage. Correct organization is also essential for cell functions including transcription, DNA repair and recombination. However, when organization is impaired this can lead to chromosomal aberrations that accompany tumour progression.
Extensive organizational structure analyses have been carried out utilizing chromosome conformation capture (Hi-C) methodology which has helped us understand the different orders of compaction of the genome. However, Hi-C is not able to take into account the differences between the exactly identical replicated sister chromatids. How DNA interactions occur between the sisters and throughout the nucleus is therefore not well understood. Newly replicated sister chromatids are held together by a mixture of DNA intertwines and the cohesin complex. As the cell cycle progresses and the DNA must be segregated, sisters are compacted by SMC proteins and disentangled by topoisomerase proteins. The interplay between the proteins and how they act to perfectly disentangle sister chromatids in order to prevent DNA damage in every cell cycle is important for us to be able to understand.
Therefore this project aimed to expand techniques that are available to distinguish sister chromatids from one another for chromosome conformation analysis. Then this technique for sister chromatid conformation analysis could be further utilised to answer crucial questions about how the conformation of DNA is controlled to prevent aberrant processes from occurring.
This work advances the field of DNA conformation analysis substantially, by developing a technique that will allow wide scale uptake for many different labs to be able to further understand the processes occurring during the cell cycle and how these processes go wrong in disease.
Extensive organizational structure analyses have been carried out utilizing chromosome conformation capture (Hi-C) methodology which has helped us understand the different orders of compaction of the genome. However, Hi-C is not able to take into account the differences between the exactly identical replicated sister chromatids. How DNA interactions occur between the sisters and throughout the nucleus is therefore not well understood. Newly replicated sister chromatids are held together by a mixture of DNA intertwines and the cohesin complex. As the cell cycle progresses and the DNA must be segregated, sisters are compacted by SMC proteins and disentangled by topoisomerase proteins. The interplay between the proteins and how they act to perfectly disentangle sister chromatids in order to prevent DNA damage in every cell cycle is important for us to be able to understand.
Therefore this project aimed to expand techniques that are available to distinguish sister chromatids from one another for chromosome conformation analysis. Then this technique for sister chromatid conformation analysis could be further utilised to answer crucial questions about how the conformation of DNA is controlled to prevent aberrant processes from occurring.
This work advances the field of DNA conformation analysis substantially, by developing a technique that will allow wide scale uptake for many different labs to be able to further understand the processes occurring during the cell cycle and how these processes go wrong in disease.
I aimed to be able to expand techniques that are available to distinguish sister chromatids from one another for chromosome conformation analysis. I chose to work with diploid immortalised human cell lines as my basis for understanding DNA conformation through the cell cycle. To complete this aim I decided to therefore modify and optimise a technique known as Sister-C in yeast for human cell application.
To distinguish two exactly identical chromatids from one another I chose to label the newly replicating DNA with a thymidine analogue. DNA normally comprises four different bases attached to the sugar phosphate backbone. These are Adenine, Guanine, Cytosine and Thymidine. In this project I needed to replace Thymidine with a different nucleoside analogue. I worked on trying different analogues to find one that would be the most suitable; incorporating efficiently and not causing toxicity to the cells. I finally chose to replace the Thymidine with a synthetic nucleoside analogue, also used in the yeast version of the technique, called 5-bromo-2′-deoxyuridine (BrdU) so that one half of the DNA duplex is labelled.
To be able to label DNA at an exact point in the cell cycle I chose to synchronise the human cells so that all cells in one experiment are going through the cell cycle together. Multiple cell lines and synchronization protocols were tested to find a method of synchronising as many cells as possible together. I synchronised the cells before the replication phase of the cell cycle which is the timepoint at which I added the BrdU. Therefore, as the cells all synchronously replicate, BrdU will be incorporated into the newly made DNA. Due to the semi conservative nature of DNA replication the half of the DNA strand which was used as a template will still contain the thymidine base and the newly synthesized other half of the duplex incorporates BrdU instead of T in most instances.
In order to study chromosome conformation I then optimised chromosome conformation capture techniques followed by next generation sequencing or long read sequencing in order to perform them on the sister chromatids. I crosslinked the cells followed by fragmenting DNA and ligating together pieces of DNA that were in close proximity to one another. Traditionally these can then be sequenced in order to build up a map of which pieces of DNA are close to one another in 3D space. However, for my project the BrdU containing DNA strands are first degraded by UV light to leave only the non-BrdU containing fragments. We sequence the remaining fragments and analyse the results to understand which sister chromatid each piece of DNA originally came from. If the remaining contacts are between the same strand of DNA (i.e. Watson-Watson) this will constitute a cis sister interaction and if the contacts are between different strands of DNA (i.e. Watson-Crick) this will come from a trans sister interaction.
I worked extensively with bioinformaticians to try to understand how best to analyse and visualise the results of the sequencing data. We built up new pipelines and computational methods for processing proximity ligated sister chromatid DNA data, to be able to simply show striking results in an easily understandable format.
This new human cell culture technique opens the world of chromatin conformation analysis to endless possibilities for further applications, including further study of human cell cycle dynamics and the role DNA conformation has in disease.
To distinguish two exactly identical chromatids from one another I chose to label the newly replicating DNA with a thymidine analogue. DNA normally comprises four different bases attached to the sugar phosphate backbone. These are Adenine, Guanine, Cytosine and Thymidine. In this project I needed to replace Thymidine with a different nucleoside analogue. I worked on trying different analogues to find one that would be the most suitable; incorporating efficiently and not causing toxicity to the cells. I finally chose to replace the Thymidine with a synthetic nucleoside analogue, also used in the yeast version of the technique, called 5-bromo-2′-deoxyuridine (BrdU) so that one half of the DNA duplex is labelled.
To be able to label DNA at an exact point in the cell cycle I chose to synchronise the human cells so that all cells in one experiment are going through the cell cycle together. Multiple cell lines and synchronization protocols were tested to find a method of synchronising as many cells as possible together. I synchronised the cells before the replication phase of the cell cycle which is the timepoint at which I added the BrdU. Therefore, as the cells all synchronously replicate, BrdU will be incorporated into the newly made DNA. Due to the semi conservative nature of DNA replication the half of the DNA strand which was used as a template will still contain the thymidine base and the newly synthesized other half of the duplex incorporates BrdU instead of T in most instances.
In order to study chromosome conformation I then optimised chromosome conformation capture techniques followed by next generation sequencing or long read sequencing in order to perform them on the sister chromatids. I crosslinked the cells followed by fragmenting DNA and ligating together pieces of DNA that were in close proximity to one another. Traditionally these can then be sequenced in order to build up a map of which pieces of DNA are close to one another in 3D space. However, for my project the BrdU containing DNA strands are first degraded by UV light to leave only the non-BrdU containing fragments. We sequence the remaining fragments and analyse the results to understand which sister chromatid each piece of DNA originally came from. If the remaining contacts are between the same strand of DNA (i.e. Watson-Watson) this will constitute a cis sister interaction and if the contacts are between different strands of DNA (i.e. Watson-Crick) this will come from a trans sister interaction.
I worked extensively with bioinformaticians to try to understand how best to analyse and visualise the results of the sequencing data. We built up new pipelines and computational methods for processing proximity ligated sister chromatid DNA data, to be able to simply show striking results in an easily understandable format.
This new human cell culture technique opens the world of chromatin conformation analysis to endless possibilities for further applications, including further study of human cell cycle dynamics and the role DNA conformation has in disease.
The project is now completed and therefore there are no more expected results. However, there are potentially endless applications for this new technique for us and others. We will be able to continue using the technique worked on here for multiple future applications outside of the scope of this current grant. We will continue to analyse the folding of chromosomes throughout the cell cycle and how this affects processes such as DNA replication, chromosome segregation and DNA transcription. WE can also follow labelled DNA through multiple cell cycles. We will also aim to understand how DNA damage is dealt with by bringing together multiple aberrations in 3D space in order for them to be resolved.
Beyond our own lab this research opens the door for many other labs in the area of chromosome conformation to start to take up the technique of labelling sister chromatids to allow for better discernment of the 3D structure of DNA. This is a rapidly growing area of research which we expect to be applied to meiosis as well as mitosis to further understand homologous recombination and complex DNA topological interactions.
Beyond our own lab this research opens the door for many other labs in the area of chromosome conformation to start to take up the technique of labelling sister chromatids to allow for better discernment of the 3D structure of DNA. This is a rapidly growing area of research which we expect to be applied to meiosis as well as mitosis to further understand homologous recombination and complex DNA topological interactions.