Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CHROMOREP (Reconstitution of Chromosome Replication and Epigenetic Inheritance)

Teaser

Each cell in our body – and our bodies contain trillions of cells – harbours all of the genetic information to make a human. However, different cells express different genes in the genome that specifically define muscle, skin, brain etc. How these expression patterns are...

Summary

Each cell in our body – and our bodies contain trillions of cells – harbours all of the genetic information to make a human. However, different cells express different genes in the genome that specifically define muscle, skin, brain etc. How these expression patterns are maintained during the proliferation of cells needed for organ development is unknown. Every time a cell divides, it must first replicate its entire genome. This involves removing all proteins from the DNA, at least transiently, unwinding the DNA duplex, and copying each strand. Thus, the machinery involved in specific gene expression patterns is transiently displaced from DNA during replication and must be quickly re-established. Amongst the proteins that are displaced during DNA replication are an abundant class called histones. Histones bind very tightly to DNA in structures called nucleosomes and we know little about how nucleosomes are disrupted during DNA synthesis. Although there are relatively few different histones and histone ‘variants’, each of the four core histones (H2A, H2B, H3 and H4) have tails that are the sites of a multitude of covalent modifications including acetylation, methylation, phosphorylation ubiquitinylation, etc. These modifications can specifically recruit proteins involved in gene activation or repression. It is believed that the displacement of nucleosomes during replication must somehow be coupled to the re-assembly of nucleosomes from the same histones behind the replication fork to ensure the accurate re-establishment of gene expression patterns. We know very little about how parental nucleosomes are re-deposited behind the replication fork or how this is coordinated with the assembly of nucleosomes from newly synthesised histones.
In addition to providing a deep understanding of a fundamentally important cellular process, our work may have important implications for understanding the aetiology of human cancer. It has emerged in recent years that most and perhaps all cancers exhibit some form of ‘replicative stress’, loosely defined as perturbation of normal replication rates and accumulation of DNA damage during replication. It is unclear whether there is a single cause of replicative stress or whether replicative stress is an umbrella term for a variety of biochemical mistakes, which may be different in different cancers. Our work will help understand the biochemical mechanism by which chromosomes are replicated. This work will synergise with other work from my lab aimed at understanding replicative pathways which are misregulated in cancer.
The aim of our project is to understand how replication forks contend with nucleosomes and how gene expression patterns are re-established during and after DNA replication using a ‘bottom-up’ approach to reconstitute this process with purified proteins, based on a DNA replication system developed in our laboratory.

Work performed

Our project comprises three overarching aims, each with two sub-aims. Significant progress has been made on the first two aims. We will soon start work on the third aim.
I. Origin choice and timing. All eukaryotic cells initiate replication from multiple ‘replication origins’ distributed along chromosomes which each have a signature efficiency and time of replication during S phase. This project is aimed at understanding how nucleosome arrays (chromatin) affect the choice of replication origins and the time at which origins are activated during S phase.
1. Origin choice – We have recently published work showing that chromatin enforces replication origin selection (Kurat et al. Mol. Cell 2017). The reconstitution of origin patterns genome-wide remains as part of the next phase of the grant.
2. Origin timing – We have established a competition assay in which plasmids containing different histone modifications are mixed together and replicated to begin to understand how early firing origins out-compete late origins for limiting replication factors. We have found that histone acetylation promotes faster assembly of the CMG helicase (Casas Delucchi, unpublished). This will give us an assay to reconstitute the timing programme.
II. Mechanisms of nucleosome displacement and potential transfer to daughter chromatids during DNA replication
1. Displacement of parental nucleosomes – We have published work showing histone ‘chaperones’ (proteins that bind histones and neutralise their charge), nucleosome remodellers (enzymes that use ATP hydrolysis to push nucleosomes around) and histone acetylation all contribute to rapid replication through chromatin (Kurat et al. Mol. Cell 2017). This work also showed parental nucleosomes were efficiently transferred behind the progressing fork. We are reconstituting the pathway by which newly synthesised histones are incorporated into replicating DNA to complement this work (Hill unpublished)
2. Pathway(s) for inheriting parental nucleosomes – When we have this pathway fully functional we will combine it with our chromatin replication system to attempt complete chromatin replication.
III. Inheritance of chromatin states during and after DNA
1. Recruitment of histone modifiers during DNA replication – We have established an approach to identify modifiers recruited to DNA in extracts and have identified several of these. We will continue this approach with nucleosomes containing different modifications.
2. Coupling chromatin replication to transcriptional silencing – We cannot begin this project until III.1 is complete, which I anticipate should happen within the next year.

Final results

This work is state of the art biochemistry; the first time replication of chromatin has been reconstituted with purified proteins. It is too early to draw any conclusions about wider societal implications, but our work has already shown that replication rates can be subtly affected by different nucleosome remodellers and modification enzymes. Since these are often mis-regulated by oncogenes, it is possible that our work may explain aspects of replicative stress in some cancers.

Website & more info

More info: https://www.crick.ac.uk/research/a-z-researchers/.