Histone exchange chromatin dynamics and chromatin function: Role of ATP-dependent chromatin remodelling factor Fun30 in epigenetics

Lead Research Organisation: Babraham Institute
Department Name: Nuclear Dynamics

Abstract

The genetic material in each cell is packaged and organized by an extremely complex, heterogeneous structure, called chromatin. The basic building block of this structure is the nucleosome, a molecular protein spool around which DNA winds in almost two turns. Nucleosomes are modified and interact with other proteins to create chromatin structures that shut down genes or allow access to the machinery that read out the information from genes. Specific chromatin structures mediate gene regulation and are required for genome stability and chromosome segregation. Such structures have to be faithfully duplicated for the maintenance of gene expression patterns through development. Defects in heterochromatin have been linked to genome instability, infertility, accelerated aging and an enhanced risk of cancer. Despite this fundamental importance, our knowledge of how specific chromatin structures, such as heterochromatin, are assembled and maintained is limited. We will study how nucleosome remodelling and modification enzymes interact to assemble a specific chromatin structure to shut down genes and to stabilise the genome. This research has implications for our understanding of normal development, but also for cancer and fertility.

Technical Summary

Specific chromatin assemblies are essential for correct gene expression, genome stability and chromosome segregation. Chromatin structures need to be maintained and need to be re assembled after DNA replication, when the replication fork perturbs chromatin in a profound way. Yet, the concept of 'chromatin structure' is not appropriate. We are starting to appreciate that chromatin is much more in flux than previously thought and that phenomena such as heterochromatin should be seen as an active process rather than a structure. What is becoming clear is that histone turnover rate is an important parameter that governs chromatin function. ATP-dependent nucleosome remodelling factors are key mediators of chromatin dynamics. We aim to unravel how specific chromatin structures are assembled and maintained for gene regulation and epigenetic inheritance by studying the role and mechanisms of ATP-dependent chromatin remodelling factor Fun30 in yeast heterochromatin. This will uncover links between chromatin function, histone ubiquitination, histone variant exchange and nucleosome remodelling. Our work indicates that Fun30 regulates the deposition of the histone variant H2AZ, a histone that is involved in transcriptional regulation and that counteracts gene silencing in yeast. We will test using established chromatin immunoprecipitation techniques if Fun30 specifically removes histone variant H2AZ for gene silencing or if it regulates histone replacement in general by mediating a 'churning over' of histones. We will use yeast genetics to dissect the link between ubiquitination, histone modifications and nucleosome remodelling, in particular we will use yeast strains that have point mutations of the ubiquitination sites and mutations of the ubiquitination/ deubiquitination machinery . We will test the direct role of Fun30 in these chromatin remodelling events by reconstitution of the reaction in vitro.
 
Description We explored a component of the molecular machinery that is involved in packaging and organising the genome. We focused on a specific factor called Fun30 that is involved in organising the basic subunit of genome organisation, the nucleosome. The nucleosome is like a spool around which the DNA wraps in two turns and is composed of proteins called histones. We showed that Fun30 affects nucleosomes genome-wide but also has an important role in supporting the function of centromeres. Centromeres are structures around the DNA of each chromosome that are critical in diviniding chromosomes between daughter cells after cell division.
Exploitation Route Our work provided basic insights into the function of highly conserved factors found in all cells of eukaryotes. We generated assays to explore centromere function that might be taken up by other groups.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our work has been cited in numerous publications.
First Year Of Impact 2011
Sector Other
 
Description MRC collaborative grant: Next generation sequencing high throughput epigenomics
Amount £943,678 (GBP)
Funding ID G0801156 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 09/2013
 
Title Expression vectors for wildtype and mutant forms of Fun30 in yeast 
Description Expression vectors for wildtype and mutant forms of Fun30 in yeast, thees vectors have been frequently requested and we deposited them to the Addgene repository. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact The vectors have been requested by other researchers, e.g. in the USA. 
URL http://www.addgene.org
 
Title genome-wide mapping data - Fun30 PLoS Genetics paper 
Description ChIPseq and RNAseq data relevant to the biological function of chromatin remodeling factor Fun30 in budding yeast, deposited to the EBI array express repository (http://www.ebi.ac.uk/arrayexpress/) 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact Our paper has been cited 11 times as to 7-11-2014. 
URL http://www.ebi.ac.uk/arrayexpress/
 
Description Collaboration with the group of Dr Nicolas Kent 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the biological frame work and published the paper.
Collaborator Contribution The group of Dr Nicolas Kent provided nucleosome mapping data.
Impact Paper: Durand-Dubief et al. PLoS Genetics, 2012. data sets submitted to repository. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002974#s4
Start Year 2010