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

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.

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.