Investigating the Regulatory Roles of Histone Chaperones in Cellular Plasticity - PROJECT SUMMARY Histone chaperones are functionally and structurally diverse proteins. They play a central role in chromatin organization and maintenance by binding to histones and facilitating nucleosome assembly during DNA replication, transcription, recombination and repair processes. Moreover, some histone chaperones evolved additional functions independent of histone binding. Histone chaperones are thus essential for cellular proliferation and organismal development. Intriguingly, circumventing this lethality in a number of cell fate change paradigms revealed roles of histone chaperones in cellular plasticity. For example, we and others have shown that the chromatin assembly factor 1 (CAF-1), a histone chaperone complex involved in replication dependent nucleosome assembly and heterochromatin regulation, prevents cellular reprogramming. More recently, we demonstrated that CAF-1 maintains lineage integrity of stem and progenitor cells by repressing the transcription of differentiation genes. In this context, CAF-1 controls chromatin accessibility at enhancer/promoter elements of lineage specific loci and prevents aberrant binding of transcription factors. In addition to these CAF-1 sensitive sites, we also identified heterochromatic loci whose accessibility is perturbed upon CAF-1 loss, albeit with unknown effects on cell fate. The influence of CAF-1 on local euchromatic and heterochromatic loci is intriguing given that CAF-1 acts in a sequence independent manner to assemble nucleosomes during DNA replication. Whether such profound effects of CAF-1 on cell fate are linked to its nucleosome assembly function or additional non-canonical functions remain unexplored. Moreover, given the growing repertoire of histone chaperones and associated histone variants, it remains unclear whether CAF-1 cooperates with other histone chaperones to maintain lineage integrity. Therefore, the functional and structural determinants of the histone chaperone network as a whole in the context of cell fate remain important open questions. To investigate the molecular mechanisms underlying the lineage specifying functions of histone chaperones, we will use well established cell fate change paradigms in combination with gene editing/RNAi, multi-omics, biochemical and functional approaches. Specifically, we propose the following two research directions: (1) Investigate the regulatory mechanisms and function of histone chaperone sensitive chromatin sites and, (2) interrogate the structure-function relationships of histone chaperones and how their domains are intimately linked to control cell fate. In the short-term, we plan to dissect the epigenome and structural determinants of CAF-1. In the long-term we plan to extend our analysis to other histone chaperones pathways and how they cooperate with CAF-1 to control cell fate. If successful, our studies will contribute to design strategies for manipulating histone chaperone pathways to control cell fate in health and disease.
