Structural investigation of the histone chaperone complex FACT using genetically encoded crosslinkers in Saccharomyces cerevisiae

Abstract

Chromatin is a highly dynamic nucleoprotein structure that stores the eukaryotic genetic information. Histones are the essential chromatin proteins that form the core octamers around which DNA is compacted within the nucleus. These small proteins regulate all aspects of chromatin biology. Histone chaperones act as guards and guides of histones and are implicated in the regulation of transcription, replication and DNA-repair. In order to understand how histone chaperones achieve this variety of functions, it is important to structurally characterize the interplay between histone chaperones and their binding partners. Most structural and functional data exists from solution studies, where their implications in vivo are indirect. Only under physiological conditions can we truly begin to delineate the dynamics and interplay between chromatin and associated proteins. In this regard, methods need to be developed to address the in vivo characterization of chromatin processes. In this thesis, I present an innovative crosslinking approach to study the interactome of the histone chaperone complex FACT (facilitates chromatin transcription) in living yeast. Using the genetically encoded crosslinker amino acid (4-Benzoyl-L-phenylalanine) pBPA at nearly two hundred different positions, I map the interactions of FACT at a single amino acid resolution, in vivo. This highly reproducible crosslinking approach reveals putative interaction surfaces for a diverse set of suggested FACT binding partners. Using this assay I show that the acidic C-terminal domain (CTD) of the FACT complex subunit Pob3 interacts with the histones H2A/H2B in a defined manner in vivo, although this domain is predicted to be structurally disordered. Furthermore, I characterize a novel nuclear localization signal at the very end of the Pob3-CTD. Thus, in the case of the FACT complex, the acidic CTD has both a role in the nuclear transport and in the histone binding. Furthermore, my observations provide evidence for a functional role of the acidic domain during replication. Based on the data presented in this thesis I can suggest that acidic domains present on many histone chaperones act as putative binding platforms for the interaction with histones in vivo.