Identifying the role of the FACT histone chaperone in epigenetics through single-molecule fluorescence imaging.

Abstract

Modifications of histones in eukaryotic cells lead to reversible changes in gene expression without altering the genetic code itself. Such epigenetic modifications are necessary for the proper functioning of cellular processes like gene expression, DNA repair, and cell division. Modified histones directly affect chromatin dynamics and promote other regulation factors such as chromatin remodelers, including histone chaperones. Histone chaperones make functional DNA templates that maintain epigenetic information by remodeling the nucleosome during essential life processes, such as transcription and replication, and by avoiding histone aggregation. The facilitates chromatin transcription (FACT) histone chaperone includes two essential proteins, Pob3 and Spt16. Previously, we found that deleting Pob3 suppresses epigenetic inheritance, indicating that FACT plays an important role in epigenetic maintenance. Therefore, we engineered Schizosaccharomyces pombe (fission yeast) cell strains that express fusions of Spt16 or Pob3 to the fluorescent protein PAmCherry and investigated the dynamics of single proteins in the FACT complex in living S. pombe cells to identify the mechanism for this epigenetic inheritance suppression, including measuring changes in interactions during transcription and replication. By analyzing the motion of Spt16-PAmCherry single-molecule trajectories with nonparametric Bayesian statistics, we were able to distinguish three types of single-molecule motion: a fast (free) mobility state, an intermediate (transiently bound) mobility state, and a slow (bound) mobility state. We are assigning these biophysical states to specific biochemical processes by constructing Spt16 and Pob3 mutants with impaired histone protein binding properties, as well as drug-susceptible strains in which we can inhibit transcription and replication to identify changes in the FACT protein mobility. Overall, our live-cell single-molecule experiments reveal how the FACT complex interacts with histones and other chaperones and indicate a mechanism for epigenetic maintenance.