Abstract
The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.
ACKNOWLEDGMENTS
We are grateful to David Jones for the use of the Horiba Fluorolog-3 spectrometer, Robert Hodges for the use of the Rayonet photochemical reactor RPR-100, Michael McAndrew for help with PCR screening of the FLAG-tagged genes, Stephanie Williams for performing PHO5 induction assays, and Elizabeth Doggett for the construction of EDY002. We thank Jean Scorgie for help with the fluorescence assay, Myrriah Chavez for help with some of the early studies, Yumeng Hao for help with plasmid construction, Steve Hahn for the gift of pLH157, and Richard Baker for the gift of pRB294 containing a NotI-HA cassette.
We acknowledge support from NIH GM 064475 and CPRIT to J.K.T. and NIH GM GM079154 to M.E.A.C.