Redundant Functions for Nap1 and Chz1 in H2A.Z Deposition

Abstract

H2A.Z is a histone H2A variant that contributes to transcriptional regulation, DNA damage response and limits heterochromatin spreading. In Saccharomyces cerevisiae, H2A.Z is deposited by the SWR-C complex, which relies on several histone chaperones including Nap1 and Chz1 to deliver H2A.Z-H2B dimers to SWR-C. However, the mechanisms by which Nap1 and Chz1 cooperate to bind H2A.Z and their contribution to H2A.Z deposition in chromatin is not well understood. Using structural modeling and molecular dynamics simulations, we identify a series of H2A.Z residues that form a chaperone-specific binding surface. Mutation of these residues revealed different surface requirements for Nap1 and Chz1 interaction with H2A.Z. Consistent with this result, we found that loss of Nap1 or Chz1 individually resulted in mild defects in H2A.Z deposition, but that deletion of both Nap1 and Chz1 resulted in a significant reduction of H2A.Z deposition at promoters and led to heterochromatin spreading. Together, our findings reveal unique H2A.Z surface dependences for Nap1 and Chz1 and a redundant role for these chaperones in H2A.Z deposition.

Figure 1

Molecular recognition and structure of H2A.Z-H2B by Chz1. (a) The six centroid structures from the refined DMD ensemble of H2A.Z-H2B-Chz1 complex are structurally aligned and displayed using cartoon representation. The centroid structures are overlaid over the yeast nucleosome structure in gray, highlighting the surface on H2A.Z-H2B that is bound by both Chz1 and DNA. (b) The H2A.Z-H2B dimer is displayed using surface representation with the residues colored according to the average number of interface contacts they form with Chz1, which is displayed with the cartoon representation. The structures are overlaid over the yeast nucleosome structure in gray. The dashed-box indicates the region of H2A.Z-H2B surface that forms the acidic patch. The asterisk indicates the DNA-binding surface of H2A.Z-H2B. (c) The acidic patch from the H2A.Z-H2B dimer is shown using surface representation forming interactions from three arginine residues from Chz1. (d) The DNA binding surface of H2A.Z-H2B is shown colored according to interface contacts. The nucleosome structure in gray is overlaid to highlight the path of DNA on this surface.

Figure 2

Biochemical analysis of H2A.Z mutants predicted to destabilize Chz1-H2A.Z interaction. (a) Table showing the list of computationally predicted mutants (HTZ1 Mutation), the region they belong to in the nucleosome structure (Structural Region), and the effect of the mutation on the computationally calculated binding energy between H2A.Z-H2B and Chz1 (ΔΔG (Mean ± S.E.)). S.E. refers to standard error of mean. (b) Phenotypic analyses of H2A.Z mutants predicted to destabilize H2A.Z-Chz1 interaction. Shown is are 5-fold serial dilutions of the indicated strains that were spotted on either control (Sc-Ura) or drug-containing plates. (c) Immunoblots showing H2A.Z levels in whole cell extracts (top) and in chromatin (bottom) from wild-type and various H2A.Z mutant strains.

Figure 3

Separation-of-function mutants of H2A.Z define residues important for Nap1 and Chz1 interaction and function (a) Immunoblots showing the ability of various H2A.Z mutants to interact with Nap1. (b) Immunoblots showing the ability of various H2A.Z mutants to interact with Chz1. (c) 5-fold serial dilution of the indicated H2A.Z mutant strains were either plated alone or in combination with either a CHZ1 or NAP1 deletion on plates containing 10 mM caffeine plates.

Figure 4

Nap1 and Chz1 function redundantly in the deposition of H2A.Z at promoters. H2A.Z levels were examined by ChIP in the indicated strains and at the promoters (PRO) and open reading frames (ORFs) of (a) STE11 (b) SRB5 and (c) SCC2.

Figure 5

Nap1 and Chz1 function in a coordinated manner to restrict the spread of heterochromatin. (a) Schematic of the telomeric and mating type locus on chromosome III showing the neighboring genes. (b) qRT-PCR analysis of the genes represented in the schematic in different mutant strains of indicated genotypes (materials and methods). SWR1 and HTZ1 deletions were used a positive control. One way ANOVA was used to measure the statistical significance.