Site-specific acetylation mark on an essential chromatin-remodeling complex promotes resistance to replication stress

Abstract

Recent work has identified several posttranslational modifications (PTMs) on chromatin-remodeling complexes. Compared with our understanding of histone PTMs, significantly less is known about the functions of PTMs on remodeling complexes, because identification of their specific roles often is hindered by the presence of redundant pathways. Remodels the Structure of Chromatin (RSC) is an essential, multifunctional ATP-dependent chromatin-remodeling enzyme of Saccharomyces cerevisiae that preferentially binds acetylated nucleosomes. RSC is itself acetylated by Gcn5 on lysine 25 (K25) of its Rsc4 subunit, adjacent to two tandem bromodomains. It has been shown that an intramolecular interaction between the acetylation mark and the proximal bromodomain inhibits binding of the second bromodomain to acetylated histone H3 tails. We report here that acetylation does not significantly alter the catalytic activity of RSC or its ability to recognize histone H3-acetylated nucleosomes preferentially in vitro. However, we find that Rsc4 acetylation is crucial for resistance to DNA damage in vivo. Epistatic miniarray profiling of the rsc4-K25R mutant reveals an interaction with mutants in the INO80 complex, a mediator of DNA damage and replication stress tolerance. In the absence of a core INO80 subunit, rsc4-K25R mutants display sensitivity to hydroxyurea and delayed S-phase progression under DNA damage. Thus, Rsc4 helps promote resistance to replication stress, and its single acetylation mark regulates this function. These studies offer an example of acetylation of a chromatin-remodeling enzyme controlling a biological output of the system rather than regulating its core enzymatic properties.

Fig. 1.

Acetylated and unacetylated RSC complexes have similar biochemical properties. (A) Purified RSC complexes with TAP-tagged Rsc2. Complex was quantified based on intensity of Sth1 (*), which is the core ATPase subunit of RSC. Stoichiometry was determined based on Rsc4 intensity (**) relative to Sth1 (*). (B) Dual-antibody Western blot with WT and rsc4 K25R RSC complexes. Sixty- (lanes 1 and 3) and 30-nM (lanes 2 and 4) RSC complexes were loaded. IR680 (red) secondary antibody recognizes anti-TAP, and IR800 (green) secondary antibody recognizes anti-AcK antibody. (C) Representative time courses for remodeling by WT and rsc4 K25R RSC, as assayed by restriction enzyme accessibility. Maximal rate constants (min⁻¹) and their variation from two repeats are reported. (D) ATPase rates with saturating DNA (n = 4; SDs shown). (E) (Left) Reaction scheme for competitive remodeling assay. PstI site is in red. (Right) Representative time course for reaction of WT RSC with a mixture of unacetylated core+78 bp (S1) and unacetylated core+55 bp (S2) nucleosomes. Proteinase K-treated substrates (uncut DNA) and products (PstI-cut DNA) are labeled “S” and “P,” respectively. DNA is stained with SYBR Gold. Lane 1, 0.5 min; lane 2, 15 min; lane 3, 60 min; lane 4, 145 min; lane 5, 240 min. (F) Ratio of remodeling efficiencies for core+55 nucleosomes vs. core+78 nucleosomes (n = 3; error bars indicate SDs). Ac/unAc, acetylated core+55/unacetylated core+78 nucleosomes; unAc, both nucleosomes are unacetylated.

Fig. 2.

Rsc4 acetylation and bromodomain function are required for resistance to DNA damage. (A) Pathway analysis of E-MAP synthetic genetic interactions. Strongest interactions to complexes (yellow) and biological processes (blue) are displayed for the rsc4 K25R mutant (P < 0.005). (B) Schematic of Rsc4 mutants. (C) Serial growth assays on YPAD medium and YPAD with varying MMS concentrations. Cells were diluted to OD₆₀₀ = 0.6 and pinned onto agar plates containing drugs (48–72 h, 30 °C). Strains contain plasmid-expressed WT or mutant rsc4, as indicated. (D) S-score values for rsc4 K25R genome interactions (y axis) are correlated with those of rsc4-2 (Left) and rsc4 _Δ4 (Right) mutants (x axis). R² = 0.31 for rsc4-2 and R² = 0.05 for rsc4_Δ4. (E) Synthetic interaction between Rsc4 K25 and INO80 complex components IES3 and NHP10. Serial growth assays were performed as in C.

Fig. 3.

BD1 is required for maintenance of Rsc4 acetylation in vivo and DNA-damage resistance. (A) Mutation of BD1 (L120P) is sufficient to reduce Rsc4 acetylation in vivo. (B) Quantification of in vivo Rsc4 acetylation levels relative to WT. In each strain acetylation levels are normalized to Rsc2 levels by the IR800/IR680 (AcRsc4/Rsc2-CBP) ratio (n = 3 independent experiments; error bars indicate SDs). (C) MMS sensitivities of BD1 and BD2 mutants and their synthetic interactions with INO80 components. Assays were performed as in Fig. 2C.

Fig. 4.

Requirement for Rsc4 acetylation in the context of replication stress. (A) The double-mutant rsc4K25R nhp10Δ has delayed S-phase progression in MMS. Shown are representative cytometry profiles (n = 3 experiments). N, DNA copy number. (B) Representative profiles for midlog (asynchronous) cultures used in A. (C) Budding indices for cells treated as in A (n = 3; error bars indicate SDs). Cells with a bud diameter <50% of that of the mother cell were scored as small-budded cells, and cells with a diameter >50% of that of the mother cell were scored as large-budded cells. (D) Rsc4 K25Ac and NHP10 are synthetically required for growth in the presence of HU. Assays were performed as in Fig. 2C with various HU concentrations.