The BAH domain of Rsc2 is a histone H3 binding domain

Abstract

Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction.

Figure 1.

Rsc2 is important for mediating silencing of rDNA. (A) Silencing was monitored in strains containing a URA3 reporter gene in the rDNA (32) by assaying survival on media lacking URA relative to survival on SC medium. (B) Survival of silencing reporter strains as in (A) on media lacking URA was quantitated, and the data are represented as the mean ± 1 SD of at least three independent experiments. Statistical analysis was performed using an unpaired t-test. Asterisks indicates P ≤ 0.01. (C) Chromatin IP analysis of Rsc2-myc at various locations across the rDNA repeat. Data shown are the mean enrichment of at least three independent experiments ± 1 SD. The right hand panel shows a schematic of the 9.1 kb rDNA repeats with an expanded view of a single repeat. The 25S, 5S and 18S coding sequences are indicated with solid arrows. The relative positions of the E-pro promoter region, cohesin-associated region and autonomously replicating sequence (ARS) are indicated. Locations of primer pairs are indicated with arrows.

Figure 2.

The Rsc2 BAH-CT1 domain directly interacts with chromatin. (A) Western blot analysis of rsc2 null strains carrying an empty vector (lane 1; EV), a plasmid with myc-tagged full-length RSC2 under the control of its own promoter (lane 2; pRsc2-myc) and an overexpression construct of myc-tagged BAH-CT1 under the control of the GAPDH promoter (lane 3; OE pBAH-CT1-myc). Westerns were analyzed with anti-myc (top two panels) or anti-H2A (bottom panel) as a loading control. (B) Full-length Rsc2 and Rsc2 BAH-CT1 are associated with chromatin in vivo. ChIP assays examining enrichment of full-length Myc-tagged Rsc2 (top panel) or Myc-tagged overexpressed BAH-CT1 (bottom panel) relative to the untagged control at the HTA1 promoter or the 18S region of the rDNA (18S-B on Figure 1C). Data shown are the mean enrichment of at least three independent experiments ± 1 SD. (C) Coomassie stained gel of recombinant His-tagged BAH-CT1 (left panel). Western blot analysis of pull-down assays with nickel agarose using anti-H2B (right panel). Lane 1 shows 10% input of yeast mononucleosomes used in pull-down assays. The total bound fraction and 10% of the unbound fraction of each pull-down reaction were analysed for the presence of H2B by western blotting. (D) Gel shift analysis using mononucleosomes assembled onto radiolabeled DNA (lane 1) with increasing concentrations of recombinant His-tagged BAH-CT1.

Figure 3.

The BAH-CT1 domain of Rsc2 interacts with histone H3. (A) Gel shift analysis using radiolabeled DNA with recombinant His-tagged BAH-CT1 or recombinant MBP-tagged Ies6 as a positive control. (B) Coomassie-stained gel of recombinant GST or GST-BAH-CT1 (left panel) used in pull-down assays with native calf thymus histones. Bound proteins were analyzed by western blotting with antibodies against each of the four core histones (right panel). (C) Pull-down assay using GST or GST-BAH-CT1 and native yeast mononucleosomes analyzed with anti-H4 antibody. (D) Pull-down assay using GST or GST-BAH-CT1 and recombinant purified histone H3, analyzed with antibodies against histone H3. (E) Co-immunoprecipitation of chromatin with wt Rsc2 is unaffected by LRS mutation A75V of histone H3. Co-IP was performed from the indicated strains containing either myc-tagged Rsc2 expression plasmid (Rsc2-myc) or empty vector with anti-myc antibody. Input and bound proteins were analyzed with anti-myc (top panel) or anti-H2A (bottom panel).

Figure 4.

Structure of the Rsc2 BAH-CT1. (A) Schematic representation of the functional domains of Saccharomyces cerevisiae Rsc2 where BD, BAH and CT indicate bromodomain, BAH domain and C-terminal conserved regions, respectively. The boxed region indicates the amino acid boundaries of the RSC2-BAH-CT1 expression construct used in this study. (B) Stereo-pair secondary structure cartoon of RSC2-BAH-CT1, colored blue to red from the visible N-terminus at residue 401 to the C-terminus at residue 633. (C) Side-by-side comparison of the BAH domain structures of Rsc2 BAH-CT1 (this study), BAF180, Sir3 and Orc1. The core canonical BAH domain fold is colored gray in each case. N- and C-terminal additions/extensions to the fold are colored blue and red, respectively, with loop insertions at two points colored in green and yellow. PDB accession codes are shown in parentheses.

Figure 5.

The Rsc2 BAH-CT1 domain differs from both Sir3 and Orc1 BAH domains in regions critical for making histone-specific contacts, indicating that the mechanism of H3 binding in Rsc2 is distinct from these proteins. (A) Molecular surface representations of Sir3 BAH (left) and Rsc2 BAH-CT1 (right) coloured by electrostatic potential. The tail of histone H4 (amino acids 13-24) bound to Sir3 is shown in stick representation, with carbon atoms colored in cyan. The equivalent path for the histone tail in Rsc2 was determined by superposing the two structures. The overall charge distribution on the surface of Rsc2 is different to Sir3, being much less acidic and more hydrophobic in nature and therefore unlikely to bind to the histone H4 tail. Furthermore, the presence of the CT1-loop (amino acids 577–584) also prevents an interaction by blocking the expected binding path. (B) Molecular details of the Sir3-BAH interaction with helix α1 and loop L1 of histone H3, including several residues comprising the LRS region of the nucleosome core particle. Potential hydrogen bonds are indicated by black dotted lines. (C) Molecular details for the equivalent region of Rsc2 BAH-CT1 as shown in (B), highlighting that the identities of the amino acids involved in H3 binding are not conserved between the two BAH domains. (D) The N-terminus of Sir3 contains a ‘basic patch’ (amino acids 28–32), which interacts with a complementarily charged ‘acidic patch’ formed between histones H2A and H2B on the surface of the nucleosome core particle, as shown by the molecular cartoon and electrostatic surface (left). Sir3 BAH is shown in gold, and superposed Rsc2 BAH-CT1 in gray. The visible N-terminus (Asp401) of Rsc2 is indicated by the short arrow. Amino acid sequence alignment and secondary structure prediction (right) indicates that a similar basic patch is not present in Rsc2, and that the protein could not interact with the acidic patch in the same manner as Sir3. BD2 = bromodomain 2. (E) As for (A), but showing the interaction between Orc1 and the tail of histone H4 (amino acids 16–23) dimethylated at lysine 20. (F) Molecular details of the Orc1 BAH H4K20me2 binding pocket. (G) Molecular details for the equivalent region of Rsc2 BAH-CT1, as shown in (F), highlighting that the identities of the amino acids involved in K20me2 binding are not conserved between the two BAH domains, and do not form a methyl-lysine binding pocket.

Figure 6.

A conserved motif on Rsc2 BAH mediates the interaction with H3. (A) Left panel: Coomassie stained gel of GST, GST-BAH domain from Rsc1 (GST-BAH^Rsc1) and GST-BAH1 from BAF180 (GST-BAH1^BAF180). Right panel: GST pull-down assay using Rsc2 BAH-CT1 (GST-BAH-CT1), GST-BAH^Rsc1 or GST-BAH1^BAF180 and recombinant histone H3. Bound protein was analyzed by western blotting using anti-H3. (B) Rsc2 GST-BAH-CT1, GST-BAH^Rsc1 and GST-BAH1^BAF180 proteins were assayed for the ability to specifically interact with histone H3 from a mixture of calf thymus core histones in pull-down assays. Bound protein was analyzed by western blotting using antibodies specific for each of the four core histones. (C) A panel of mutant Rsc2 GST-BAH-CT1 proteins was assayed for the ability to interact with histone H3 in pull-down assays. Bound protein was analyzed by western blotting using anti-H3 antibody. Coomassie stained gel of the GST-BAH-CT1 proteins used in the pull-down assays (bottom panel). (D) GST-BAH-CT1 or GST-BAH-CT1-K437E was used in pull-down assays containing the indicated amount of recombinant H3 and bound protein was analysed by western blotting using anti-H3. (E) Molecular details of the β4–β5 connecting loop of Rsc2 BAH-CT1 showing the positions of Trp436, Lys437 and Trp444. (F) GST pull-down assay using wt, W436A and W436L GST-BAH-CT1 constructs. Bound protein was analyzed by western blotting using anti-H3. (G) Sequence alignment of the region of the BAH domain encompassing β4, β5 (indicated by arrows below the alignment) and the connecting loop. The positions of Trp436 and Trp444 in Rsc2 are indicated by arrows above the alignment.

Figure 7.

Mutation of the conserved motif in the Rsc2 BAH domain that is important for H3 binding in vitro disrupts some Rsc2 functions in vivo. (A) Western blot analysis of whole cell lysates prepared from rsc2 null strains carrying empty vector or expression constructs with full-length Myc-tagged Rsc2 (wt, W436A, W436L or K437E mutant) using anti-myc (top panel) or anti-H2A (bottom panel) as a loading control. (B) Silencing was monitored as in Figure 1B using the rsc2 null strain carrying either empty vector, wt Rsc2 or mutant Rsc2 constructs, and the data are represented as the mean ± 1 SD of at least three independent experiments. (C) The W436A and W436L mutant Rsc2 proteins are unable to fully complement the hypersensitivity of an rsc2 null strain. Serial dilutions of mid-log cultures were plated onto media containing the indicated drug and incubated for 2–3 days at 30°C before imaging. (D) Survival of the W436A mutant strain compared with wt Rsc2. Three independent cultures of each strain were grown and plated onto media containing the indicated amount of HU. Survival was calculated relative to media lacking HU and the data are shown ± 1 SD. (E) Western blot analysis of whole cell lysates prepared from rsc2 null strains carrying either empty vector or overexpression BAH-CT1-myc constructs as indicated using anti-myc (top panel) or anti-H2A (bottom panel) as a loading control. (F) W436A and W436L mutations in Rsc2 BAH-CT1 impair chromatin association in vivo. ChIP assays examining enrichment of Myc-tagged overexpressed BAH-CT1 relative to the untagged control at the 18S region of the rDNA. Data shown are the mean enrichment of at least three independent experiments ± 1 SD.