Residues in the Nucleosome Acidic Patch Regulate Histone Occupancy and Are Important for FACT Binding in Saccharomyces cerevisiae

Abstract

The essential histone chaperone FACT plays a critical role in DNA replication, repair, and transcription, primarily by binding to histone H2A-H2B dimers and regulating their assembly into nucleosomes. While FACT histone chaperone activity has been extensively studied, the exact nature of the H2A and H2B residues important for FACT binding remains controversial. In this study, we characterized the functions of residues in the histone H2A and H2B acidic patch, which is important for binding many chromatin-associated factors. We found that mutations in essential acidic patch residues cause a defect in histone occupancy in yeast, even though most of these histone mutants are expressed normally in yeast and form stable nucleosomes in vitro Instead, we show that two acidic patch residues, H2B L109 and H2A E57, are important for histone binding to FACT in vivo We systematically screened mutants in other H2A and H2B residues previously suspected to be important for FACT binding and confirmed the importance of H2B M62 using an in-vivo FACT-binding assay. Furthermore, we show that, like deletion mutants in FACT subunits, an H2A E57 and H2B M62 double mutant is lethal in yeast. In summary, we show that residues in the nucleosome acidic patch promote histone occupancy and are important for FACT binding to H2A-H2B dimers in yeast.

Keywords: H2A-H2B dimer; Nap1; Pob3; Spt16; nucleosome assembly.

Figure 1

(A) Phenotypes of mutants in essential acidic patch residues. Yeast cells containing the indicated WT or mutant histone genes present on a LEU2 plasmid were spotted on SC-LEU or SC-LEU+FOA media. A URA3 plasmid containing WT histone genes was shuffled out by 5-FOA selection. (B) Growth curve analysis in a Gal shutdown system. WT histone expression was repressed upon shift to glucose media (see Materials and Methods) at time 0, allowing only the indicated histone mutants to be expressed. A representative growth curve is shown. (C and D) ChIP-qPCR analysis of histone occupancy in a two-plasmid system. HA-H2A or FLAG-H2B WT (black) or mutant (white) histones were coexpressed with WT untagged histones and immunoprecipitated using the corresponding anti-HA or anti-FLAG antibody. Untagged WT histones (gray) were used as a negative control. Data are shown as percent IP normalized to input whole-cell extracts. P-values were calculated for the represented triplicate experiment using an unpaired t-test. Mean ± SD is depicted. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Figure 2

H2B L109A mutant causes a defect in H2B binding to FACT. (A) Co-IP analysis using anti-myc beads of yeast whole-cell extracts containing myc-tagged Spt16 or Nap1. FLAG-tagged mutant histones were coexpressed with untagged WT histones and separated by Western blot. The anti-myc Western blot detects multiple bands (likely representing Spt16-myc degradation products), as observed previously (Mao et al. 2016), because the lane is overloaded to detect the relatively low levels of soluble histones bound to Spt16. (B) Quantification of the percent IP of the tagged band relative to the untagged band. Graphs represent the mean and SEM of at least three independent replicates. (C–E) Nuclease treatment does not improve H2B L109A binding to (C) Spt16 or (D) Nap1. Cell extracts were treated with 0.32 U/μl Pierce universal nuclease prior to IP to release chromatin-bound histones and increase the soluble histone pool. (E) Quantification of the percent IP of the tagged band relative to the untagged band. Graphs represent the mean and SEM of at least three independent replicates. * P < 0.05.

Figure 3

The acidic patch residue H2A E57 is important for binding FACT. (A) Co-IP analysis using anti-myc beads of nuclease-treated whole-cell extracts containing Spt16-myc and HA-tagged WT or indicated mutant histones. Untagged WT histones are coexpressed. (B) Quantification of the percent IP of the tagged band relative to the untagged band. Graphs represent the mean and SEM of at least three independent replicates. ** P < 0.01.

Figure 4

Analysis of FACT binding to histone mutants identified from structural studies. (A) Published crystal structures of FACT in complex with histones pdbID#4KHA (left) and pdbID#4WNN (right). Residues identified as important for FACT binding (e.g., H2A R78, H2B Y45, and H2B M62) are indicated. Image was created using PyMOL. (B and C) H2A R78A does not inhibit binding to FACT. (B) Co-IP analysis using anti-myc beads of nuclease-treated Spt16-myc yeast whole-cell extracts containing HA-tagged WT or H2A R78A mutant histones. An untagged WT is coexpressed. (C) Quantification of the percent IP of the tagged band relative to the untagged band. Graphs represent the mean and SEM of at least three independent replicates. (D and E) H2B M62E significantly inhibits FACT binding. (D) Co-IP analysis using anti-myc beads of yeast whole-cell extracts containing myc-tagged Spt16 and FLAG-tagged WT H2B, H2B M62E, or H2B Y45A mutant histones coexpressed with untagged WT histones. (E) Quantification of the percent IP of the tagged band relative to the untagged band. Graphs represent the mean and SEM of at least three independent replicates. ** P < 0.01.

Figure 5

Histone H2A and H2B residues important for FACT binding have yeast phenotypes in common with FACT mutants. (A) Serial dilutions of the indicated histone mutant strains were spotted onto SC plates with 0 or 150 mM HU. (B) FLO8-HIS3 assay (see Materials and Methods) to measure cryptic transcription activation in the indicated histone mutants. Growth on SC-His media indicates the histone mutant strain activates a cryptic promoter within the FLO8 gene, leading to HIS3 expression. (C) The H2A E57A H2B M62E double mutant is lethal in yeast. WT or mutant histone genes are present on a LEU2 plasmid, and WT histone genes are on a URA3 plasmid that is shuffled out by 5-FOA selection. (D) Model of FACT binding to the H2A-H2B dimer. Our data suggest that FACT may target both a small hydrophobic patch near the DNA-binding interface that is comprised of H2B M62 and Y45 residues (highlighted blue) and residues H2A E57 and H2B L109 in the acidic patch (highlighted red). H2A-H2B structure was visualized from pdbID#1ID3 using PyMOL.