The FACT histone chaperone guides histone H4 into its nucleosomal conformation in Saccharomyces cerevisiae

Abstract

The pob3-Q308K mutation alters the small subunit of the Saccharomyces cerevisiae histone/nucleosome chaperone Facilitates Chromatin Transactions (FACT), causing defects in both transcription and DNA replication. We describe histone mutations that suppress some of these defects, providing new insight into the mechanism of FACT activity in vivo. FACT is primarily known for its ability to promote reorganization of nucleosomes into a more open form, but neither the pob3-Q308K mutation nor the compensating histone mutations affect this activity. Instead, purified mutant FACT complexes fail to release from nucleosomes efficiently, and the histone mutations correct this flaw. We confirm that pob3-T252E also suppresses pob3-Q308K and show that combining two suppressor mutations can be detrimental, further demonstrating the importance of balance between association and dissociation for efficient FACT:nucleosome interactions. To explain our results, we propose that histone H4 can adopt multiple conformations, most of which are incompatible with nucleosome assembly. FACT guides H4 to adopt appropriate conformations, and this activity can be enhanced or diminished by mutations in Pob3 or histones. FACT can therefore destabilize nucleosomes by favoring the reorganized state, but it can also promote assembly by tethering histones and DNA together and maintaining them in conformations that promote canonical nucleosome formation.

Keywords: FACT suppressor; histone mutations; histone rearrangement; nucleosome reorganization.

Figure 1

Suppression of pob3-Q308K by H3-H4 mutations. 8264-17-3 pTF237 (Table 1) was transformed with linear vector and a histone gene fragment derived from pQQ18 (Ahn et al. 2005) by PCR using Pfu polymerase under standard conditions. (B) After recovery of candidate plasmids with only the mutations shown, retransformation of 8264-17-3 pTF237, and loss of pTF237, viability was compared under the conditions indicated (see Materials and Methods). (C and D) The same plasmids were tested as the sole source of histones in strains with the spt16-11 mutation (DY10004) or normal FACT (DY9999). YPAD is rich medium; HU and NaCl indicate the concentrations of hydroxyurea or NaCl added to YPAD (mM).

Figure 2

Locations of the residues involved in pob3-Q308K suppression in a nucleosome. Residues that gave rise to suppression when mutated are mapped on the yeast nucleosome structure (Protein Data Bank, I1D3, White et al. 2001, images rendered in Pymol). (Left) Intact nucleosome with the histones shown as surfaces and the DNA as sticks. The suppressor mutations cluster in the region where the N-terminal tail (NT) of H4 (green) meets the globular core of the nucleosome. The first 17 residues of the H4 NT are unstructured. (Middle) A closer view in the same orientation as the left panel, with histones shown as cartoons with the mutated residues as spheres. Basic residues are rendered in blue (H4-R23 and H4-R55), hydrophobic in magenta (H3-L65, H4-L22, and H4-I26), and polar in orange (H4-N25). H4-D24 (gray) was not identified in the screen and mutation to alanine did not produce suppression (not shown). (Right) The same as the middle panel, except rotated ∼90° about the vertical (dyad) axis.

Figure 3

Effect of mutations after integration into the genome. The H4-R23S and H4-N25D mutations were integrated at both HHF1 and HHF2, and then the markers were deleted using the scheme outlined in the top panel (with KAN as the KanMX marker and H4* denoting the desired allele of H4). Dilutions of strains lacking the markers (Table 1) were tested as in Figure 1 with −Lys indicating synthetic medium lacking lysine to reveal the Spt⁻ phenotype.

Figure 4

Effect of mutations on total and soluble histone H3 levels. Histone levels were measured by Western blotting (Materials and Methods) using whole cell extracts (total) or after removing chromatin by centrifugation to measure the free pool of histones (soluble). Signals were normalized in each case to the level observed with strains carrying wild-type POB3 and H4; the absolute level of soluble histone was ∼0.6% of the total level. Error bars indicate the standard deviation from three independent measurements.

Figure 5

Mutant histones form nucleosomes in vitro with normal stability and normal reorganization. (A) Nucleosomes with the H4 sequence indicated were incubated for 30 min at different final concentrations of NaCl and then subjected to native PAGE. The fraction of the total DNA migrating in the nucleosomal position was calculated. (B) Nucleosomes were incubated at 30° or 65° for 1 hr either in the absence or presence of sheared genomic DNA (no DNA or +DNA) and then tested for integrity as in A. (C) The initial rate of DraI digestion was measured using normal and mutant nucleosomes with normal and mutant FACT. (D) The fraction of nucleosomes incorporated into complexes with FACT was determined by native PAGE after incubating with 40 nM Spt16-Pob3 with 3 µM Nhp6. Error bars indicate the standard deviation among samples tested in triplicate.

Figure 6

Pob3-Q308K releases from normal nucleosomes inefficiently, and this is reversed by the suppressor histone mutations. (A) The outline for the experiments shown in B–D. (B) Samples of normal and mutant nucleosomes were mixed with 3 µM Nhp6 and 50 nM (+) or 200 nM (++) Spt16-Pob3 or Spt16-Pob3-Q308K, incubated for 10 min at 30°, challenged with competitor DNA, and then subjected to native PAGE. The migration of FACT–nucleosome complexes (FACT:Nuc), nucleosomes (Nuc), and free DNA were detected by scanning for Cy5 (DNA). (C) WT nucleosomes were treated as in B using Spt16-Pob3-Q308K and then separated by native PAGE without (Ø) or after incubating at 30° with competitor DNA for the time shown. The DNA signal in the resistant complexes as a fraction of the total signal in the lane is given. Sequestration of the Nhp6 alters migration of the complexes, so the resistant complexes do not comigrate with the Nuc + FACT complexes. The “DNA + Nhp6” complexes in lane 2 (Ø) contain saturating levels of Nhp6, whereas those in lanes 3–6 (and in D) contain one or two molecules of Nhp6 due to incomplete sequestration of the Nhp6 (Ruone et al. 2003; Rhoades et al. 2004). (D) As in B, except Spt16-Pob3 contained normal subunits (WT), Pob-Q308K (QK), or Spt16 lacking the first 468 amino acids (ΔN; Vandemark et al. 2008).

Figure 7

The pob3-Q308K effects are partially dominant, consistent with a gain of function. Diploid strains (Table 1) were constructed and tested as described in Figure 1. The top panel shows the five viable combinations of normal (POB3), mutated (pob3-Q308K), and deleted (pob3-Δ) versions of the POB3 locus. The bottom panel shows the three viable combinations that also lack HPC2.

Figure 8

Effects of pob3-T252E support the importance of balanced interaction between FACT and nucleosomes. (A) and (B) Strains (Table 1) with the relevant genotypes shown were grown to saturation and tested as described in Figure 1.