Replication-independent histone deposition by the HIR complex and Asf1 - PubMed (original) (raw)

Comparative Study

Replication-independent histone deposition by the HIR complex and Asf1

Erin M Green et al. Curr Biol. 2005.

Abstract

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.

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Figures

Figure 1

Figure 1

The four Hir proteins exist as a complex in vivo and co-purify with Asf1. (A) TAP purifications were performed using wild-type (PKY028) and ASF1-STAP (PKY3121) cells (left panel) and from wildtype (PKY028) and HIR2-STAP cells (PKY3062) (right panel). The Asf1-STAP purification was performed at 100 mM KCl and the Hir2-STAP purification was performed at 300 mM KCl. Proteins eluted from the second TAP affinity resin were analyzed on a SDS-PAGE gel and detected by silver staining. Polypeptides identified by mass spectrometry, immunoblotting or molecular weight (histone H3) are indicated. *indicates an unidentified polypeptide. (B) Summary of Asf1-STAP and Hir2-STAP mass spectrometry data. The percent sequence coverage is indicated in the table, with the number of unique peptides shown in parentheses. Although histone H3 peptides were not detected in all Asf1-STAP preparations by mass spectrometry, H3 was always apparent on the silver-stained protein gels. (C) Efficient association between Asf1 and the HIR complex is dependent upon all four Hir proteins. Asf1-TAP complexes were purified from wildtype (PKY3121), _hir1_Δ (PKY3906), _hir2_Δ (PKY3908), _hpc2_Δ (PKY3912) and _hir3_Δ (PKY3928) cells, analyzed on SDS-PAGE gels and detected by silver staining. Purified Hir2-STAP complex is shown for comparison. The Hir2-STAP subunit migrates more slowly than Rad53, which is still present in the complexes from hir mutant cells. *indicates an unidentified polypeptide. (D) A summary of Asf1-TAP mass spectrometry data. The percent sequence coverage of each protein is shown, with the number of unique peptides in parentheses.

Figure 2

Figure 2

Histone deposition by HIR and Asf1/H3/H4 complexes. (A) 30 fmol of a 250 bp radiolabeled DNA probe was incubated with 100 fmol of histones H3/H4 (lanes 2–4), 100 fmol of pre-formed Asf1/H3/H4 complex (lanes 5–9), 100 fmol of Asf1 (lanes 10–12) and either 1.75 μL (lane 6), 3.5 μL (lane 7), or 7 μL (lanes 3, 8, 11, 13) of HIR complex or HIR dialysis buffer (7 μL; lanes 4, 9, 12, 14). Reaction products were resolved on a native 4% polyacrylamide gel and detected by autoradiography. The upper and lower arrows indicate two distinct shifted species. (B) The shifted species contain histone H3. The EMSA was performed as described above, except that 5 μL of HIR complex or HIR dialysis buffer was used. Anti-histone H3 (0.125 μg, Abcam) was added to lanes 6–10. The arrow indicates the supershifted species. (C) Plasmid DNA pre-relaxed with human topoisomerase I was incubated with CBP-TAP purified HIR complex (20 μL) pre-incubated with 0.4 (lanes 2 and 4) and 0.8 (lanes 3 and 5) pmol of histones H3/H4. DNA was analyzed by agarose gel electrophoresis and visualized by SYBR Gold (Molecular Probes) staining.

Figure 3

Figure 3

The HIR complex and Asf1 assemble chromatin independently of DNA replication or CAF-1. (A) SV40 replication reactions containing α-32P-dATP. CAF-1 (10 ng, lane 2 or 20 ng, lane 3) or CBP-TAP purified HIR complex (2.25 μL, lanes 4 and 6 or 4.5 μL, lanes 5 and 7) were performed as indicated. Asf1/H3/H4 complexes (0.9 pmol) were added in lanes 6–8. Chromatin assembly was detected by the appearance of supercoiled DNA (indicated by arrow) on an agarose gel. Total DNA was visualized by staining with ethidium bromide (upper panel). Replicated molecules that incorporated α-32P-dATP were detected by autoradiography (lower panel). The values shown indicate the percentage of supercoiled DNA in each lane for both the total DNA and the replicated DNA. (B) The HIR complex does not stimulate replication dependent, CAF-1-mediated chromatin assembly. SV40 replication reactions were performed as in (A) in the presence of CAF-1 (7.5 ng, lanes 5–8, 10), histones H3/H4 (5 ng, lanes 2–4 and 6–8), HIR complex (4 μL, lanes 3, 7), HIR dialysis buffer (4 μL, lanes 4, 8), and pre-formed Asf1/H3/H4 complex (3.6 pmol, lanes 9, 10).

Figure 4

Figure 4

Asf1-H36AD37A impairs histone deposition by the HIR/Asf1 complex. Histone deposition was assayed as in Figure 2C. Plasmid DNA pre-relaxed with human topoisomerase I was incubated with 0.4 and 0.8 pmol histones H3 and H4 (lanes 2–3), 0.8 and 1.6 pmol Asf1 (lanes 4–5) or Asf1-H36AD37A (lanes 7–8) pre-incubated with 0.4 and 0.8 pmol histones H3/H4. CBP-TAP-purified HIR complex (15 μL) was added to reactions in lanes 10–16.

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