Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant

Abstract

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.

Fig. 1.

Nucleosomes are not restored to repressed Hsp70 genes in H3.3-deficient cells. (A) Schematic depiction of the nucleosomal Hsp70 gene. The location of PCR amplicons and the Northern probe are shown. (B) DNA survival after MNase digestion. Chromatin was purified from salivary glands before (NoHS), during heat-shock (HS), or 2 h into recovery after heat-shock (HS+2hr). Blue, DNA survival in wild-type controls; red, DNA survival in H3.3 knock-down glands. PCR Cycle-to-threshold (Ct) values were normalized to a background intergenic amplicon and to NoHS values. DNA protection is presented in a log₂ scale, and the SD for each measurement is indicated. (C) Northern detection of Hsp70 transcripts in control and H3.3 knock-down salivary glands. The amount of the Hsp70 signal was calculated relative to the 18S signal (stained with ethidium bromide) after subtraction of background.

Fig. 2.

The Xnp and Hira assembly factors are recruited to nucleosome-depleted chromatin. Polytene chromosomes were prepared from larvae heat-shocked at 37° for 20 min with or without recovery at 25°, and imaged with a 100× objective. The location of the Hsp70 genes at cytological positions 87A and 87C are indicated (white lines). DAPI is shown in red. (A) Heat-shock of wild-type larvae causes the rapid recruitment of elongating RNA polymerase II (blue) to the Hsp70 loci. RNA polymerase leaves within 60 min of recovery as the genes are repressed. Xnp, Hira, and ASF1 are rapidly recruited to induced Hsp70 genes, and leave during recovery. (B) Polytene chromosomes from H3.3 knock-down salivary glands. Elongating RNA polymerase II (blue) and ASF1 label the induced Hsp70 loci, and leave within 60 min of recovery. Xnp and Hira are also recruited to induced Hsp70 genes, but remain bound 2 h after recovery. (C) ChIP of assembly factors from wild-type (blue) and H3.3-deficient (red) salivary glands. The association of factors at the “C” amplicon in Fig. 1 was assessed before, during and after heat-shock.

Fig. 3.

Xnp and Hira are required for H3.3 nucleosome replacement. Deposition of H3.3^core-GFP (green) in polytene chromosomes after recovery from heat-shock. Polytene spreads were imaged with a 100× objective. DAPI is in red, elongating RNA Polymerase II is in blue, and the nucleolus is outlined with a dotted line. (A) H3.3^core-GFP is efficiently deposited along chromosome arms in wild-type, and the nucleolus is devoid of soluble tagged protein. (B) xnp⁴⁰³/Df(3R)Exel6202 and (C) Hira^HR1 mutants show reduced H3.3^core-GFP signals along chromosome arms, and increased accumulation of the tagged protein within nucleoli. (D) Deposition of H3.3^core-GFP is completely blocked in Hira^HR1;xnp⁴⁰³/Df(3R)Exel6202 double-mutants and the tagged protein accumulates within the nucleolus.

Fig. 4.

Step-wise model for recognition of chromatin gaps and delivery of predeposition H3.3•H4 histones. Old nucleosomes (red) are disrupted by diverse processes on chromatin, displacing histones. Xnp and Hira independently bind exposed DNA at chromatin gaps. A separate predeposition complex containing ASF1 and new H3.3•H4 heterodimers (green) is recruited to chromatin-bound Xnp and Hira. In the final step, Xnp and Hira pry histones off ASF1 and wrap them with DNA to rebuild nucleosomes.

Fig. 5.

Hira is required for H3.3 nucleosome replacement at active rDNA genes. Assembly factor localization and H3.3 deposition in stage 10 somatic follicular epithelia of adult ovaries, imaged with a 60× objective. DNA is in red. The nucleolus is a clear area within each nucleus. (A) Hira-GFP (green) stains chromatin throughout the nucleus and labels foci within the nucleolus. (B) Xnp (green) broadly stains chromatin but does not localize within the nucleolus. (C–E) Hira but not Xnp is required for H3.3 deposition (green) at active rDNA genes. (C) A pulse of H3.3-GFP rapidly accumulates in nucleolar foci in wild-type follicle nuclei. (D) A pulse of H3.3-GFP deposits within the nucleolus in xnp⁴⁰³/Df(3R)Exel6202 cells. (E) H3.3-GFP does not deposit within the nucleolus in Hira¹ adults.

Fig. 6.

Xnp and Hira mark chromatin defects in H3.3-deficient cells. Magnifications of the nucleolus (Left) and the nucleosome-depleted TAGA satellite block (Right) from polytene chromosome spreads imaged with a 100× objective. Chromosomes were prepared from larvae carrying the In(1)w^m4 inversion to easily visualize the TAGA satellite block away from the compacted chromocenter. Images for wild-type (Upper) and H3.3-deficient (Lower) cells are shown stained with anti-Xnp or anti-Hira antibodies. DAPI is in red. Xnp does not normally associate with nucleolar chromatin in wild-type spreads, but is strongly enriched on nucleolar chromatin in H3.3-deficient glands. Hira stains the nucleolus in both wild-type and H3.3-deficient spreads. Xnp associates with the nucleosome-depleted satellie block in both wild-type and H3.3-deficient cells, but the area of the block expands in H3.3-deficient cells. The area of the Xnp-stained block (± SD) is shown in arbitrary units. Hira does not normally associate with the nucleosome-depleted TAGA satellite block, but is strongly enriched at the TAGA satellite block in H3.3-deficient cells.