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. 2011 Jun 28;108(26):10574-8.
doi: 10.1073/pnas.1017882108. Epub 2011 Jun 13.

Four amino acids guide the assembly or disassembly of Arabidopsis histone H3.3-containing nucleosomes

Leilei Shi  1 , Jing Wang, Fang Hong, David L Spector, Yuda Fang
Affiliations

Four amino acids guide the assembly or disassembly of Arabidopsis histone H3.3-containing nucleosomes

Leilei Shi et al. Proc Natl Acad Sci U S A. .

Abstract

The histone variant H3.3 and the canonical histone H3.1, which differ in only 4- to 5-aa positions, are coexpressed in complex multicellular eukaryotes from fly to human and plant. H3.3 is mainly associated with active chromatin by replacing H3.1 through chaperones such as histone regulator A, death domain associated protein DAXX, thalassemia/mental retardation syndrome X-linked homolog ATRX, or proto-oncogene protein DEK and plays important roles in the germline, epigenetic memory, and reprogramming. However, the signals within H3.3 that serve as a guide for its dynamic deposition or depletion in plant chromatin are not clear. Here, we show that Arabidopsis histone H3.3 differs from H3.1 by 4-aa sites: amino acids 31, 41, 87, and 90. Although histone H3.1 is highly enriched in chromocenters, H3.3 is present in nucleolar foci in addition to being diffusely distributed in the nucleoplasm. We have evaluated the function of the 4 aa that differ between H3.1 and H3.3. We show that amino acid residue 87, and to some extent residue 90, of Arabidopsis histone H3.3 are critical for its deposition into rDNA arrays. When RNA polymerase I-directed nucleolar transcription is inhibited, wild type H3.3, but not H3.3 containing mutations at residues 31 and 41, is depleted from the rDNA arrays. Together, our results are consistent with a model in which amino acids 87 and 90 in the core domain of H3.3 guide nucleosome assembly, whereas amino acids 31 and 41 in the N-terminal tail of Arabidopsis H3.3 guide nucleosome disassembly in nucleolar rDNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Multiple alignments of the typical histone H3.1 and H3.3 proteins from Arabidopsis thaliana (At), Drosophila melanogaster (Dm), and Homo sapiens (Hs). The different amino acid residues between H3.1 and H3.3 at the sites 31, 41, 87, 89, and 90 are highlighted by red rectangular boxes. The consensus amino acids and conservation level are shown at the top of the aligned sequence.
Fig. 2.
Fig. 2.
Distinct localization patterns between the plant histone H3.1/HTR1 and H3.3/HTR4. (A) H3.1/HTR1-GFP in a nucleus of a leaf epidermal cell. (B) DAPI staining of the nucleus; dense chromocenters are resolved (pseudocolored red). (C) Overlay of A and B. H3.1/HTR1-GFP colocalizes with DAPI-dense chromocenters in addition to diffuse signal in the nucleoplasm. (D) H3.3/HTR4-GFP in the nucleus of a leaf epidermal cell. (E) DAPI staining of the nucleus (pseudocolored red). (F) Overlay of D and E. H3.3/HTR4-GFP is enriched in nucleolar foci in addition to a diffuse signal in the nucleoplasm. (Scale bar: 10 μm.)
Fig. 3.
Fig. 3.
Roles of the amino acids residues 31, 41, 87, and 90 of plant histone H3.3/HTR4 in its deposition to or depletion from rDNA. (A) WT H3.1/HTR1 and H3.3/HTR4 and sites-mutated forms of HTR4 including HTR4-T31A, HTR4-Y41F, HTR4-T31A-Y41F, HTR4-H87S, HTR4-L90A, and HTR4-H87S-L90A were fused to GFP and transiently expressed in tobacco leaf cells; representative images are shown. (B) Quantitative analysis of these GFP-tagged proteins (A); the percentages of nuclei having fluorescent foci in the nucleoli were scored for these fusions. Error bars indicate SDs (n = 9). (C) Transcription of tobacco rRNA precursor and an actin gene was examined by RT-PCR and the amplified fragments were analyzed in an ethidium bromide-stained gel. The leaf discs were treated with actinomycin D every 1 h up to 3 h. Samples incubated in water were used as controls. (D) WT H3.3/HTR4 and sites-mutated forms of HTR4 including HTR4-T31A, HTR4-Y41F, HTR4-T31A-Y41F, HTR4-H87S, and HTR4-L90A were fused to GFP and transiently expressed in tobacco leaf cells. The leaf discs were treated with actinomycin D; representative images are shown. Samples incubated in water were used as controls. (E) Quantitative analysis of these GFP-tagged proteins (D); the percentages of nuclei having fluorescent foci in the nucleoli were scored for these fusions. Error bars indicate SDs. For HTR4, HTR4-T31A, HTR4-Y41F, and HTR4-T31A-Y41F treated with water and actinomycin D, n = 7. For HTR4-H87S and HTR4-L90A treated in water, n = 9. For HTR4-H87S and HTR4-L90A treated in actinomycin D, n = 10. (Scale bars: 10 μm.)
Fig. 4.
Fig. 4.
A model for the dynamics of plant H3.3/HTR4 in nucleoli. The amino acid residues 87 and 90 in the core domain of H3.3 guide its deposition to active nucleolar chromatin, whereas the amino acid residues 31 and 41 in the N-terminal tail of H3.3 guide its depletion from the rDNA arrays when transcription is silenced. Dashed lines mean that the switches between active chromatin and silenced chromatin likely include multiple steps in addition to the loading and unloading of histone H3.3.
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