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. 2007 Aug;19(8):2403-16.
doi: 10.1105/tpc.107.053579. Epub 2007 Aug 17.

A WD40 domain cyclophilin interacts with histone H3 and functions in gene repression and organogenesis in Arabidopsis

Hong Li  1 , Zengyong He, Guihua Lu, Sung Chul Lee, Jose Alonso, Joseph R Ecker, Sheng Luan
Affiliations

A WD40 domain cyclophilin interacts with histone H3 and functions in gene repression and organogenesis in Arabidopsis

Hong Li et al. Plant Cell. 2007 Aug.

Abstract

Chromatin-based silencing provides a crucial mechanism for the regulation of gene expression. We have identified a WD40 domain cyclophilin, CYCLOPHILIN71 (CYP71), which functions in gene repression and organogenesis in Arabidopsis thaliana. Disruption of CYP71 resulted in ectopic activation of homeotic genes that regulate meristem development. The cyp71 mutant plants displayed dramatic defects, including reduced apical meristem activity, delayed and abnormal lateral organ formation, and arrested root growth. CYP71 was associated with the chromatin of target gene loci and physically interacted with histone H3. The cyp71 mutant showed reduced methylation of H3K27 at target loci, consistent with the derepression of these genes in the mutant. As CYP71 has close homologs in eukaryotes ranging from fission yeast to human, we propose that it serves as a highly conserved histone remodeling factor involved in chromatin-based gene silencing in eukaryotic organisms.

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Figures

Figure 1.
Figure 1.
Analysis of cyp71 Mutants and the CYP71 Protein. (A) Wild-type and cyp71 mutant plants grown in the greenhouse for 6 weeks. (B) Schematic presentation of T-DNA insertions in the CYP71 gene (arrowheads). Exons and untranslated regions are indicated by closed rectangles, and lines between the exons denote introns. (C) Levels of CYP71 transcripts determined by RT-PCR. The ACTIN transcript level was taken as a control. ACTIN is shown at top and CYP71 is shown at bottom. (D) Domain structure of the CYP71 protein with four WD40 repeats and a cyclophilin (CYP) domain. The numbers indicate amino acid positions.
Figure 2.
Figure 2.
Defects of Leaf Organogenesis, Morphology, and Vein Pattern in cyp71. (A) and (B) Fourteen-day-old seedlings of Col-0 (A) and cyp71 (B) grown in a greenhouse. The age refers to days after germination. (C) A cotyledon and sequential rosette leaves from Col-0 (top) and cyp71 (bottom) plants. (D) and (E) Scanning electron microscopy images of leaf primordia in Col-0 (D) and cyp71 (E) plants. (F) and (G) Vein pattern of the first rosette leaf in the wild type (F) and the cyp71 mutant (G). (H) and (I) Changes in the vein pattern and fusion of sepals in cyp71 (I) compared with the wild type (H). (J) to (L) Phyllotaxy pattern of the wild type (J) and the cyp71 mutant ([K] as normal and [L] as abnormal). Bars = 1 mm in (A) to (C), (F), (G), and (J) to (L) and 200 μm in (D), (E), (H), and (I).
Figure 3.
Figure 3.
Analysis of the Inflorescence Apex, Siliques, SAM Structure, and Root Morphology in the Wild Type and cyp71 Mutants. (A) A main inflorescence of a wild-type plant. (B) Terminated main inflorescence of a cyp71 mutant plant. (C) A main inflorescence of cyp71 with a few flowers produced before termination. (D) Siliques from the wild type (left) and cyp71 (right). (E) to (J) Scanning electron microscopy images of wild-type and cyp71 inflorescences. (E) A wild-type inflorescence with a cluster of early floral buds. (F) An enlarged view of a shoot apex with floral primordia in a wild-type plant. (G) A cyp71 inflorescence with a few floral primordia irregularly initiated on the tip. (H) A cyp71 shoot apex with two flower primordia. (I) and (J) A cyp71 mutant shoot apex without floral primordium. (K) to (P) Longitudinal SAM sections in the wild type and cyp71. (K) and (L) SAM of a 3-d-old wild-type (K) or cyp71 (L) seedling. The age refers to days after germination. (M) and (N) SAM of a 7-d-old wild-type (M) or cyp71 (N) seedling. (O) and (P) SAM of a 9-d-old wild-type (O) or cyp71 (P) seedling. (Q) to (Y) Defects in the root development of cyp71. (Q) A typical 10-d-old wild-type (left) and cyp71 (right) seedling grown on half-strength Murashige and Skoog agar medium. (R) and (S) Part of the primary root from a wild-type (R) or a mutant (S) seedling. (T) and (U) Cross sections through the elongation zone of a wild-type (T) or mutant (U) root. Arrowheads point to xylem cells. (V) to (Y) Confocal images of SCR-GFP expression pattern in the primary roots of wild-type (V) or mutant ([W] to [Y]) plants. Propidium iodide (red) was used to visualize the cell wall. Bars = 2 mm in (A) to (D) and (Q), 100 μm in (E), (G) to (I), (R), and (S), and 50 μm in (F), (J) to (P), and (T) to (Y).
Figure 4.
Figure 4.
Expression Pattern of CYP71 and Nuclear Localization of the CYP71 Protein. (A) GUS activity pattern in a 3-d-old seedling. The age refers to days after germination. (B) GUS activity in the root tip. (C) GUS activity in a 14-d-old seedling. (D) and (E) Expression of CYP71 mRNA in the meristem and leaf primordia as indicated by in situ hybridization using an antisense probe. Color development in the cyp71 mutant (E) served as a background control. (F) to (I) GUS activity was detected in a young flower bud (F), a stamen (G), and the vascular tissues of sepal (H) and petal (I). (J) to (M) Nuclear localization of the CYP71-GFP fusion protein transiently expressed in Agrobacterium-infiltrated tobacco leaf protoplast. (J) Bright-field image of a protoplast. (K) 4′,6-Diamidino-2-phenylindole fluorescence. (L) GFP fluorescence. (M) Merged image of (K) and (L). Bars = 0.2 mm in (A) to (C) and (F) to (I) and 10 μm in (D) to (E) and (J) to (M).
Figure 5.
Figure 5.
Class I KNOX Genes and Floral Homeotic Genes Are Ectopically Expressed in cyp71 Mutant Leaves. (A) Left, RT-PCR analysis of KNAT1, KNAT2, STM, AS1, AS2, and ACTIN in rosette leaves of the wild type and cyp71. Right, RT-PCR analysis of mRNA levels of AG, AP2, LFY, WUS, and ACTIN in rosette leaves of Col-0 and cyp71. The Col-0 seedling was used as a control. (B) Quantitative PCR analysis of KNAT1, STM, AG, and AP2 expression in leaves of the wild type and cyp71. The results are shown as means ± se from three experiments. (C) KNAT1-GUS expression in the wild type (top) and cyp71 (bottom). From left to right are seedlings, cotyledons, and the first pair of true leaves. Note that the leaf of cyp71 is lobed (arrow) and that GUS activity typically accumulated at the tip of cyp71 leaves (arrowhead). Bars = 1 mm.
Figure 6.
Figure 6.
Association of CYP71 with the STM and KNAT1 Chromatin Loci. (A) Protein gel blot analysis of total protein in wild-type and transgenic plants using anti-GFP or anti-HA serum. (B) Schematic presentation of the KNAT1, STM, and AP1 genes, indicating the positions of the PCR fragments amplified from the ChIP products. PF1 to PF3, PCR fragments 1 to 3. Exons and introns are indicated by closed and open rectangles, respectively. (C) ChIP assay revealed the association of CYP71 with the KNAT1 and STM loci, but not with AP1. Results from three lines of 35S-CYP71-GFP and one line of 35S-CYP71-HA transgenic plants are presented. ChIP assay did not detect an association of FKBP53 with KNAT1 and STM loci in the 35S-FKBP53-GFP plants. Samples were from leaves of transgenic plants for 28 d. The input is chromatin before immunoprecipitation. C, 35S-FKBP53-GFP transgenic plants; 1 to 3, three lines of 35S-CYP71-GFP plants; −AB, ChIP product with no antibody; GFP-AB, ChIP product with antibody to GFP; HA-AB, ChIP product with antibody to HA. The experiments were repeated three times using independent materials, and results from one experiment are shown.
Figure 7.
Figure 7.
CYP71 Interacts with H3 and Is Required for the Methylation of H3K27. (A) Protein pull-down assays were performed using recombinant GST or GST-CYP71 with different inputs (chicken histones, recombinant H3, or histone-enriched nuclear extract from Arabidopsis). Polypeptides were resolved by SDS-PAGE and visualized by Coomassie blue staining (for core histones in the top panel) or protein gel blot with anti-H3 antibody (for the bottom three panels with chicken histones, H3, or histone-enriched nuclear extract). (B) Schematic representation of full-length and truncated forms of CYP71. (C) Coomassie blue staining of GST-CYP71 and GST-truncated forms (N1, M1, and C1) that were expressed and purified from E. coli and used to examine the interaction with the H3 peptide in (D). (D) The H3 peptide pulled down by full-length or truncated forms (N1, M1, and C1) of CYP71 was analyzed by protein gel blot with anti-H3 antibody. (E) ChIP analysis using antibodies against H3K27 (me2 and me3). Samples were from leaves of 28-d-old wild-type and cyp71 mutant plants. Representative images from three independent experiments are shown. The positions of amplified PCR fragments are shown in Figure 6. Input is described in the Figure 6C legend, and mock indicates the control sample without antibody. (F) and (G) Relative levels of H3K27me2 (F) or H3K27me3 (G) in the cyp71 mutant versus the wild type, normalized by the level of ACTIN2. The se of three experiments is indicated. (H) ChIP analysis using antibodies against H3K4me3 and H3K9me2. ACTIN served as an inner control for H3K4me3; Ta3 served as an inner control for H3K9me2.
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