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. 2018 Mar 16;46(5):2356-2369.
doi: 10.1093/nar/gky017.

WOX11 recruits a histone H3K27me3 demethylase to promote gene expression during shoot development in rice

Saifeng Cheng  1 , Feng Tan  1 , Yue Lu  1 , Xiaoyun Liu  2 , Tiantian Li  3 , Wenjia Yuan  1 , Yu Zhao  1 , Dao-Xiu Zhou  1   4
Affiliations

WOX11 recruits a histone H3K27me3 demethylase to promote gene expression during shoot development in rice

Saifeng Cheng et al. Nucleic Acids Res. .

Abstract

WUSCHEL-related homeobox (WOX) genes are key regulators of meristem activity and plant development, the chromatin mechanism of which to reprogram gene expression remains unclear. Histone H3K27me3 is a chromatin mark of developmentally repressed genes. How the repressive mark is removed from specific genes during plant development is largely unknown. Here, we show that WOX11 interacts with the H3K27me3 demethylase JMJ705 to activate gene expression during shoot development in rice. Genetic analysis indicates that WOX11 and JMJ705 cooperatively control shoot growth and commonly regulate the expression of a set of genes involved in meristem identity, chloroplast biogenesis, and energy metabolism in the shoot apex. Loss of WOX11 led to increased H3K27me3 and overexpression of JMJ705 decreased the methylation levels at a subset of common _targets. JMJ705 is associated with most of the WOX11-binding sites found in the tested common _targets in vivo, regardless of presence or absence of the JMJ705-binding motif. Furthermore, wox11 mutation reduced JMJ705-binding to many _targets genome-wide. The results suggest that recruitment of JMJ705 to specific developmental pathway genes is promoted by DNA-binding transcription factors and that WOX11 functions to stimulate shoot growth through epigenetic reprogramming of genes involved in meristem development and energy-generating pathways.

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Figures

Figure 1.
Figure 1.
WOX11 is involved in plant shoot development. (A–C) In situ hybridization detection of WOX11 transcripts in developing embryo (4 days after fertilization) (A) and 5-day-old shoot apex (B) with an antisense or a sense probe (C). Embryonic SAM in (A) is indicated by a red arrow. Bar = 50 μm. (D) Phenotype of wild type (HY) and wox11 at 7 days after germination (left), shoot height measures are shown on the right. Bar = 1 cm. **P < 0.01, t-test, two-sided (N = 30). (E) Morphology of HY and wox11 at heading stage in the field. (F and G) Shoot apex sections of HY and wox11 at 5 days after germination. Black line traces the SAM contour between leaf primordia P1 and P2. Red arrow indicates P1 leaf primordia. Bar = 20 μm. (H) Statistical analysis of HY and wox11 SAM size determined by measuring the dome area delimited by drawing a straight line between the basal edges of the two opposing youngest leaf primordia. *P < 0.05, t-test, two-sided (N = 10). (I) qRT-PCR analysis of OSH genes expression (relative to Actin transcripts) in shoot apex of HY, wox11, jmj705 and the double mutant (wox11/jmj705). Bar indicates mean ± SD from three replicates. *P< 0.05, **P< 0.01, n, not significant, t-test, two-sided.
Figure 2.
Figure 2.
WOX11 interacts with the histone H3K27me3 demethylase JMJ705 in vitro and in vivo. (A) Detection of WOX11 interaction with JMJ705 by yeast two-hybrid assay (left). Schematic structures of full length and truncated domains of JMJ705 and WOX11 were indicated on the right. (B) Interaction of WOX11 and JMJ705 in rice protoplasts. Representative cells are shown, as imaged by laser-scanning confocal microscopy. Detection in rice protoplasts of YN:WOX11 and JMJ705:YC interaction shown as yellow signal. The empty vector YC was co-expressed with YN:WOX11 and used as a control. Bar = 10 μm. (C) Pull-down assay of WOX11 and JMJ705. WOX11–6XHis was incubated with GST or JMJ705N+C-GST in GST beads and was pulled down from the JMJ705N+C-GST conjugated GST beads. (D) In vivo co-immunoprecipitation assay of WOX11 and JMJ705 interaction in tobacco. 35S-WOX11 construct was transiently transfected into tobacco leaf cells alone or co-transfected with 35S-FLAG-JMJ705-N+C-GFP. Nuclei isolated from leaves were inspected for expression of WOX11 and JMJ705 protein and then precipitated with the anti-WOX11 antibody. Anti-FLAG was used to detect the JMJ705 protein by western blots. (E) In vivo co-immunoprecipitation assay of WOX11 and JMJ705 interaction in rice. Nuclei isolated from wild type (ZH11) and JMJ705-FLAG (oxJ5–5) calli were precipitated with anti-WOX11 (left) and anti-FLAG antibodies (right) and analysed by immunoblots with anti-FLAG to detect the JMJ705 protein (left) and with the anti-WOX11 antibody to detect the WOX11 protein (right).
Figure 3.
Figure 3.
JMJ705 is involved in shoot development. (A) qRT-PCR analysis of JMJ705 transcript levels (relative to Actin transcripts) in SAM, 2-day germinated embryo (embryo), root, 14-day-old seedling (seedling), flag leaf, panicle and endosperm. (B) Shoot apex sections of 5 days HY, wox11, jmj705, and the double mutant (wox11/jmj705) (left). Statistical analysis of SAM sizes is shown on the right. Black line traces the SAM contour between leaf primordia P1 and P2. Red arrow indicates P1 leaf primordia. Bar = 20 μm. *P < 0.05, **P < 0.01, t-test, two sided (N = 10). (C) Phenotype of HY and jmj705 mutant at 7 days after germination. Bar = 1 cm. Statistical analysis is shown on the right. *P < 0.05, **P < 0.01, t-test, two sided (N = 25). (D) Morphology of HY and jmj705 at heading stage in the field. (E) Phenotype of the segregated wild type (WT), the JMJ705-FLAG in wild type background (oxJ5), the wox11 mutant in wild type background (wox11) and JMJ705-FLAG in wox11 background (wox11/oxJ5), from F2 of the crosses between wox11 and oxJ5–5 (left) at 7 days after germination. Bar = 1 cm. Statistical analysis of phenotypes are shown on the right. *P < 0.05, **P < 0.01, n, not significant, t-test, two sided (N = 30).
Figure 4.
Figure 4.
WOX11 and JMJ705 regulate common sets of genes. Venn diagrams showing overlaps between differentially expressed genes in wox11 and jmj705 mutants by RNA-seq. ‘up’ and ‘down’ indicate genes up- and down-regulated in the mutants plants compared to the wild type. Significant enrichments of the overlaps are indicated. **P < 0.01, hypergeometric test.
Figure 5.
Figure 5.
Tests of JMJ705 and WOX11 binding and H3K27me3 levels of selected putative common _target genes. (A) ChIP assays with anti-FLAG and anti-IgG of 5 days seedling chromatin isolated from wild type (ZH11) and Flag-tagged WOX11 transgenic plants (OXF1). Pw indicates primers contain WOX11 binding site. Actin was used as an internal control. (B) ChIP assays with anti-H3K27me3 of 5 d seedling chromatin isolated from wild type (HY) and wox11 mutant Actin was used as an internal control. (C) ChIP assays with anti-FLAG and anti-IgG of 5 d seedling chromatin isolated from wild type (ZH11) and JMJ705-FLAG plants (oxJ5–5). The first six genes contain both the WOX11 (Pw) and JMJ705-binding (Pz) sites, and the other three genes (bZIP47,Os11g13890, U2AF) contain only WOX11 binding sites. For ERF3 (with Pw and Pz sites), there was no proper primer for Pw and only Pz was detected and presented in the inner box. Actin was used as an internal control. (D) ChIP assays with anti-H3K27me3 of 5 days seedling chromatin isolated from wild type (ZH11) and oxJ5–5 seedlings. Y-axis means assay-site fold enrichment of the signal from immunoprecipitation over the background. Bar indicates mean ± SD from three replicates. Significances of differences are indicated. *P <0.05; **P <0.01, n, not significant, t-test, two-sided.
Figure 6.
Figure 6.
WOX11 modulates JMJ705 binding to _targeted genes. ChIP assays with anti-FLAG (anti-IgG as controls) of 5-day-old seedling chromatin isolated from the segregated wild type (WT), the JMJ705-FLAG in wild type background (oxJ5), and JMJ705-FLAG in wox11 background (wox11/oxJ5), from F2 of the crosses between wox11 and oxJ5–5. Seven representative genes were tested. OSH3, OSH15, OSH71, LHY and ERF3 contain both the WOX11 (Pw) and JMJ705-binding (Pz) sites, bZIP47 and Os11g13890 contain only the WOX11-binding site. The Os12g02210 locus that contains only JMJ705-binding site was used as an internal control. Gene structures, Pw and Pz sites were indicated. Three biological replicates of independent chromatin preparations were performed. One of the biological replicate is shown here. The other two biological replicates are in shown in Supplementary Figure S8. Y-axis means assay-site fold enrichment of the signal from immunoprecipitation over the background. Bar = means ± SD from three technical repetitions.
Figure 7.
Figure 7.
WOX11-dependent recruitment of JMJ705 to genomic loci. (A) Venn diagram displaying significant overlaps between genes occupied by JMJ705 in oxJ5 and wox11/oxJ5. (B) Mean density of JMJ705 occupancy at all JMJ705-associated genes in oxJ5 (1115 genes) as compared to genes with wox11/oxJ5 (946). Numbers of sequenced tags (Y-axis) per 5% of the genic region (black box) or per 100 bp intervals in the 2-kb genomic region flanking the gene (line, X-axis) are shown. Arrow indicates the direction of transcription. (C) Mean density of JMJ705 occupancy at all JMJ705-associated sites (peaks found in oxJ5) in oxJ5 and wox11/oxJ5. The average signal within 2-kb genomic regions flanking the center of the JMJ705 peaks is shown. (D) The Integrative Genomics Viewer (IGV) views of JMJ705 signal on representative genes in oxJ5 and wox11/oxJ5. Gene structures and name are shown underneath. Red lines represent tested regions in ChIP-qPCR. The scale was identical for different tracks, and the Y-axis scales represent normalized signal of shifted merged MACS tag counts for every 10-bp window. Bar = 1 kb. (E) ChIP-qPCR detection of JMJ705 occupancy with anti-IgG (negative control) and anti-FLAG at genes loci listed in (D) in WT, oxJ5 and wox11/oxJ5 apex. Os05g32290 was used as a negative control. Y-axis means assay-site fold enrichment of the signal from immunoprecipitation over the background.
Figure 8.
Figure 8.
A working model of WOX11 and JMJ705 regulated shoot development in rice.
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