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Cell-type-dependent histone demethylase specificity promotes meiotic chromosome condensation in Arabidopsis

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Abstract

Histone demethylation is crucial for proper chromatin structure and to ensure normal development, and requires the large family of Jumonji C (JmjC)-containing demethylases; however, the molecular mechanisms that regulate the substrate specificity of these JmjC-containing demethylases remain largely unknown. Here, we show that the substrate specificity of the Arabidopsis histone demethylase JMJ16 is broadened from Lys 4 of histone H3 (H3K4) alone in somatic cells to both H3K4 and H3K9 when it binds to the meiocyte-specific histone reader MMD1. Consistent with this, the JMJ16 catalytic domain exhibits both H3K4 and H3K9 demethylation activities. Moreover, the JMJ16 C-terminal FYR domain interacts with the JMJ16 catalytic domain and probably restricts its substrate specificity. By contrast, MMD1 can compete with the N-terminal catalytic domain of JMJ16 for binding to the FYR-C domain, thereby expanding the substrate specificity of JMJ16 by preventing the FYR domain from binding to the catalytic domain. We propose that MMD1 and JMJ16 together in male meiocytes promote gene expression in an H3K9me3-dependent manner and thereby contribute to meiotic chromosome condensation.

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Fig. 1: JMJ16 interacts with MMD1 in vitro and in vivo.
Fig. 2: Demethylase activity assay of JMJ16.
Fig. 3: MMD1 promotes the substrate specificity of JMJ16 to H3K9me3.
Fig. 4: The effect of MMD1 on the substrate specificity of JMJ16.
Fig. 5: Structural modelling analysis of JMJ16 substrate specificity.
Fig. 6: JMJ16 promotes meiocyte gene expression and chromosome condensation in male meiocytes.
Fig. 7: A model of cell-type-dependent regulation of gene expression by the JMJ16–MMD1 module.

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Data availability

All data are available in the paper and its Supplementary Information. RNA-seq data of atjmj16 and atmmd1 were deposited to the SRA under accession numbers SRR3584322 (ref. 36) and PRJNA530269. Source data are provided with this paper.

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Acknowledgements

We thank staff at the ABRC at Ohio State University for providing the Arabidopsis mutant seeds; J. Gu and Q. Yao at Fudan University for sharing experimental technology; and A. Dong at Fudan University for discussions. This research was supported by grants from the National Natural Science Foundation of China (nos. 31925005 and 31870293) the State Key Laboratory of Genetic Engineering and Fudan University.

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Author notes

  1. Hang Dai

    Present address: Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham, NC, USA

  2. These authors contributed equally: Jun Wang, Chaoyi Yu.

Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering and Institute of Biodiversity Sciences, Institute of Plants Biology, Center for Evolutionary Biology, School of Life Sciences, Fudan University, Shanghai, China

    Jun Wang, Chaoyi Yu, Juanying Ye, Hang Dai, Hongkuan Wang, Jiyue Huang, Jinbiao Ma & Yingxiang Wang

  2. Department of Biology, Eberly College of Science, The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA

    Jun Wang & Hong Ma

  3. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Shuaibin Zhang & Xiaofeng Cao

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  1. Jun Wang
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Contributions

Conceptualization: Y.W., H.M. and J.M. Methodology: J.W., C.Y., S.Z., J.Y., H.D., H.W., J.H. and X.C. Investigation: J.W., C.Y., S.Z., J.Y., H.D., H.W. and J.H. Writing, original draft: Y.W. and J.W. Review and editing, H.M., Y.W. and J.M. Supervision: H.M., Y.W., J.M. and X.C.

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Correspondence to Jinbiao Ma, Hong Ma or Yingxiang Wang.

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Peer review information: Nature Plants thanks Jose Gutierrez-Marcos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Interaction assays of MMD1 and JMJ16.

a, Self-activation detection of full length MMD1, PHD domain (601–680aa) and MMD1ΔPHD domain (1–600aa) with the blank AD. The blue growth yeast stains in QDO/X-gal/ABA (QDO/A/g) indicate self-activation. 3 replicates were done with the similar results. b, The functional enrichment analysis of the isolated proteins by the MMD1ΔPHD as a bait. c, Detection of interaction of MMD1 with JMJ14, JMJ18 and JMJ19 in tobacco leaves using bimolecular fluorescence complementation (BiFC). DAPI staining represents the nucleus. The merged panel (right panel) is the overlay of DAPI (left, blue) and YFP (middle, green). 12 cells were observed in each experiment. d, Showing the divide fragments for JMJ16 and MMD1 for the subsequent examination. e, Y2H assay of MMD1 truncations and FYR-C domain in the QDO/ABA (QDO/A) plates. The growth yeast indicates a positive interaction. The experiment was repeated for 3 times with similar results. f, Pull-down assay shows the interactions between FYR-C domain (978–1209aa) and MMD1-9 truncations staining by Coomassie blue. g, The MMD-9 interacts with the FYR-C domain by GST-pull down staining by Coomassie blue. The interaction between full-length MMD1 and JMJ16 proteins is a positive control, and between FYR-C and GST serves as a negative control. Recombinant His tagged full-length JMJ16 was purified from insect cells. GST tagged truncated MMD-9 and full length MMD1, and His tagged FYR-C were expressed in and purified from E. coli. The experiments were repeated three times. h, The core conserved domain in the MMD1ΔPHD regions and its homologues in plants was named the MMD domain. Alignment of MMD domain in land plants shows the conserved MMD domain highlighted in the red box. i, Comparison of FYR-C domain highlighted in green box in the PKDM7 family shows the similarity and diversity of FYR-C domains.

Source data

Extended Data Fig. 2 The co-localization of MMD1 and JMJ16 in male meiocytes, and purification of JMJ16 and MMD1 proteins.

a, MMD1 and JMJ16 partially co-localize in the pachytene chromosome of meiocytes isolated from transgenic plants JMJ16 fused HA driven by a native promoter. DAPI represents chromosome, green signals represent JMJ16 and red signals represent MMD1. Merged panel in the row 4 is the overlay of MMD1 and JMJ16. The co-localization regions of JMJ16 and MMD1 show color in yellow. JMJ16 and MMD1 are marked by HA and MMD1 primary antibodies and Alex Fluor 480 or 555 secondary antibody, respectively. Scale bar = 5 μm. b, The quantification of co-localization ratio of MMD1 and JMJ16 in by Image J in a (7 and 10 biologically independent leptotene and pachytene cells were used, respectively). The data was represented by mean ± SD and P-value (Pachytene/Leptotene) = 6.71 × 10−7. c, The HA-IP-purified JMJ16 protein from the JMJ16-YFP-HA transgenic Arabidopsis leaves was verified by western blot with anti-GFP antibody. d, His-purified full-length JMJ16- from insect cells (SF9) was stained by Coomassie (left) and verified by Western blot with anti-His antibody (right). e, MMD1-GST purified from E. coli (left panel) and after its cleavage (middle panel) was stained by Coomassie blue staining and verified by western blot with anti-MMD1 antibody (right panel). The Western blotting or Coomassie blue staining in each experiment was repeated for 3 times.

Source data

Extended Data Fig. 3 Immunostaining of specific histone modification in WT, mmd1 and jmj16 male meiocytes, and methylation assay of specific histone modification in WT, mmd1 and jmj16 inflorescences by WB.

a–g, Immunostaining of specific histone modification (CENH3, H3K4me1, H3K4me2, H3K9me1, H3K9me2, H3K27me1 and H3K9Ac) on pachytene chromosome in WT mmd1 and jmj16. DAPI was observed in 340 nm and methylated histones were recorded in 555 nm. The merged panel (bottom) is the overlay of DAPI (above, blue) and methylated histones (middle, red). The sample sizes for each experiment were shown in Fig. 3b. h, i, H3 methylation levels in inflorescences by western blot (h) and their quantification in WT, mmd1 and jmj16 (i). Scale bar = 5μm. P-value (jmj16/WT) = 0.0369, * P < 0.05, ** P < 0.01, two-tailed Student’s t test with two-sided. Data are represented as mean ± SEM from three independent experiments.

Source data

Extended Data Fig. 4 Interaction examination of JMJ16-N and -C termini, and structural modeling of JMJ16 catalytic domain with zinc-finger and helical domain.

a, Illustration of FRET receptor bleaching in tobacco. The single-direction black arrow represents energy transfer in the top figure. We hypothesize that the closed distance or interaction between the JMJ16 N- and C-termini would result in energy transfer from CFP (donor) to YFP (receptor). Therefore, in the basal state, when YFP is inactivated using photo-bleaching, the energy will remain at CFP. If MMD1 can disrupt the self-interaction of JMJ16 in the bottom part, YFP should be always undetectable if the laser is only given to CFP. CFP will remain unchanged with the photo-bleaching of YFP. b, As the control to Fig. 4i, the laser was given to both CFP and YFP. Then, the YFP was inactivated by photo-bleaching, which demonstrates the normal function of the YFP protein in the presence of MMD1, and the photo-bleaching was also able to reduce the YFP activity. Four biological independent cells were used in the statistics analysis and the data was represented by mean ± SEM. c, The structure model of JMJ16 [catalytic domain Jumonji with C5HC2 zinc-finger and helical domain (HD)]. The Jumonji domain is colored in green, while the HD plus C5HC2-Zinc finger part is in marine. d–f, Structural comparison of JMJ16 model and peptide-bound Jumonji domains. d, JMJ14-H3K4me3 complex (PDB code: 5YKO): The Jumonji domain and HD -C5HC2 domain are colored in violet and yellow, respectively. e, Human JMJD2A-H3K9me3 complex (PDB code: 2OQ6): The Jumonji domain and the extended helixes region (294-355aa) are colored in wheat and gray, respectively. f, Mouse JMJD3-H3K27me3 complex (PDB code: 4EZH): The Jumonji domain and the C-helix and GATAL zinc-finger are colored in light blue and light pink, respectively. JMJ16 is colored in the same scheme as in Supplementary Fig. 4c. The H3 peptides are shown in space-filling representation.

Extended Data Fig. 5 Analyses of the target genes regulated by MMD1 and JMJ16.

a, The histogram shows up and down-expressed genes regulated by JMJ16 in meiocytes. b, Venn diagram shows genes co-regulated by both MMD1 and JMJ16. c, Validation of JMJ16 expression by anti-HA antibody in JMJ16-HA transgenic plants driven with native promoter. The experiment was repeated for 3 times with similar results. d, Analysis of condensin subunit gene expression in leaves of WT and jmj16. Genes with a reduction in jmj16 meiocytes as shown in Fig. 6b exhibit elevated expression or no significant alternation in leaves. P-values are 0.00686 for CAPE1, 1.91 × 10−4 for CAPE2, 9.36 × 10−4 for CAPG, 1.43 × 10−4 for CAPG2, 0.033 for CAPH and 0.031 for CAPC. e, f, Selected genes such as FLC show significantly increased expression in jmj16 leaves (e), but no significant changes in jmj16 meiocytes (f). The SMC3 gene is a control. P-values are 8.35 × 10−5 for FLC, 0.00144 for COL1, 0.0202 for UNE15, 0.021 for PPME1, 0.011 for PRF5, 0.013 for GRP16), 1.27 × 10−3 for IAA2, 9.37 × 10−4 for IAA7, 2.79 × 10−4 for IAA19, 0.044 for IAA30, 1.45 × 10−3 for AT3G38790, 0.0133 for AT3G28840, 0.0103 for AT3G28980 and 0.016 for AT3G28830 in Extended Data Fig. 5e; P-value (SMC3) = 0.0098 in Extended Data Fig. 5f. g, h, Detection of H3K4me3 (g) and H3K9me3 (h) occupancy in condensin genes in inflorescences of WT and mmd1. P-value (CAPH-P) = 1.94 × 10−4. * P < 0.05, ** P < 0.01, two-tailed Student’s t test with two-sided. Data are represented as mean ± SEM from three independent experiments.

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Extended Data Fig. 6 The Arabidopsis jmj16 mutant shows defects in fertility and meiotic chromosome condensation.

a, Illustration of the gene, protein organizations and T-DNA insertional positions of JMJ16. b, The JMJ16 expression level was analyzed by RT-qPCR in WT and jmj16 mutants. P1-4 represents primers’ position on JMJ16 marked in a. Data are represented as mean ± SEM from three independent experiments. c, d, Morphology of fertility was examined and homozygous mutants show partial sterility with abnormally short siliques, scale bar = 5 cm and inviable pollen grains (red pollens are alive and dark/green pollens are dead) by Alexzander staining of anthers, scale bar = 10μm. Independent plants for WT (n = 45), jmj16-1(n = 40), jmj16-2 (n = 25) and JMJ16 complementation transgenic lines (n = 20) were used with similar results. Twenty independent anthers for each genotype were used with similar results. e, Genetic analyses of meiotic chromosome condensation defects in jmj16, mmd1 and mmd1 jmj16 double mutants. Diplotene cells for WT (n = 21), mmd1 (n = 20), jmj16-1 (n = 25) and mmd1 jmj16-1 (n = 30) were used with similar results. Diakinesis cells for WT (n = 25), mmd1 (n = 30), jmj16-1 (n = 31) and mmd1 jmj16-1 (n = 20) were used with similar results; Metaphase I cells for WT (n = 20), mmd1 (n = 24), jmj16-1 (n = 30) and mmd1 jmj16-1 (n = 20) were used with similar results. f, The estimation of metaphase I chromatin areas (circled by yellow lines) in WT (n = 17), mmd1 (n = 11), jmj16-1 (n = 12) and mmd1 jmj16-1 double mutants (n = 16). Data are represented as mean ± SEM. Scale bar = 5μm. P-values are 1.34 × 10−13 (mmd1/ WT), 1.26 × 10−8 (jmj16-1/WT), 5.65 × 10−15 (mmd1 jmj16-1/WT), 1.18 × 10−4 (mmd1/jmj16-1), 1.88 × 10−6 (jmj16-1/mmd1 jmj16-1) and 0.296 (mmd1/mmd1 jmj16-1). * P < 0.05, ** P < 0.01, two-tailed Student’s t test.

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Extended Data Fig. 7 The expression of synthetic JMJ16-N with MMD PHD domain partially recues meiotic chromosome condensation defect in mmd1 in vivo.

a, Illustration of synthetic constructs between the PHD domain of MMD1 (PHDMMD1) and the JMJ16-N without (Construct A) or with (Construct B) ZF (zinc finger domain). b, The expression level of JMJ16 in transgenic plant meiocytes was analyzed by RT-qPCR using the primers in the JJMJ16-N. The data are represented by mean ± SEM by three replicates with similar results. P-values are 1.51 × 10−4 (Construct A-1/WT), 1.19 × 10−5 (Construct A-2/WT), 4.54 × 10−4 (Construct B-1/WT) and 4.39 × 10−5 (Construct B-2/WT). c, Meiotic prophase chromosome behaviors of WT, mmd1 and constructs A and B transgenic plants are stained by DAPI in zygotene, pachytene, diakinesis and anaphase II, respectively. Zygotene cells for WT (n = 20), mmd1 (n = 20), Construct A-1 (n = 22), Construct A-2 (n = 21), Construct B-1 (n = 21) and Construct B-2 (n = 20) were used with similar results; Pachytene cells for WT (n = 15), mmd1 (n = 14), Construct A-1 (n = 14), Construct A-2 (n = 10), Construct B-1 (n = 12) and Construct B-2 (n = 20) were used with similar results; Diakinesis cells for WT (n = 20), mmd1 (n = 21), Construct A-1 (n = 20), Construct A-2 (n = 15), Construct B-1 (n = 21) and Construct B-2 (n = 19) were used with similar results; Anaphase II cells for WT (n = 20), mmd1 (n = 24), Construct A-1 (n = 22), Construct A-2 (n = 20), Construct B-1 (n = 12) and Construct B-2 (n = 20) were used with similar results. d, Estimation of chromatin areas of metaphase I chromosome in male meiocytes in Fig. 6i. The areas of chromatins in construct A and construct B were defined by circle drawing tools and area detection by Image J software and the number of cells is labeled in the column. We used cm2 as the area unit that is calculated by the pictures enlarged by the microscope in the centi-meter level, however, the real size of chromatins is micro-meter. P-values are 3.17 ×10−9 (mmd1/WT), 7.7 × 10−11 (Construct B-1/WT) and 5.33 × 10−19 (Construct B-2/WT). The data are represented as mean ± SD. e, RT-qPCR analyses of the CAP-D3 expression in meiocytes of WT, mmd1 and transgenic lines. The data are represented as mean ± SEM with three biological independent replicates. P-values are 3.09 ×10−4 (mmd1/WT), 7.37 × 10−5 (Construct B-1/WT) and 7.74 × 10−7 (Construct B-2/WT). f, The binding affinity of construct A and B. Well 1 and 2 are controls from WT to show H3K4me3 and H3K9me3. FLAG IP was conducted in WT (Well 3), A (Well 4) and B (Well 5) with anti-FLAG. The IP lysates were detected by H3K4me3, H3K9me3 and FLAG antibodies. Construct B only binds H3K4me3 and Construct A binds to both H3K9me3 and H3K4me3. Two replicates for Western blotting were performed. g–i, The complementation of full length MMD1 and JMJ16 in mmd1 mutants. ACTIN::MMD1-FLAG and ACTIN::JMJ16-HA are transformed into mmd1 heterozygotes. g, In the offspring of mmd1 homozygotes, MMD1 and JMJ16 were detected by MMD1 and HA antibodies. h, The siliques used for WT (n = 120), mmd1 (n = 145) and JMJ16 + MMD1(mmd1) (n = 168). i, Meiotic metaphase I chromosome phenotypes used for WT (n = 24), mmd1 (n = 20) and JMJ16 + MMD1(mmd1) (n = 27). Scale bar = 5μm. * P < 0.05, ** P < 0.01, two-tailed Student’s t test.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3. Supplementary Table 1: summary of JmjC demethylases towards multiple substrate specificities in plants and animals. JmjC-containing demethylates in plants and animals showed multiple demethylation activities for different histone modifications. Supplementary Table 2: peptides used in this study. Peptides used in the MALDI–TOF-MS and FP are listed in this table with amino-acid sequences, catalogue numbers and providers. Supplementary Table 3: primers used in this study. Primer names, sequences and applications were noted in this table.

Reporting Summary

Supplementary Data

Genes with downregulated and upregulated expression in jmj16 meiocytes. Sheet 1: 1,633 upregulated genes in jmj16 male meiocytes (log2[fdc] ≥ 1). Sheet 2: 4,626 downregulated genes in jmj16 male meiocytes (log2[fdc] ≤ −1). Sheet 3: 543 coregulated genes with upregulated expression in mmd1 (stage 4–7 anthers) (log2[fdc] ≥ 1) and jmj16 male meiocytes (log2[fdc] ≥ 1). Sheet 4: 802 coregulated genes with downregulated expression in mmd1 (stage 4–7 anthers) (log2[fdc] ≤ −1) and jmj16 male meiocytes (log2[fdc] ≤ −1). Sheet 5: biological processes annotation in upregulated genes or downregulated genes in jmj16 meiocytes. The transcriptome data were obtained from two replicates. For each replicate, about 20,000 male meiocytes were isolated from anthers (see Methods). The statistical significance (P value) of the differential expression of genes was calculated using a two-tailed Student’s t-test; FDR-adjusted P ≤ 0.05 was considered to be significant. The GO enrichment analysis was conducted by AgriGO (http://bioinfo.cau.edu.cn/agriGO/analysis.php), and statistics were compiled by FDR correction; FDR-adjusted P ≤ 0.05 was considered to be significant.

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Wang, J., Yu, C., Zhang, S. et al. Cell-type-dependent histone demethylase specificity promotes meiotic chromosome condensation in Arabidopsis. Nat. Plants 6, 823–837 (2020). https://doi.org/10.1038/s41477-020-0697-0

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