Physical coupling of H3K4me3 demethylases and Polycomb repressive complex 2 to accelerate flowering in rice

Two H3K4me3 demethylases physically interact with the Polycomb repressive complex 2, thereby altering methylation of a key flowering locus and promoting rice flowering.

A comprehensive genetic network for flowering time regulation has been established in rice, like the early heading date1 (Ehd1) pathway (Doi et al. 2004;Lee and An 2015).OsLFL1 suppresses Ehd1 expression under long day conditions (Peng et al. 2007;Itoh et al. 2010).PRC2 complex and its accessorial plant homeodomain protein, rice leaf inclination 2/VIN3-LIKE 2 (OsLC2/VIL2), repress OsLFL1 expression by depositing H3K27me3 at its chromatin (Wang et al. 2013;Yang et al. 2013;Xie et al. 2015).Exploring OsLFL1 regulation will improve our understanding of rice floral transition.
To identify factors that cooperate with PRC2, we used EMF2b to screen a library containing 1,588 predicted rice transcription factors by the yeast two-hybrid assay (Y 2 H; Fig. 1A; Shi et al. 2021).Twenty candidate proteins were identified, including JMJ704 (LOC_Os05g23670; Supplementary Table S1), which belongs to the H3K4me3 demethylase KDM5 family.JMJ704 and EMF2b association supplies an opportunity to address the cooperated function of H3K4me3 demethylase with PRC2.We confirmed JMJ704-EMF2b protein interaction and mapped their interaction surface by Y 2 H (Fig. 1, B-D).The interaction was mainly mediated by the N-terminal of JMJ704, containing the JmjN and JmjC domains and the VRN2-EMF2-FIS2-SU(Z)12 (VEFS) box of EMF2b (Fig. 1, C  and D).The interaction was further confirmed by split firefly luciferase complementation (SFLC) assay in Nicotiana benthamiana leaves and co-immunoprecipitation (Co-IP) in rice protoplast (Fig. 1, E and F).
LC2-PRC2 complex binds to the promoter of OsLFL1, thereby catalyzing H3K27me3 to repress its expression (Wang et al. 2013;Yang et al. 2013;Xie et al. 2015).We measured the expression levels of OsLFL1 and its downstream genes Ehd1, Heading date 3a (Hd3a), and RICE FLOWERING TIME LOCUS T1 (RFT1) in a series of developmental times.OsLFL1 was continuously up-regulated (Fig. 2E); Ehd1, Hd3a, and RFT1 displayed lower expression in jmj703 jmj704-1 before flowering (Fig. 2, F-H).The global level of H3K4me3 was increased in jmj703 jmj704, and H3K27me3 level was decreased (Fig. 2I), supporting that H3K4me3 inhibits PRC2 activity (Pasini et al. 2008;Schmitges et al. 2011).We also detected increased H3K4me3 and reduced H3K27me3 at the OsLFL1 locus in jmj703 jmj704-1 plants (Fig. 2, J-L), suggesting H3K4me3 demethylation potentially promotes H3K27me3 deposition at OsLFL1.Previous studies show that zinc finger (ZnF) domains can bind DNA in plants (Ciftci-Yilmaz and Mittler 2008;Cui et al. 2016).We purified the Maltose binding protein (MBP)-tagged C-terminal of JMJ704 (MBP-JMJ704-C) containing ZnF and FY-rich (Phenylalanine/tyrosine-rich, FYR) domains, and found that it could bind to these probes from OsLFL1 (P1, P2, and P3) but not the probe of OsACTIN1 (LOC_Os03g50885), which is not likely to be a target for PRC2 and H3K4me3 demethylases, detected by electrophoretic mobility shift assay (EMSA; Fig. 2, J and M, and Supplementary Fig. S6A).MBP-JMJ704-C had relatively higher affinity against P2 (Fig. 2M).The binding activity at P1 and P2 of OsLFL1 was disrupted by deleting the ZnF domain (MBP-JMJ704-C ΔZnF; Supplementary Fig. S6A).The ZnF domain alone could bind to these two probes with slightly reduced levels (Supplementary Fig. S6A).These data support that JMJ704 binds to OsLFL1 DNA mainly through the ZnF domain in vitro.JMJ703 also binds to OsLFL1 DNA, and the binding activity is also mainly dependent on ZnF domain (Supplementary Fig. S6B).JMJ704/JMJ703 and AtJMJ14 belong to KDM5/ JARID1 group proteins with a conserved C5HC2-type ZnF domain (Supplementary Fig. S6C).A previous study suggested that the DNA motif DOF (DNA-binding with one finger) is one of the enriched motifs in the binding regions of AtJMJ14 (Fig. 2N; Wang et al. 2023).We only found the DOF motif in P2, and the lack of the DOF motif (P2ΔDOF) reduced the binding level of JMJ704-ZnF (Fig. 2, J and N).Our data suggest that JMJ703/JMJ704 bind to OsLFL1, reducing H3K4me3 and promoting H3K27me3 levels at its chromatin, thereby promoting OsLFL1 in flowering time regulation.
As H3K4me3 inhibits PRC2 activity, understanding how H3K4me3 is coordinately removed during PRC2 silencing is critical.To address the cooperated function of JMJ703/ JMJ704 and PRC2, we got an emf2b mutant in Dongjin (DJ) background (Xie et al. 2015).Mutation of EMF2b resulted in reduced H3K27me3 and increased H3K4me3 globally (Fig. 2O).Like jmj703 jmj704, emf2b plants displayed higher expression of OsLFL1; increased H3K4me3 and decreased H3K27me3 at the OsLFL1 chromatin (Fig. 2, P-R).These suggest that JMJ703/JMJ704-PRC2 is functionally coupled in repressing OsLFL1.Our data show that JMJ703 and JMJ704 physically associate with PRC2 to reduce H3K4me3 and increase H3K27me3 globally and at the OsLFL1 locus to control flowering time (Fig. 2S).

Figure 1 .Figure 2 .
Figure 1.PRC2 associates with H3K4 demethylase JMJ704.A) The scheme of mating-based yeast two-hybrid (Y 2 H) screening.EMF2b bait yeast strain was mated with a rice TFs library containing 1,588 TFs in 96-well plates.After mating, the cultures were plated on selective solid medium plates.The positive clones like JMJ704 were pricked out.AD, activation domain; BD, binding domain; TF, transcription factor.B) JMJ704 interacts with EMF2b in yeast cells.T, SV40 large T-antigen; Lam, Lamin C. C) Schematic illustrations of the JMJ704 and EMF2b domains.The numbers indicate the positions of the amino acid residues in the constructs used for Y 2 H. ZnF, Zinc finger; FYR, FY-rich; VEFS, VRN2-EMF2-FIS2-SU(Z)12; aa, amino acid.D) Rough mapping of JMJ704 and EMF2b interaction domains.The fusion proteins or truncated proteins were constructed as shown in C).In B and D), the pairs of indicated plasmids were co-transformed and grown on selective solid media lacking tryptophane, leucine, and histidine (SD-WLH).Representative pictures from three independent experiments are shown.E) SFLC assay shows the physical association of JMJ704 with EMF2b in Nicotiana benthamiana.The pairs of co-infiltrated plasmids are presented in each quadrant.Images are representative of three independent experiments.F) EMF2b-HA is co-immunoprecipitated with JMJ704-Venus in rice protoplasts.Hemagglutinin (HA)-tagged EMF2b (EMF2b-HA) and Venus-tagged JMJ704 (JMJ704-Venus), or EMF2b-HA alone served as a negative control, were expressed.Proteins were extracted and subjected to Co-IP.Samples before and after immunoprecipitation were detected by anti-HA and anti-GFP.Images are representative of two independent experiments.

Figure 2 .
Figure 2. (Continued) level of OsLFL1 E), Ehd1 F), Hd3a G), and RFT1 H) in Nip and the jmj703 jmj704.RNA was isolated from the second leaf blades collected from the top of the indicated plants.Values are mean ± s.d.(n = 3 biological replicates).The OsACTIN1 gene was used as an internal reference control (*P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant, P ≥ 0.05; unpaired two-tailed t-tests).I) The H3K4me3 and H3K27me3 levels in indicated genotypes.Protein was extracted and detected by corresponding antibodies showing on the left.The H3 level was used as the loading control.J) Genomic structure of OsLFL1 and regions tested in chromatin immunoprecipitation (ChIP) and EMSA assays.Green vertical line indicates DNA-binding with one finger (DOF) motif.P1, probe 1; P2, probe 2; P3, probe 3; DOF, DNA binding with one finger.K and L) Levels of H3K4me3 K) and H3K27me3 L) at the OsLFL1 chromatin assayed by ChIP-qPCR.Values are mean ± SEM (n = 3 biological replicates).Each examined region is shown below locus in J). (*P < 0.05; **P < 0.01; ns, no significant, P ≥ 0.05; unpaired two-tailed t-tests).M) EMSA testing JMJ704 binding to OsLFL1 DNA fragments.The purified MBP-JMJ704-C (487-971; 2 μg) were incubated with 10 nmol carboxyfluorescein (FAM)-labeled probes.Probe ACTIN was used as a negative control.N) The ZnF domain of JMJ704 binds to OsLFL1 P2 fragment through the DOF motif.The purified MBP-JMJ704-ZnF (487-682; 2 μg) were incubated with 10 nmol FAM-labeled probes.O) Comparison of the H3K4me3 and H3K27me3 levels between wild-type DJ and emf2b plants.Protein was extracted and detected by corresponding antibodies showing on the left.The H3 level was used as the loading control.P) Relative expression level of OsLFL1 in DJ and emf2b.Values are mean ± s.d.(n = 4 biological replicates).The OsACTIN1 gene was used as an internal reference control (***P < 0.001; unpaired two-tailed t-tests).Q and R) Levels of H3K4me3 Q) and H3K27me3 R) at the OsLFL1 chromatin assayed by ChIP-qPCR.Values are mean ± SEM (n = 3 biological replicates).Each examined region is shown below locus in J) (**P < 0.01; ns, no significant, P ≥ 0.05; unpaired two-tailed t-tests).S) Model for regulatory network controlling flowering time by JMJ703/JMJ704-PRC2 in rice.
, Hubei Hongshan Laboratory, and a startup fund from Wuhan University.