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. 2019 Feb 1;31(2):430–443. doi: 10.1105/tpc.18.00693

Figure 2.

Figure 2.

JMJ16 is an H3K4 Demethylase.

(A) JMJ16-YFP-HA exhibits H3K4me1/2/3 demethylase activity in N. benthamiana leaves.

(B) Mutations in conserved Fe (II) binding amino acids (JMJ16m-YFP-HA) abolish its H3K4me1/2/3 demethylase activity in N. benthamiana leaves. In (A) and (B), JMJ16-YFP-HA and JMJ16m-YFP-HA expression in N. benthamiana nuclei (arrows) was visualized by monitoring YFP fluorescence (green, middle). The histone methylation status was analyzed by immunostaining with specific histone methylation antibodies (red, right). The location of nuclei was visualized using 4′,6-diamidino-2-phenylindole staining (blue, left). Scale bars = 5 μm.

(C) Statistical analysis of the levels of fluorescent signals in (A) and (B). At least 25 pairs of JMJ16-YFP-HA- or JMJ16m-YFP-HA-expressing versus nonexpressing nuclei in the same field of view were analyzed. Error bars = ±SD. Different letters indicate significant differences based on one-way ANOVA with Tukey’s honestly significant difference test (P < 0.01).

(D) GST-JMJ16670 exhibits H3K4me1/2/3 demethylase activity in vitro. E. coli-expressed and affinity-purified GST-JMJ16670 was incubated with calf thymus histone, and the H3K4 methylation status was determined using methylation-specific antibodies. GST-JMJ14596 was used as a positive control and anti-H3 was used as a loading control.

(E) Global H3K4 methylation status of 10-d–old plants. JMJ14 OE and JMJ16 OE indicate the expression of JMJ14-YFP-HA and JMJ16-YFP-HA driven by the 35S promoter in Col-0, respectively. Methylation-specific antibodies were used to determine the global H3K4 methylation status. Anti-H3 was used as a loading control.

(F) Quantification analysis of (E). Immunoblots of three biological replicates were quantified; for each replicate, immunoblots were performed using freshly prepared protein extracts from Col-0, JMJ14-OE, and JMJ16 OE (#14-3 and #18-3). Error bars = ±sd. Different letters indicate significant differences among genotypes based on one-way ANOVA with Tukey’s honestly significant difference test (P < 0.05).