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Research Article

Jumonji domain protein JMJD5 functions in both the plant and human circadian systems

Matthew A. Jones, Michael F. Covington, Luciano DiTacchio, Christopher Vollmers, Satchidananda Panda, and Stacey L. Harmer
PNAS December 14, 2010 107 (50) 21623-21628; https://doi.org/10.1073/pnas.1014204108
Matthew A. Jones
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Michael F. Covington
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Luciano DiTacchio
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Christopher Vollmers
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Satchidananda Panda
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Stacey L. Harmer
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  • For correspondence: slharmer@ucdavis.edu
  1. Edited* by Steve A. Kay, University of California at San Diego, La Jolla, CA, and approved November 9, 2010 (received for review September 21, 2010)

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Abstract

Circadian clocks are near-ubiquitous molecular oscillators that coordinate biochemical, physiological, and behavioral processes with environmental cues, such as dawn and dusk. Circadian timing mechanisms are thought to have arisen multiple times throughout the evolution of eukaryotes but share a similar overall structure consisting of interlocking transcriptional and posttranslational feedback loops. Recent work in both plants and animals has also linked modification of histones to circadian clock function. Now, using data from published microarray experiments, we have identified a histone demethylase, jumonji domain containing 5 (JMJD5), as a previously undescribed participant in both the human and Arabidopsis circadian systems. Arabidopsis JMJD5 is coregulated with evening-phased clock components and positively affects expression of clock genes expressed at dawn. We found that both Arabidopsis jmjd5 mutant seedlings and mammalian cell cultures deficient for the human ortholog of this gene have similar fast-running circadian oscillations compared with WT. Remarkably, both the Arabidopsis and human JMJD5 orthologs retain sufficient commonality to rescue the circadian phenotype of the reciprocal system. Thus, JMJD5 plays an interchangeable role in the timing mechanisms of plants and animals despite their highly divergent evolutionary paths.

  • KDM8
  • JMJ31
  • TOC1
  • coexpression

Circadian rhythms are endogenous oscillations that attune the behavior and physiology of organisms to regular changes in their environment, such as dusk and dawn. It is estimated that 1/10th of mammalian genes and one-third of Arabidopsis genes are regulated in a circadian fashion (1, 2). Not surprisingly, the circadian clock modulates a broad range of processes, ranging from the regulation of growth and flowering time in plants to the control of body temperature and the sleep-wake cycle in mammals (3, 4). The coordination of these molecular and physiological processes with the external environment has been shown to provide an adaptive advantage in organisms as diverse as cyanobacteria, higher plants, and insects (5–7).

Although it is thought that circadian clocks have arisen multiple times during eukaryotic evolution (8), molecular oscillators in a diverse range of lineages share a broad overall structure consisting of interlocking transcriptional and posttranslational feedback loops (3, 4). In plants, the morning-phased Myb-like transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of TIMING OF CAB1 EXPRESSION 1 (TOC1) by binding to a conserved motif within its promoter known as the evening element (9–11). TOC1, in turn, promotes expression of CCA1 and LHY via an indirect mechanism, forming a negative feedback loop (12). This central CCA1/LHY/TOC1 loop interlocks with additional morning- and evening-phased circuits to provide more robustness and flexibility to the circadian mechanism (reviewed in 3, 13).

Because genes that act together are often coregulated (14), we have characterized a gene that is coexpressed with TOC1 on both developmental and daily time scales. This gene, JMJD5, contains a jumonji-C (jmjC) domain that is often found in proteins with histone demethylase activity (15); indeed, its human ortholog (also known as KDM8) has recently been shown to have this function (16). Although originally described as a permanent modification, histone methylation marks are now acknowledged to be dynamic alterations that can fine-tune transcription in a broad range of eukaryotic phyla (15). Components of histone methyltransferase complexes have been associated with mammalian circadian clock function (17, 18), and changes in histone methylation have been correlated with modifications in circadian gene expression in plants (19).

Here, we report that jmjd5 mutant plants have a short-period circadian phenotype and demonstrate that JMJD5 works in concert with TOC1 to promote CCA1 and LHY expression. Remarkably, we also found that mammalian cells deficient for the human JMJD5 ortholog have a short-period phenotype very similar to that of jmjd5 plants and that both JMJD5 orthologs retain sufficient commonality to rescue the circadian phenotype of the reciprocal system. These data suggest that JMJD5 may play a similar role in both the Arabidopsis and human circadian systems despite the wide evolutionary distance separating plants and mammals.

Results

TOC1 and JMJD5 Are Coexpressed.

Despite recent advances (12), the mode of action of the central plant clock gene TOC1 is currently enigmatic. Coexpressed genes frequently share biological function (14), prompting us to identify genes that are developmentally coregulated with TOC1. We therefore obtained microarray expression data generated from 79 different Arabidopsis samples (representing a wide variety of developmental stages and diverse organs) (20) and calculated the Pearson correlation coefficients between TOC1 and each gene on the ATH1 array. The gene most highly correlated with TOC1 (r = 0.77) was EARLY FLOWERING 3 (ELF3; Table S1), a gene involved both in light input to the clock and in central clock function (21–23), thereby validating our approach. Because genes involved in the circadian clock are themselves frequently expressed with a circadian oscillation (3, 13), we examined the daily patterns of expression of our candidate genes using previously published microarray data (2). Of the 10 genes most highly correlated with TOC1, only ELF3 and a previously uncharacterized gene, JMJD5 (At3g20810), were expressed with a circadian rhythm (Fig. 1A). The expression patterns of TOC1 and JMJD5 were the third most highly correlated across all genes on the microarray (r = 0.75; Fig. 1B and Table S1). Empirical resampling of randomly permuted data shows that the chance for this level of correlation occurring between two randomly selected genes is only 1.3% (2).

Fig. 1.
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Fig. 1.

JMJD5 is coexpressed with TOC1 over developmental and diurnal timescales. (A) Daily expression patterns of the 10 genes most highly developmentally correlated with TOC1. TOC1, JMJD5, and ELF3 are shown in black, with the remaining genes shown in gray. Accession numbers for each of these genes are listed in Table S1. Expression data were obtained from the work of Covington and Harmer (51). (B) Scatter plot of JMJD5 compared with TOC1 mRNA expression levels across a range of developmental time points. Each symbol represents the mean of each different biological sample; error bars represent SEM. Data were obtained from the work of Schmid et al. (20). (C) qRT-PCR analysis of JMJD5 expression under constant red light. WT Columbia (Col), jmjd5-1, jmjd5-2, and toc1-2 seedlings were compared using oligos spanning intron 1 (Fig. S1A and Table S4). Plants were entrained to 12/12-h light/dark cycles for 6 d before being moved to constant conditions with 40 μmol·m−2·s−1 red light. JMJD5 mRNA levels were normalized to PP2a. Data are the mean of three technical replicates; SE is shown. Presented results are representative of three independent experiments.

We next investigated whether JMJD5 expression was regulated by known clock genes. In toc1-2 mutants, JMJD5 expression levels gravitated toward a median level relative to the peak of expression in WT plants (Fig. 1C). In contrast, JMJD5 mRNA was not detectable by quantitative RT-PCR (qRT-PCR) in seedlings overexpressing CCA1 (Fig. S1D). This repression was similar to but more pronounced than the reduction of TOC1 expression previously reported in these CCA1-OX plants (9) (Fig. S1E). Like TOC1, the JMJD5 promoter contains an evening element sequence immediately upstream of the transcriptional start site (TSS) in addition to two within the 5′ UTR (Fig. S1F), suggesting that CCA1 might bind to this region and directly repress JMJD5 expression. These data demonstrate that not only are JMJD5 and TOC1 coregulated across developmental and circadian time but they are both repressed by the morning-phased clock protein CCA1.

jmjd5, Like toc1, Is Conditionally Arrhythmic.

To determine whether JMJD5 is simply an output of the clock or if it is involved in the clock mechanism, we introduced the CCR2::LUC luciferase reporter construct into two lines with T-DNA insertions within the At3g20810 locus (jmjd5-1 and jmjd5-2; Fig. S1A). qRT-PCR using oligos spanning the first exon–exon junction revealed no detectable full-length mRNA in either mutant but a low level of a truncated form of the message in jmjd5-1 (Fig. 1C and Fig. S1B). Because the conserved jmjC domain is located at the C terminus of the protein (Fig. S1A), both alleles are likely loss-of-function mutations. Consistent with this possibility, circadian rhythms in luciferase activity in both mutants were ≈0.5–1.0 h shorter than in WT (Fig. 2 A and B; P < 0.05, Student's t test). This phenotype was highly reproducible and was observed when plants were grown in a variety of light conditions (constant red light, constant blue light, or constant red plus blue light; Table S2). Neither insertion allele displayed an altered rhythm under constant darkness, indicating that JMJD5 function is light-dependent (Table S2). Similar light-dependent circadian phenotypes have been reported for other clock mutations (24, 25).

Fig. 2.
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Fig. 2.

jmjd5 mutants have circadian phenotypes. Luciferase activity of CCR2::LUC plants monitored in continuous red light. (A) WT Columbia (Col), jmjd5-1, and jmjd5-2 seedlings were entrained to 12/12-h light/dark cycles for 6 d before being moved to constant conditions with 30 μmol·m−2·s−1 monochromatic red light. The jmjd5-2 alleles displayed reduced luciferase activity despite expressing normal levels of CCR2 mRNA (Fig. S1C). Similar phenomena have previously been described in other T-DNA lines (49). Error bars indicate SEM and are displayed every 20 h for clarity (n ≥ 20). (B) Scatter plot of data shown in A comparing circadian period with relative amplitude error, a measure of rhythmic robustness, with a perfect cosine wave having a value of 0 as calculated by Fourier fast transform-nonlinear least squares (50). Data points are presented for WT Col, jmjd5-1, and jmjd5-2. Plants were treated as described in A. (C) Bioluminescence of WT, jmjd5-1, and toc1-2 seedlings maintained in higher intensity red light. Plants were entrained to 12/12-h light/dark cycles for 6 d before being moved to constant conditions with 120 μmol·m−2·s−1 red light. Data are representative of two independent experiments. Error bars are plotted every 20 h and indicate SEM (n ≥ 11).

Loss-of-function toc1-2 mutants have a strong short-period phenotype in most conditions but become arrhythmic in constant red light (26). We therefore examined clock regulation of luciferase activity in jmjd5 mutants maintained in different fluence rates of constant red light. When held under low levels of red light (∼30 μmol·m−2·s−1), jmjd5 mutants had a short period but preserved robust rhythms (Fig. 2 A and B). Intriguingly, at higher fluence rates of monochromatic red light (120 μmol·m−2·s−1), jmjd5 seedlings became arrhythmic or had greatly dampened oscillations compared with WT plants, which had robust rhythms under these conditions (Fig. 2C and Fig. S2A). The jmjd5 mutants continued to cycle on days 4 and 5 of free-run under a high fluence rate of white light (Fig. S2B), suggesting that this phenotype was specific to red light. Thus, the jmjd5 and toc1 mutant phenotypes are similar: Both have short free-running periods in constant blue or white light but become arrhythmic in constant red light.

JMJD5 Is a Positive Regulator of CCA1 and LHY Expression.

Although it is known that TOC1 promotes CCA1 and LHY expression, the underlying mechanism is not yet fully understood (9, 12). TOC1 and JMJD5 are coexpressed and have comparable mutant phenotypes; thus, we speculated that JMJD5, like TOC1, might play a role in the regulation of CCA1 and LHY expression. To investigate this possibility, we used qRT-PCR to examine the expression of core circadian genes in toc1-2 and jmjd5 mutants. Because both the toc1 and jmjd5 phenotypes were most severe in monochromatic red light (Fig. 2C and Fig. S2A), we chose to examine clock gene expression in plants grown in high-intensity constant red light, collecting samples over a full 2 d. Consistent with the luciferase data, qRT-PCR analysis revealed that expression of the evening-phased genes GIGANTEA (GI) and TOC1 was disrupted, with a decrease in rhythmic amplitude (Fig. 3 A and B).

Fig. 3.
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Fig. 3.

qRT-PCR analysis of circadian clock-regulated genes. WT Columbia (Col), jmjd5-1, and toc1-2 mutants were compared by qRT-PCR. Levels of TOC1 (A), GI (B), CCA1 (C), and LHY (D) mRNA were assessed. Plants were entrained to 12/12-h light/dark cycles for 6 d before being moved to constant conditions with 120 μmol·m−2·s−1 red light. mRNA levels for each gene were normalized to PP2a. Data are the mean of three technical replicates; SE is shown. Results are representative of three independent replicates.

We next examined expression of CCA1 and LHY, factors known to repress GI and TOC1 expression (9, 27). In jmjd5 mutants, peak levels of CCA1 and LHY mRNA were reduced relative to WT (Fig. 3 C and D and Fig. S3), similar to the reduced expression seen in toc1-2 plants (Fig. 3 C and D). These data indicate that like TOC1, JMJD5 promotes expression of the morning-phased clock genes CCA1 and LHY.

JMJD5 and TOC1 Interact Genetically.

Given that TOC1 and JMJD5 are coregulated and have related mutant phenotypes, we speculated they might act at a similar step in the clock mechanism and would show genetic interactions. We therefore generated plants mutant for both genes and assessed circadian function in multiple light conditions. When monitored in constant red plus blue light, jmjd5-1 toc1-2 double mutants had a free-running period ≈1.5 h shorter than toc1-2 single mutants; in comparison, jmjd5-1 single mutants had a period only 0.5 h shorter than WT controls (Fig. 4A). Therefore the combination of jmjd5-1 and toc1-2 mutations had a synergistic effect on period length. When maintained under constant red light of a moderate fluence rate (30 μmol·m−2·s−1), jmjd5-1 toc1-2 mutants also showed a synergistic effect on rhythmicity. Under this condition, jmjd5-1 single mutants were robustly rhythmic, whereas ≈70% of toc1-2 mutants had a detectable rhythm in luciferase activity (Fig. 4C). In the double mutants, only 30% of plants had a significant circadian rhythm. Thus, TOC1 and JMJD5 showed synergistic genetic interactions for multiple clock phenotypes. In contrast, only additive effects on period and rhythmicity were observed in jmjd5-1 lhy-100 double mutants, with the double mutants having a free-running period 0.5 h shorter than the lhy-100 single mutants (Fig. 4 B and D).

Fig. 4.
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Fig. 4.

JMJD5 and TOC1 interact genetically. (A) Period estimates of WT Columbia (Col), jmjd5-1, toc1-2, and jmjd5-1 toc1-2 genotypes. Plants were entrained to 12/12-h light/dark cycles for 6 d before being held in continuous 35 μmol·m−2·s−1 red and blue light. Each genotype had a significantly different period length (a–d; P < 0.001, Tukey range test). Error bars represent SE (n ≥ 11). Presented data are representative of three independent experiments. (B) Period estimates of WT Col, jmjd5-1, lhy-100, and jmjd5-1 lhy-100 genotypes. Seedlings were treated and analyzed as described in A. Error bars represent SE (n ≥ 30). Presented data are representative of three independent experiments. (C) Rhythmicity of jmjd5-1 toc1-2 seedlings held under 30 μmol·m−2·s−1 red light. Percent rhythmicity was defined as the fraction of seedlings that returned a period estimate with a relative amplitude error <1 as determined by Fourier fast transform-nonlinear least squares (50). Seedlings and entrainment conditions were as described in A. Populations of toc1-2 and jmjd5-1 toc1-2 double-mutant seedlings had significantly worse rhythms than WT or toc1-2 populations, respectively (*P < 0.05, Student's t test). Error bars indicate SE from three independent experiments. (D) Rhythmicity of jmjd5-1 lhy-100 seedlings held under 30 μmol·m−2·s−1 red light. Seedlings and entrainment conditions were as described in C. Error bars indicate SE from three independent experiments.

Human Cells Deficient for JMJD5 also Have a Short-Period Phenotype.

Although the core circadian transcription factors are not shared across higher taxa, some proteins (such as casein kinase and cryptochrome) and protein modifications (such as histone acetylation and poly-ADP ribosylation) do play a role in the circadian clocks of diverse eukaryotes (11, 28, 29 and reviewed in 30, 31). JMJD5 orthologs can be found in many eukaryotic genomes (Fig. S4A and Table S3). For example, the human JMJD5 protein (also known as KDM8) (16) is highly related to the Arabidopsis protein, with 39% identity and 56% similarity at the amino acid level and similar predicted secondary and tertiary protein structures (Fig. S4 B and C). Amino acid similarity rises to 68% within the conserved jmjC domain. Given the similarities between the human and Arabidopsis JMJD5 genes, we investigated whether the human ortholog might also be involved in clock function. We designed an siRNA construct against the human JMJD5 gene and transfected this into U2OS cells expressing luciferase under the control of the BMAL1 promoter (U2OS-B6) (32). Transformation with the siRNA construct specifically knocked down endogenous levels of JMJD5 to 20% of WT (Fig. S5 A and B). Transformed cells had a significantly shorter period than U2OS-B6 cells transfected with a scrambled siRNA control (Fig. 5 A and B; P = 0.022, Student's t test) but continued to show robust rhythmicity (Fig. 5A). This period shortening was strikingly similar to the circadian phenotype observed for Arabidopsis jmjd5 mutants in most light conditions (Table S2).

Fig. 5.
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Fig. 5.

Arabidopsis and human JMJD5 have conserved functions. (A) Circadian rhythmicity of U2OS-B6 cells stably expressing a Bmal1 promoter-driven luciferase reporter construct. Average luciferase activity of cells transfected with either scrambled siRNA or JMJD5 siRNA was assayed following synchronization with a serum shock as described by Vollmers et al. (32) (n = 5). Data are representative of three independent experiments. (B) Circadian period of human U2OS-B6 cells stably transformed with the plant JMJD5 ortholog. U2OS-B6 cells expressing the plant JMJD5 ortholog (U2OS + AtJMJD5) had a significantly longer period than WT U2OS-B6 cells when transfected with a human JMJD5 siRNA (**P = 0.0026, Student's t test). Error bars indicate SEM (n = 5). Cells were treated as described in A. Data are representative of two independent experiments. (C) Circadian period of jmjd5-1 plants transformed with either the plant or human JMJD5 gene. Plants were entrained to 12/12-h light/dark cycles for 6 d before being moved to continuous 40 μmol·m−2·s−1 red light. T1 plants transformed with either the plant or human JMJD5 gene had a significantly longer period than jmjd5-1 mutants (*P < 0.05, mixed-effect linear model). Data are pooled from three independent experiments. Error bars show SEM (n ≥ 20).

JMJD5 Homologs Act in Both Plant and Human Circadian Systems.

Because loss of either ortholog caused similar effects on clock function in plants and human cells, we speculated that the proteins might share a conserved cellular function. To test this possibility, we created a cell line (U2OS + AtJMJD5) that stably expressed the Arabidopsis gene in a U2OS-B6 background and monitored its circadian periodicity following knockdown of the endogenous human gene (Fig. 5B). Remarkably, U2OS + AtJMJD5 cells transfected with a human JMJD5 siRNA had a significantly longer period than U2OS-B6 cells (Fig. 5B; P = 0.0026, Student's t test), indicating that the plant protein is able to function within the human circadian system. This rescue is highly significant but not complete, because a modest decrease in period length was observed in U2OS + AtJMJD5 cells when transfected with JMJD5 siRNA compared with a scrambled siRNA control (Fig. 5B).

We also investigated whether the human ortholog was functional in planta. To test this hypothesis we introduced either the Arabidopsis or the human JMJD5 gene, expressed under the control of the plant JMJD5 promoter (Fig. S4D), into the Arabidopsis jmjd5-1 mutant background and tested for rescue of the period defect. The circadian periods of T1 seedlings transformed with either the human or plant gene were then compared with those of WT and jmjd5-1 plants (Fig. 5C and Fig. S4E). Intriguingly, seedlings transformed with either JMJD5 ortholog displayed a significantly lengthened circadian period compared with the untransformed parental control (P = 0.0013 and 0.0210 for the Arabidopsis and human genes, respectively). Use of T1 plants permitted characterization of at least 20 independent insertion events for each transgenic population but necessitated the use of hygromycin selection. Such treatment is unlikely to alter circadian period, however, because hygromycin-resistant homozygous T3 transgenics demonstrated no difference in period length in the presence or absence of hygromycin (Fig. S4F). In combination, these cross-kingdom rescue experiments demonstrate that JMJD5 functions in both the Arabidopsis and human circadian clocks via a conserved molecular mechanism.

Discussion

Characterization and Placement of JMJD5 Within the Arabidopsis Circadian System.

We have successfully used a data-mining strategy to identify JMJD5, an evening-phased putative histone demethylase that acts within the Arabidopsis central clock. Similar to TOC1, JMJD5 expression is strongly repressed by CCA1 (9) (Fig. S1 D and E). Indeed, it appears that the effect of CCA1 overexpression on JMJD5 expression levels may be more pronounced than that on TOC1; unlike TOC1, JMJD5 mRNA was not detectable by qRT-PCR in seedlings overexpressing CCA1 (9) (Fig. S1 D and E). Although JMJD5 expression was altered in a toc1-2 mutant background (Fig. 1C), this might be an indirect consequence of reduced CCA1 and LHY mRNA levels in this genetic background (9). Similarly, mutation of the TOC1 homologs PSEUDORESPONSE REGULATOR 5, 7, and 9 (PRR5, PRR7, and PRR9) profoundly increased expression of CCA1 and LHY and decreased expression of JMJD5, suggesting the PRRs may indirectly regulate JMJD5 expression (33). TOC1 expression was also significantly reduced in the prr5, prr7, and prr9 mutants but to a lesser extent than that of JMJD5 (33).

We found that JMJD5 and TOC1 showed a synergistic genetic interaction, whereas the interactions between JMJD5 and LHY were additive (Fig. 4). Formally, synergism may arise either when two genes share overlapping functions within the same process or when they act in convergent signaling pathways. The reduced CCA1 and LHY expression seen in both jmjd5 and toc1 single mutants (Fig. 3 C and D and Fig. S3) suggests that they may act together to promote expression of these morning-phased clock genes. Similar synergistic effects on regulation of gene expression have previously been reported between transcription factors and chromatin-modifying enzymes (34–36) and between the mammalian clock genes CLOCK and BMAL1 (37).

jmjd5 Phenotype Is Exacerbated by Increased Fluence Rates.

Although we observed a significant shortening of circadian rhythms in a range of light conditions (Fig. 2A and Table S2) and arrhythmia at higher fluence levels of monochromatic red light (Fig. 2C), we did not find a difference in circadian rhythms between WT and jmjd5 mutants in constant darkness (Table S2). Similar light-dependent circadian phenotypes have been reported for elf3 mutants (24), a gene that we similarly found to be coexpressed with TOC1 (Fig. 1A). The exacerbation of the jmjd5 phenotype at increased fluence rates of monochromatic red light (Fig. 2C) was correlated with reduced expression of CCA1 and LHY (Fig. 3 and Fig. S3). In contrast, TOC1 expression levels were not appreciably reduced in jmjd5 (Fig. 3A).

Intriguingly, JMJD5 shares these differential effects on clock gene expression with LUX ARRHYTHMO (LUX), ELF3, and ELF4, which are all evening-phased genes that have yet to be ascribed specific positions within the circadian circuitry (21, 23, 38, 39). Like JMJD5, loss of ELF3, ELF4, or LUX causes decreased expression of CCA1 and LHY without strongly affecting TOC1 expression levels (38, 40–42). It is possible that these coexpressed genes act in concert in some manner to promote morning-phased clock gene expression.

JMJD5 Functions in Both the Human and Plant Circadian Systems.

Orthologs of JMJD5 are found across eukaryotes (Fig. S4A and Table S3). Phylogenetic analyses suggest that these homologs originated before the separation of the animal and plant phyla, ≈1.5 billion y ago (43). Despite this ancient divergence, the human JMJD5 gene can rescue the circadian defect of Arabidopsis jmjd5 mutants. Given the apparent independent origins of the plant and human circadian clocks (8, 44), it would appear that JMJD5 has been recruited independently into each of these molecular oscillators.

The human JMJD5 protein has recently been shown to demethylate histone H3 dimethylated at lysine 36 (H3K36me2), both in vitro and in vivo (16). Although the enzymatic activity of the Arabidopsis ortholog is not yet determined, our data suggest that human and plant proteins can fulfill similar functions within the plant and human circadian systems, respectively (Fig. 5 B and C and Fig. S4E). All the cofactor-binding residues thought necessary for histone methylase activity (15) are found in the Arabidopsis JMJD5 protein (Fig. S4B), suggesting that it may also have this biochemical function. Despite this conservation, we cannot exclude the possibility that the human JMJD5 acts simply as a scaffold to facilitate the function of endogenous plant protein complexes rather than supplying needed enzymatic activity.

Although H3K36me2 modifications are predominantly found toward the 3′ end of actively transcribed eukaryotic genes (45–47), enrichment of this modification is also found near the TSS and within exons, where it acts as a repressive mark to suppress erroneous transcription (45, 47, 48). Both CCA1 and LHY loci carry extensive H3K36me2 marks within their coding regions (48), suggesting that JMJD5 may directly demethylate the CCA1 and LHY loci and thereby promote their transcription. Alternately, JMJD5 could promote gene expression less directly by modifying the combination of histone modifications present at the affected loci. Histone modifications are increasingly acknowledged to act in concert to direct transcriptional activity (15, 16, 47).

In summary, we have identified a previously undescribed component of both the Arabidopsis and human circadian systems that may link the clock with dynamic histone modification and thereby provide additional regulation of gene expression. Our coexpression and genetic analyses indicate that Arabidopsis JMJD5 acts in concert with TOC1 to positively regulate expression of CCA1 and LHY. Although we do not presume that the inclusion of JMJD5 in the molecular oscillators of both the Arabidopsis and human circadian systems is a retained ancestral function of this protein, it is intriguing that it functions in the clocks of these highly divergent species. At the very least, this suggests that histone methylation is an important regulatory mechanism in two very different eukaryotes. Future work will further elucidate the relationship between JMJD5 and the core clock components of both animals and plants.

Materials and Methods

Plant Materials and Growth Conditions.

Details on the construction of the binary vectors and the mutants used in this study are included in SI Materials and Methods.

Mammalian Cell Culture.

U2OS cell lines used in this study are described in SI Materials and Methods.

Luciferase Assays.

Luciferase assays were performed as described (32, 49, 50). Additional information is provided in SI Materials and Methods.

PCR Techniques.

RNA isolation, cDNA preparation, and qRT-PCR techniques are described in SI Materials and Methods.

Gene Expression Correlation and Phylogenetic Analyses.

Details on the statistical and phylogenetic tools used in this study are included in SI Materials and Methods.

Acknowledgments

We thank Julin Maloof for statistical advice and Reetika Rawat (University of California, Davis, CA) for seed stocks. Mutants were obtained from the Arabidopsis Biological Resource Center and the European Arabidopsis Stock Centre. This study was supported by National Institutes of Health Grant GM069418 (to S.L.H.).

Footnotes

  • 1To whom correspondence should be addressed. E-mail: slharmer{at}ucdavis.edu.
  • Author contributions: M.A.J., M.F.C., L.D., C.V., S.P., and S.L.H. designed research; M.A.J., L.D., and C.V. performed research; M.A.J., M.F.C., L.D., C.V., and S.L.H. contributed new reagents/analytic tools; M.A.J., M.F.C., L.D., and S.P. analyzed data; and M.A.J. and S.L.H. wrote the paper.

  • The authors declare no conflict of interest.

  • ↵*This Direct Submission article had a prearranged editor.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1014204108/-/DCSupplemental.

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Jumonji domain protein JMJD5 functions in both the plant and human circadian systems
Matthew A. Jones, Michael F. Covington, Luciano DiTacchio, Christopher Vollmers, Satchidananda Panda, Stacey L. Harmer
Proceedings of the National Academy of Sciences Dec 2010, 107 (50) 21623-21628; DOI: 10.1073/pnas.1014204108

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Jumonji domain protein JMJD5 functions in both the plant and human circadian systems
Matthew A. Jones, Michael F. Covington, Luciano DiTacchio, Christopher Vollmers, Satchidananda Panda, Stacey L. Harmer
Proceedings of the National Academy of Sciences Dec 2010, 107 (50) 21623-21628; DOI: 10.1073/pnas.1014204108
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