the jumonji c domain-containing protein jmj30 regulates ...(tsukada et al., 2006). the name jumonji...

The Jumonji C Domain-Containing Protein JMJ30Regulates Period Length in the ArabidopsisCircadian Clock1[W][OA]

Sheen X. Lu, Stephen M. Knowles, Candace J. Webb, R. Brandon Celaya, Chuah Cha2,Jonathan P. Siu3, and Elaine M. Tobin*

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles,California 90095

Histone methylation plays an essential role in regulating chromatin structure and gene expression. Jumonji C (JmjC) domain-containing proteins are generally known as histone demethylases. Circadian clocks regulate a large number of biologicalprocesses, and recent studies suggest that chromatin remodeling has evolved as an important mechanism for regulating bothplant and mammalian circadian systems. Here, we analyzed a subgroup of JmjC domain-containing proteins and identifiedArabidopsis (Arabidopsis thaliana) JMJ30 as a novel clock component involved in controlling the circadian period. Analysis ofloss- and gain-of-function mutants of JMJ30 indicates that this evening-expressed gene is a genetic regulator of period length inthe Arabidopsis circadian clock. Furthermore, two key components of the central oscillator of plants, transcription factorsCIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL, bind directly to the JMJ30 promoter to repressits expression, suggesting that JMJ30 regulates the pace of the circadian clock in close association with the central oscillator.JMJ30 represents, to our knowledge, the first JmjC domain-containing protein involved in circadian function, and we envisionthat this provides a possible molecular connection between chromatin remodeling and the circadian clock.

Circadian rhythms are endogenous biologicalrhythms with a period of approximately 24 h. Thecircadian clock, found in organisms ranging from cy-anobacteria to mammals, allows organisms to antici-pate regular environmental changes thus enhancingevolutionary fitness. The common mechanism of theeukaryotic circadian clock involves multiple inter-locked feedback loops. In Arabidopsis (Arabidopsisthaliana), the circadian clock regulates processes suchas gene expression, photoperiodic flowering, and leafmovement (McClung, 2006). Several components ofthe Arabidopsis clock have been identified, althougha complete understanding of how the clock gener-ates self-sustaining rhythms is lacking. CIRCADIANCLOCK ASSOCIATED1 (CCA1) and LATE ELON-GATED HYPOCOTYL (LHY) are morning-expressedMYB transcription factors that are suggested to form afeedback loop in the circadian clock with the evening-expressed pseudoresponse regulator TIMINGOFCAB

EXPRESSION1 (TOC1; Schaffer et al., 1998; Wang andTobin, 1998; Strayer et al., 2000). CCA1 and LHYdirectly repress TOC1 expression by binding to theevening element (EE) in its promoter (Alabadı et al.,2001); TOC1 is in turn believed to activate transcriptionof CCA1 and LHY through CCA1 HIKING EXPEDI-TION and other unknown mechanisms (Pruneda-Pazet al., 2009). Other key clock components that interactwith the CCA1/LHY/TOC1 loop include GIGANTEA(GI) and PSEUDORESPONSEREGULATORs (PRRs) 7and 9 (Farre et al., 2005; Locke et al., 2006).

The methylation status of histones controls chroma-tin remodeling and gene expression in eukaryotes.Histone modifications have also been implicated in theregulation of the circadian clock in Arabidopsis. Forexample, the expression of TOC1 is affected by clock-controlled cycles of histone acetylation, although theresponsible histone deacetylase(s) is/are not known(Perales and Mas, 2007). Jumonji C (JmjC) domain-containing proteins have been shown to be involved inchromatin remodeling, acting as histone demethylases(Tsukada et al., 2006). The name jumonji (which meanscruciform in Japanese) was originally derived from amouse mutation that affected neural tube develop-ment and produced a cross-like structure on the neuralplate (Takeuchi et al., 1995). The JmjC domain is thecatalytic domain, and these proteins catalyze Lysdemethylation through an oxidative reaction that re-quires Fe(II) and a-ketoglutarate as cofactors. TheJmjC domain-containing proteins are involved in abroad range of processes, such as neural stem celldifferentiation (Jepsen et al., 2007), X-linked mental

1 This work was supported by the National Institutes of Health(grant no. GM23167 to E.M.T.).

2 Present address: 1571 N. Salisbury St., Porterville, CA 93257.3 Present address: 432 E. Poplar Ave., Vallejo, CA 94592.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Elaine M. Tobin ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.110.167015

906 Plant Physiology�, February 2011, Vol. 155, pp. 906–915, www.plantphysiol.org � 2010 American Society of Plant Biologists

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retardation (Iwase et al., 2007), the posterior develop-ment of animals (Lan et al., 2007), and embryonic stemcell self-renewal (Loh et al., 2007).In Arabidopsis, there are 21 JmjC domain-containing

proteins (Lu et al., 2008; Hong et al., 2009), althoughfew have been characterized. EARLY FLOWERING6(ELF6) andRELATIVEOFELF6 (REF6) proteins,whichcontain both JmjC and zinc-finger domains, regulateflowering time in Arabidopsis (Noh et al., 2004). ELF6represses photoperiodic flowering, whereas REF6 re-presses FLOWERING LOCUS C (FLC) expression. Inaddition, they have been shown to modulate geneexpression regulated by brassinosteroids by interact-ing with BRASSINOSTEROID-INSENSITIVE1-EMS-SUPPRESSOR1, a transcription factor that binds topromoters of genes that respond to brassinosteroids(Yu et al., 2008). Two other JmjC proteins, MATERNALEFFECT EMBRYO ARREST27 and INCREASED EX-PRESSION OF BONSAI METHYLATION1 are involvedin gametophyte development and repression of cytosinemethylation, respectively (Pagnussat et al., 2005). JMJ14,which contains a JmjN and zinc-finger domain in addi-tion to the JmjC domain, prevents early flowering byrepressing the expressionofFLOWERINGLOCUST (FT),and its homolog TWIN SISTER OF FT, APETALA1,SUPPRESSOROFOVEREXPRESSIONOFCONSTANS1(SOC1), and LEAFY (Lu et al., 2010; Yang et al., 2010).More recently, JMJ14 has been shown to be involvedin RNA silencing and the maintenance phase of DO-MAINS REARRANGED METHYLTRANSFERASE2-mediated RNA-directed DNA methylation (Searleet al., 2010).In the work presented here, we have identified

JMJ30 as, to our knowledge, the first JmjC domain-containing protein involved in circadian function.Since little is known about histone methylation andcircadian clock function, our work paves the way tothe exploration of a specific pathway linking histonemethylation and circadian regulation.

RESULTS

Expression of JMJ30 Oscillates with a Circadian Rhythm

Based on the sequence similarity between JmjCdomains, the 21 JmjC domain-containing proteinscan be classified into five groups (Hong et al., 2009).JMJ30, encoded by At3g20810, belongs to the JmjCdomain-only group that contains four members (Sup-plemental Fig. S1). JMJ30 is ubiquitously expressedin different tissues (Lu et al., 2008) and has the con-served Fe(II)- and a-ketoglutarate-binding amino acidsrequired for histone demethylation, indicating thatJMJ30 might be a functional histone demethylase. Inaddition, YFP-JMJ30 primarily localizes to the nucleuswith a low GFP signal detected in the cytoplasm (Sup-plemental Fig. S2). Its inclusion in the nucleus furthersupports our hypothesis that JMJ30 is a histone de-methylase in vivo. From the publicly available micro-

array data, we identified JMJ30 as the only protein ofthe 21 members that shows a robust circadian rhythmof expression (Mockler et al., 2007; Michael et al., 2008;Fig. 1A). The peak of rhythmic JMJ30 transcript ex-pression occurs around dusk (Zeitgeber time [ZT]-12)under both diurnal and constant white light (LL) con-ditions (Fig. 1B).

Loss of JMJ30 Function Affects the Free-RunningCircadian Period

To elucidate the biological roles of JMJ30 in Arabi-dopsis, we obtained two mutants, jmj30-1 and jmj30-2,in which T-DNA was inserted into the third intron(jmj30-1) or the second exon (jmj30-2) of JMJ30, re-spectively (Fig. 2A). JMJ30 full-length RNA was not

Figure 1. The JMJ30 transcript level exhibits circadian regulation. A,RNA levels of JmjC domain only containing genes in continuous light(LL) conditions. Data from the DIURNAL Project (http://diurnal.cgrb.oregonstate.edu/) microarray repository were used to compare RNAlevels of JMJ30 (At3g20810), JMJ20 (At5g63080), JMJ31 (At5g19840),and JMJ32 (At3g45880). Raw data from experimental set LL12(LDHH)were normalized to the mean of all points to compare relativeexpression levels among these family members. B, JMJ30 expressionoscillates in wild-type Arabidopsis under diurnal and LL conditions.Ten-day-old seedlings were analyzed and qRT-PCR data are presentedas the mean of two biological replicates 6 SD. Day, night, andsubjective night are denoted by white, black, and hatched bars,respectively.

JMJ30 Functions in the Circadian Clock

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detected in either of these two mutants by real-time(RT)-PCR (Fig. 2B), indicating that they are loss-of-function mutants for JMJ30. Neither of the mutantsdiffered from wild-type Columbia (Col) plants inhypocotyl elongation (data not shown) and floweringtime (Supplemental Fig. S3), two physiological re-sponses that can be affected by the circadian clock. Todetermine directly whether loss of JMJ30 affects thecircadian clock, we first examined leaf movement, awell-established circadian response in Arabidopsis(Hicks et al., 1996). Leaf movement rhythms of jmj30mutant plants had a short period in LL (Fig. 2C; wild-type plants had a period of 24.6 6 0.3 h, jmj30-1 of23.4 6 0.2 h, and jmj30-2 of 23.6 6 0.3 h). To assess therobustness of the circadian rhythms in individualseedlings, relative amplitude error (RAE) was mea-

sured. RAE values can range between 0 (perfect fittedrhythm) and 1 (rhythm not significant). Both of themutants had RAE values of approximately 0.1, similarto those of the wild type (Fig. 2, C and D).

Luciferase (LUC) activity under the control of acircadian promoter is a noninvasive method for mon-itoring clock function and integrity (Millar et al., 1995).The effect of the JMJ30 mutations on the circadianclock was also analyzed using the circadian reporterCHLOROPHYLL A/B BINDING PROTEIN2::LUC(CAB2::LUC; Millar et al., 1995; Knowles et al., 2008).Consistent with the period shortening observed in leafmovement rhythms, CAB2::LUC oscillations in thejmj30mutants had shorter periods than the oscillationsin wild-type plants (Fig. 2E; wild-type plants had aperiod of 25.2 6 0.7 h, jmj30-1 of 23.7 6 0.5 h, and

Figure 2. The jmj30 mutation shortens the circadian period under LL conditions. A, JMJ30 genomic structure. Exons arerepresented by black boxes, untranslated regions are represented by gray boxes, and introns are represented by lines. Trianglesindicate T-DNA insertions. B, RT-PCR analysis of JMJ30 full-length transcript in wild-type and jmj30 mutants. Ten-day-oldseedlings were sampled at ZT-12, and Actin transcript was used as a control. C and D, Assay of circadian leaf movement under LLconditions. C, Normalized positions of primary leaves for wild type (n = 12), jmj30-1 (n = 10), and jmj30-2 (n = 7) are shown. D,Period length and RAE were estimated using fast Fourier transform-nonlinear least-squares analysis. E and F, Assay of CAB2::LUCactivity under LL conditions. E, Mean bioluminescence traces of groups of approximately 20 seedlings for wild type (n = 7),jmj30-1 (n = 8), and jmj30-2 (n = 9) are shown. F, Period length and RAE estimates of the CAB2::LUC bioluminescence rhythmsshown in E. G to I, qRT-PCR analysis of CCA1 (G), LHY (H), and TOC1 (I) expression in wild-type and jmj30-1 plants under LLconditions. Ten-day-old seedlings that were entrained in a 12L/12D cycle, transferred to LL, and harvested for 3 d at 3-h intervalswere analyzed, and the mean of two biological replicates6 SD is shown. Day, night, and subjective night are denoted by white,black, and hatched bars, respectively. All experiments were done at least twice with similar results.

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908 Plant Physiol. Vol. 155, 2011



jmj30-2 of 23.8 6 0.6 h). The CAB2::LUC rhythmswere as robust as the leaf movement rhythms (Fig. 2, Eand F).While jmj30 affects the period of various output

rhythms (leaf movement and CAB2::LUC activity), wealso tested whether jmj30 affects the expression ofcentral oscillator genes. As shown in Figure 2, G to I,jmj30 mutation shortened the expression period ofCCA1, LHY, and TOC1, which function as the centraloscillator components (Alabadı et al., 2001). Althoughjmj30 caused period shortening of central oscillatorgenes, it has little effect on the amplitude of expression(Fig. 2, G–I). This phenotype is consistent with thefinding that the jmj30 mutation affects period lengthbut does not alter the amplitude and robustness of thecircadian output rhythms, such as leaf movementrhythms and CAB2::LUC rhythms during free-runningconditions (Fig. 2, C–F). Therefore JMJ30 is involved inregulating period length, rather than amplitude androbustness in the Arabidopsis circadian clock.

Constitutive Expression of JMJ30 Affects Flowering Time

To further investigate the role of JMJ30 in the circa-dian system, we generated transgenic plants over-expressing the JMJ30 gene. Two homozygous lineswith different epitope tags (YFP-JMJ30 ox and Myc-JMJ30 ox) were obtained. Quantitative RT-PCR (qRT-PCR) showed that the expression level of JMJ30 inoverexpressing plants is 30 to 50 times higher than thatin wild-type plants (Fig. 3A). We further demonstratedthat both JMJ30 RNA and protein are constitutivelyexpressed in the overexpressing line under diurnalconditions (Supplemental Fig. S4). In contrast to jmj30,the two lines overexpressing JMJ30 showed delayedflowering (Fig. 3, B and C) in both long-day (LD) andshort-day (SD) conditions, although the hypocotyllengths of JMJ30 ox were similar to that of wild type(data not shown). We therefore examined the expres-sion of CONSTANTS (CO) and FT, two of the key genesin the photoperiodic flowering pathway. Although therhythmic expression of CO was almost unaffected inJMJ30 ox compared with that of the wild type, expres-sion of FTwas greatly reduced in JMJ30 ox plants (Fig.3, D and E). It is known that FLC directly binds to FTand SOC1 chromatin to repress their expression(Helliwell et al., 2006).We then examined FLC and SOC1expression, and found that the transcript levels of FLCremained unchanged but the SOC1 transcript levelswere strongly reduced in JMJ30 ox relative to wildtype (Fig. 3, F and G). This suggests that the delayed-flowering phenotype resulted from down-regulationof FT and SOC1 in the JMJ30 ox plants.

Constitutive Expression of JMJ30 Negatively RegulatesIts Own Expression and Affects the Free-RunningCircadian Period

Clock components are often controlled by negativefeedback regulation. We tested whether JMJ30 regu-

lates its own expression, as other circadian genes do(Schaffer et al., 1998; Wang and Tobin, 1998). Althoughthe endogenous JMJ30 transcript still showed robustoscillations in JMJ30 ox plants, there was a significantreduction in amplitude compared with that in wild-type plants (Fig. 4A). This finding suggests that JMJ30negatively regulates its own expression. To examinethe effect of constitutive expression of JMJ30 on thecircadian clock, a CAB2::LUC reporter was introducedinto the JMJ30 ox plants. Robust circadian rhythms inCAB2::LUC activity with a short period were observedin JMJ30 ox plants (Fig. 4, B and C; wild-type plants

Figure 3. Constitutive expression of JMJ30 affects flowering time. A,qRT-PCR analysis of JMJ30 expression in wild-type and JMJ30 ox plants.Ten-day-old seedlings were sampled at ZT-12. B and C, Flowering timeof wild-type and JMJ30 ox plants under LD (16L/8D; B) and SD (8L/16D; C) conditions. Flowering time was expressed as mean rosette leafnumber 6 SEM (n = 20–25). Asterisk indicates a significant differenceby Student’s two-tail t test (P , 0.01). D to G, qRT-PCR analysis of CO(D), FT (E), FLC (F), and SOC1 (G) expression in wild-type and JMJ30 oxplants under diurnal and LL conditions. Day, night, and subjective nightare denoted by white, black, and hatched bars, respectively. All qRT-PCR data are presented as the mean of two biological replicates 6 SD.All experiments were done at least twice with similar results.





had a period of 25.2 6 0.7 h, YFP-JMJ30 ox of 23.8 60.5 h, and Myc-JMJ30 ox of 23.9 6 0.6 h). A similarshort-period phenotype was also observed in the ex-pression of central oscillator genes (Fig. 4, D–F). Obser-vation of both mutants and overexpressing plants withshort-period phenotypes was also made in character-ization of the clock component GI (Mizoguchi et al.,2005). However, the mechanism of this phenomenon isnot known. Together, this data shows that JMJ30 is acircadian clock component involved in controlling thecircadian period.

CCA1 and LHY Negatively Regulate JMJ30 Expression

JMJ30 was originally identified among genes rep-resented on a microarray whose expression wasrepressed by CCA1 and LHY (data not shown), twokey components of the central oscillator of plants.To investigate the effect of the transcription factorsCCA1 and LHY on the expression of JMJ30, we firstmeasured the expression of JMJ30 in CCA1ox-38(Wang and Tobin, 1998) and lhy-1 (Schaffer et al.,1998), which overexpress CCA1 and LHY under thestrong cauliflower mosaic virus 35S promoter, respec-

tively. The expression of JMJ30 was dramatically re-duced in both the CCA1 and LHY overexpressing lines(Fig. 5, A and B), confirming that CCA1 and LHY actnegatively on JMJ30 expression. To further evaluatethe role of CCA1 and LHY in regulating the expressionof JMJ30, we examined the expression of JMJ30 in thecca1-1 (Green and Tobin, 1999), lhy-20 single mutant(Michael et al., 2003), and cca1-1/lhy-20 double mutant(Yakir et al., 2009). The expression profile of JMJ30 inthe cca1-1 mutant is indistinguishable from that in thewild-type plants (Fig. 5C). Since the functions of CCA1and LHY are partially redundant (Mizoguchi et al.,2002), this result suggests that LHY can probablycompensate the loss of CCA1 on the regulation ofJMJ30. In contrast to cca1-1, the peak of JMJ30 expres-sion in the lhy-20 occurs 4 h earlier than in the wild-type plants (Fig. 5D). This phase shift is not due to theshort-period phenotype of the lhy-20 because short-period mutant cca1-1 and toc1-2, another short-periodmutant (Strayer et al., 2000), did not display the sameeffect (Fig. 5F). These results indicate that CCA1 andLHY are not redundant in the regulation of JMJ30expression and that LHY has a greater effect thanCCA1. In the cca1-1/lhy-20 double mutant, JMJ30

Figure 4. Constitutive expression of JMJ30 affects the circadian period under LL conditions. A, qRT-PCR analysis of endogenousJMJ30 expression in wild-type and JMJ30 ox plants under diurnal and LL conditions. B and C, Assay of CAB2::LUC activity underLL conditions. B, Mean bioluminescence traces of groups of approximately 20 seedlings for wild type (n = 7), YFP-JMJ30 ox (n =7), andMyc-JMJ30 ox (n = 5) are shown. C, Period length and RAE estimates of the CAB2::LUC bioluminescence rhythms shownin B. D to F, qRT-PCR analysis of CCA1 (D), LHY (E), and TOC1 (F) expression in wild-type and YFP-JMJ30 ox plants under LLconditions. Ten-day-old seedlings that were entrained in a 12L/12D cycle, transferred to LL, and harvested for 3 d at 3-h intervalswere analyzed. Day, night, and subjective night are denoted by white, black, and hatched bars, respectively. All qRT-PCR dataare presented as the mean of two biological replicates 6 SD. All experiments were done at least twice with similar results.

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levels rise early (comparing the level at ZT-4 in dif-ferent mutants) and reach a peak at ZT-8 (Fig. 5E),indicating that CCA1 is required to repress JMJ30expression in the early morning in the absence of LHY.Taken together, the results indicate that CCA1 andLHY have a negative effect on JMJ30 expression,although LHY has a greater effect than CCA1.

JMJ30 Is a Direct Target of CCA1 and LHY in Vivo

JMJ30 has three EEs in the region 2169/261 up-stream of the translational start codon (Fig. 6A). Thiselement has been found in several circadian-regulatedgenes with evening expression and is known to bebound by CCA1 and LHY (Alabadı et al., 2001; Peralesand Mas, 2007). To examine whether CCA1 and LHYbind to the EE-containing region of the JMJ30 in vivo,chromatin immunoprecipitation (ChIP) was performed

using either an anti-CCA1 antibody or anti-LHY anti-body. Figure 6B demonstrated that an anti-CCA1antibody efficiently immunoprecipitated a fragmentof JMJ30 that contains the EEs but not another onelocated in the 3# untranslated region. Similar resultswere observed with the anti-LHY antibody (Fig. 6C),indicating that JMJ30 is a direct target of CCA1 andLHY in vivo.

Effects of Pulses of CCA1 and LHY on the Expressionof JMJ30

To examine the effect of CCA1 and LHY binding tothe JMJ30 promoter, we utilized the ethanol-inducible

Figure 5. CCA1 and LHY regulate JMJ30 expression. A to F, qRT-PCRanalysis of JMJ30 expression in wild type (Col-WT, Landsberg erecta-WT) and CCA1-ox (line o38), LHY-ox (lhy-1), cca1-1, lhy-20, cca1-1lhy-20, and toc1-2 plants under diurnal conditions. Ten-day-old seed-lings were analyzed and the mean of two biological replicates 6 SD isshown. Day and night are denoted by white and black bars, respec-tively. All experiments were done at least twice with similar results.

Figure 6. JMJ30 is a direct target of CCA1 and LHY. A, Schematicdrawing of genomic JMJ30 and the regions examined by ChIP. Exonsare represented by black boxes, untranslated regions are represented bygray boxes, and introns are represented by lines. EE elements (e) arerepresented by white boxes. B and C, Binding of CCA1 and LHY to theJMJ30 promoter in vivo. B, ChIP assays were performed with wild-type(Col), cca1-1, and CCA1-ox (line o38) seedlings collected at ZT-2. C,ChIP assays were performed with wild-type (Landsberg erecta), lhy-11,and LHY-ox (lhy-1) seedlings collected at ZT-2. qRT-PCR was per-formed on the precipitates by two primer sets. As indicated in A, Jmj-Pamplifies the region that contains three EEs and Jmj-N amplifies aregion in the 3# untranslated region. Results were normalized to theinput DNA and Actin control. The mean of two biological replicates6SD is shown. All of these experiments were done at least twice withsimilar results.





system (Knowles et al., 2008). The ethanol-induciblesystem provides a relatively simple way to test theeffect of a pulse of virtually any gene on a biologicalpathway. It has been shown that a pulse of CCA1 orLHY in Alc::CCA1 or Alc::LHYplants can reset the clockand repress the expression of evening-phased genessuch as TOC1, LUX, and GI (Knowles et al., 2008). Toexamine the effect of a pulse of CCA1 or LHY on theexpression of JMJ30, ethanol treatment was performedinAlc::CCA1 orAlc::LHYplants (Knowles et al., 2008). Apulse of either CCA1 or LHY at circadian time 8 whenthe JMJ30 RNA level is increasing resulted in a com-plete inhibition of the increase within 2 h of the ethanoltreatment (Fig. 7). The rapidity of the response furthersupports the notion that CCA1 and LHY bind directlyto the JMJ30 promoter to repress its expression.

DISCUSSION

In this study, we report the identification of a JmjCdomain-containing protein JMJ30 involved in theArabidopsis circadian clock. JMJ30 oscillations peakat dusk (ZT-12) and persist under both diurnal andcontinuous light conditions (Fig. 1), a hallmark ofgenes that exhibit circadian expression (McClung,2006). The levels of endogenous JMJ30 mRNA arereduced in plants overexpressing JMJ30 (Fig. 4A),suggesting that its accumulation is autoregulated atthe level of transcription, another hallmark of clockgenes involved in the negative feedback loop. TwoT-DNA mutants, jmj30-1 and jmj30-2, exhibited short-ened periods in rhythms of leaf movement, CAB2::LUC activity, and expression of central oscillator genes(Fig. 2, C–I), indicating that JMJ30 is important formaintaining correct free-running periods of the circa-dian clock. Interestingly, two overexpressing lines,YFP-JMJ30 ox and Myc-JMJ30 ox, also showed short-ened periods in rhythms of CAB2::LUC activity and

expression of central clock genes (Fig. 4, B–F). Appar-ently, this is not due to the cosuppression of JMJ30in the overexpressing lines because JMJ30 transcriptlevels in overexpressing plants are much higher thanthat in wild-type plants (Fig. 3A; Supplemental Fig.S4). Although it is unlikely that the YFP and Myc tagsfused to the N-terminal of JMJ30 interrupt the functionof the JmjC domain that resides at the C terminus, wecannot rule out the possibility that the addition of thetags cause a dominant negative effect and repress theendogenous JmjC activity. The expression of JMJ30was dramatically reduced in both the CCA1 and LHYoverexpressing lines (Fig. 5, A and B), indicating thatCCA1 and LHY negatively regulate JMJ30 expression.ChIP assays conclusively demonstrate that CCA1 andLHY bind to the JMJ30 promoter in vivo (Fig. 6) andthe use of the ethanol-inducible system has allowed usto further demonstrate the repression of JMJ30 expres-sion by CCA1 and LHY in real time (Fig. 7).

JMJ30 contains a JmjC domain, which has beenimplicated in histone demethylation (Lu et al., 2008).To carry out histone demethylation, JmjC domain-containing proteins require the cofactors Fe(II) anda-ketoglutarate. Since JMJ30 contains the two con-served His and one Asp residue required for Fe(II)binding, and a conserved Thr and Lys required fora-ketoglutarate binding, it has the necessary aminoacid residues to carry out a histone demethylation reac-tion (Supplemental Fig. S1). In addition, YFP-JMJ30localizes primarily to the nucleus (Supplemental Fig.S2), suggesting it may be a functional histone demeth-ylase. An in vitro histone demethylation assay will be akey experiment to determine whether JMJ30 has histonedemethylase activity.

The jmj30 mutants and overexpressing plants differin their flowering phenotypes. Jmj30 mutants do notexhibit any flowering phenotype in either LD or SDconditions (Supplemental Fig. S3), whereas in JMJ30ox plants, flowering is delayed in LD and SD (Fig. 3, Band C). Since there are 21 JmjC domain-containingproteins in Arabidopsis, it is possible that multipleJmjC domain-containing proteins function redun-dantly in the control of flowering time. Therefore theloss of JMJ30 may not have a measurable effect onflowering time. In plants constitutively expressingJMJ30, FT and SOC1 are expressed at much lowerlevels while the expression of CO and FLC are unaf-fected (Fig. 3, D–G), suggesting that the delayed-flowering phenotype in JMJ30 ox may not be throughthe photoperiodic flowering pathway. FT expression isenhanced by increased H3K4me3 methylation andrepressed by H3K27me3 methylation (Jiang et al.,2008). A recent study showed that JMJ14 is a histoneH3 Lys 4 demethylase that represses FT expression toinhibit the floral transition in Arabidopsis (Yang et al.,2010). It is possible that JMJ30 functions as a histonedemethylase to repress FT and SOC1 expression in asimilar manner to JMJ14.

Circadian clock regulation of the transcriptome is awidespreadphenomenon.More than10%ofmammalian

Figure 7. A pulse of CCA1 or LHY expression represses the accumu-lation of JMJ30 RNA. Control, Alc::CCA1, and Alc::LHY seedlings weregiven an ethanol pulse at 32 h in LL (circadian time 8). qRT-PCRanalysis of JMJ30 expression at the time of induction (0 h), 1, 2, and 4 hafter ethanol treatment are represented. The mean of two biologicalreplicates 6 SD is shown. All of these experiments were done at leasttwice with similar results.

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transcripts (Duffield et al., 2002) and about 25% ofArabidopsis transcripts oscillate in a circadian manner(Michael et al., 2008). Recent advances in the field haverevealedanunexpected linkbetweencircadian-regulatedgene expression and dynamic changes in chromatin. InNeurospora, rhythmic histone acetylation at the promoterof the central clock gene FREQUENCY was observed(Belden et al., 2007). The CLOCK protein, an essentialcomponent of themammalian circadian system,has beenshown to be a histone acetyltransferase (Doi et al., 2006).In Arabidopsis, histone acetylation (H3K9Ac) and di-methylation (H3K4Me2) at theCCA1, LHY,TOC1, andGIpromoters positively correlates with their expression (Niet al., 2009). We have examined the expression of knownclock genes in both JMJ30 mutants and overexpressingplants; no significantdifference in their expressionprofilewas observed (Supplemental Fig. S5; data not shown).The identificationof its targetgeneswill allowelucidationof the function of JMJ30 within the circadian clock and islikely to reveal the importance of histone methylation incircadian clock function.Taken together, our study shows that Arabidopsis

JMJ30 is a novel circadian clock component involvedin controlling the circadian period. In addition, CCA1and LHY directly bind to the JMJ30 promoter torepress its expression, indicating that JMJ30 regulatesthe pace of the circadian clock in a close associationwith the central oscillator. JMJ30, a putative histonedemethylase, represents, to our knowledge, the firstJmjC domain-containing protein involved in circadianfunction.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana; Col ecotype) was used for all experi-

ments described unless stated otherwise. jmj30-1 (SAIL_811_H12) and jmj30-2

(GK_454C10) are two T-DNA insertion alleles of JMJ30. For JMJ30 over-

expression, the full-length coding sequence of JMJ30, including the stop

codon, was amplified by specific primers (Supplemental Table S1) and

cloned into the pEarleyGate 104 (35S-YFP-JMJ30) and pEarleyGate 203 (35S-

Myc-JMJ30) vectors (Earley et al., 2006) using the GATEWAY recombination

system (Invitrogen). Seedlings were grown under a 12-h fluorescent light

(30 mmol m22 s21):12-h dark (12L:12D) photoperiod at a constant temper-

ature of 22�C, unless otherwise stated.

Analysis of Circadian Rhythms

Arabidopsis plants homozygous for jmj30-1, jmj30-2, YFP-JMJ30 ox, Myc-

JMJ30 ox, and wild type (Col) were transformed with the CAB2::LUC reporter

(Knowles et al., 2008). T2 seedlings from three to six independent transformed

lines were entrained for 6 d with 12L/12D cycles before being transferred to

LL conditions. Bioluminescence rhythms of groups of approximately 20

seedlings were analyzed as previously described (Knowles et al., 2008). For

leaf movement analysis, seedlings were entrained for 10 d under a 12L/12D

cycle and then transferred to continuous white light. Seedlings were individ-

ually transferred to the wells of upright 24-well tissue culture plates and the

positions of the primary leaves were recorded every 20 min for 7 d using a

CCD camera (model: LTC 0335) from Bosch. Leaf movement was assessed by

measuring the vertical position of the primary leaves using the publicly avail-

able software ImageJ. Oscillation properties were analyzed with the BRASS

(available from http://www.amillar.org) using the fast Fourier transform-

nonlinear least-squares analysis program (Plautz et al., 1997).

Measurement of Flowering Time

Arabidopsis plants were grown on soil under either LD (16-h light/8-h

dark) or SD (8-h light/16-h dark) conditions. Flowering time was scored by

counting the number of rosette leaves at flowering. Data are presented as

mean 6 SEM (n = 10–25).

RNA Extraction and qRT-PCR

For all experiments, 1- to 2-week-old seedlings grown on plates were used.

For circadian experiments, samples were collected every 3 to 4 h either during

the light/dark cycle or in continuous white light. RNA extraction and qRT-

PCR were carried out as previously described (Knowles et al., 2008). Actin2

was used as a noncycling reference, and expression levels were normalized to

the level of the control. All reactions were performed in triplicate.

ChIP

ChIP was performed as previously described (Ni et al., 2009) using affinity-

purified anti-CCA1 antibody (Wang and Tobin, 1998) or affinity-purified anti-

LHY antibody (Lu et al., 2009) for immunoprecipitation.

Ethanol Pulse

Ethanol treatment was performed as previously described (Knowles et al.,

2008). Control plants contained the regulator construct 35S::AlcR:TNOS. Two-

week-old seedlings were treated with 10 min of ethanol vapor of 1% or 3%

(v/v) ethanol. Samples were collected at the time of induction (0 h), 1, 2, and

4 h after ethanol treatment.

Tobacco Infiltration and Confocal Microscopy

For transient expression in tobacco (Nicotiana benthamiana), leaves were

transfected with 35S::YFP-JMJ30 as described previously (Lu et al., 2009) and

analyzed 2 d after infiltration. For stable transgenic Arabidopsis, the leaves of

8-d-old transgenic 35S::YFP-JMJ30 seedlings grown on plates were analyzed.

All imaging was done using a Carl Zeiss 510 Meta laser-scanning confocal

microscope with a Plan-Apochromat 633/1.4 oil differential interference

contrast objective. YFP was excited with an argon laser at 25% to 50% of its

output that was attenuated to 7% to 9% at 514 nm. The emission was sent

through a 530 to 600 nm band-pass filter for detection of YFP.

Plant Protein Extracts and Immunoblot Analysis

Proteins were extracted in 13 extraction buffer (50 mM Tris, pH 8.0, 150 mM

NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM phenylmethylsul-

phonyl fluoride, 50 mM MG115, 50 mM MG132, and protease inhibitor cocktail

[Roche]). Immunoblotting was performed as described (Lu et al., 2009) with

the appropriate primary antibody (anti-GFP [Santa Cruz biotechnology,

sc-8334], anti-Actin [MP Biomedicals, Clone C4]).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Ge-

nome Initiative data library using the following accession numbers: JMJ30

(At3g20810), JMJ20 (At5g63080), JMJ31 (At5g19840), JMJ32 (At3g45880), CAB2

(At1g29920), FT (At1g65480), CO (At5g15840), FLC (At5g10140), SOC1

(At2g45660), ACT2 (At3g18780), ACT7 (At5g09810), CCA1 (At2g21660), LHY

(At1g01060), TOC1 (At5g61380), and PRR3 (At5g60100).

Supplemental Data

Supplemental Figure S1.Alignment of jmjC domains among jmjC domain

only containing proteins.

Supplemental Figure S2. YFP-JMJ30 is localized in the nucleus and

cytosol.

Supplemental Figure S3. jmj30 mutants have no significant flowering

phenotype.





Supplemental Figure S4. Both JMJ30 RNA and protein are constitutively

expressed in the overexpression lines.

Supplemental Figure S5. Loss of JMJ30 does not affect the expression of

several clock genes.

Supplemental Table S1. List of PCR primer sequences.

Note Added in Proof

Another report of the involvement of the JMJ30 gene (At3g20810) in

circadian rhythms in plants (and in animals) was recently published, with the

gene designation of JMJD5 (Jones MA, CovingtonMF, DiTacchio L, Vollmers

C, Panda S, Harmer SL [2010] Jumonji domain protein JMJD5 functions in

both the plant and human circadian systems. Proc Natl Acad Sci USA 107:

21623–21628).

ACKNOWLEDGMENTS

We acknowledge the assistance of the Arabidopsis Biological Resource

Center, which provided jmj30-1. We thank Angelique Deleris and Steve

Jacobsen for the jmj30-2 seeds, Steve Kay for toc1-2 seeds, and Rachael Green

for the lhy-20 and cca1-1/lhy-20 seeds.

Received October 6, 2010; accepted December 2, 2010; published December

7, 2010.

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the jumonji c domain-containing protein jmj30 regulates ...(tsukada et al., 2006). the name jumonji...

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