Regulatory link between DNA methylation and activedemethylation in ArabidopsisMingguang Leia, Huiming Zhanga,b, Russell Juliana, Kai Tanga, Shaojun Xiea,b, and Jian-Kang Zhua,b,1

aDepartment of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47906; and bShanghai Center for Plant Stress Biology,Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 200032, China

Contributed by Jian-Kang Zhu, February 4, 2015 (sent for review December 20, 2014; reviewed by Jerzy Paszkowski and Yijun Qi)

De novo DNA methylation through the RNA-directed DNA meth-ylation (RdDM) pathway and active DNA demethylation playimportant roles in controlling genome-wide DNA methylationpatterns in plants. Little is known about how cells manage thebalance between DNA methylation and active demethylationactivities. Here, we report the identification of a unique RdDMtarget sequence, where DNA methylation is required for main-taining proper active DNA demethylation of the Arabidopsis ge-nome. In a genetic screen for cellular antisilencing factors, weisolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles,as well as many RdDM mutants, which showed drastically reducedROS1 gene expression and, consequently, transcriptional silencingof two reporter genes. A helitron transposon element (TE) in theROS1 gene promoter negatively controls ROS1 expression, whereasDNA methylation of an RdDM target sequence between ROS1 5′ UTRand the promoter TE region antagonizes this helitron TE in regulatingROS1 expression. This RdDM target sequence is also targeted byROS1, and defective DNA demethylation in loss-of-function ros1 mu-tant alleles causes DNA hypermethylation of this sequence and con-comitantly causes increased ROS1 expression. Our results suggestthat this sequence in the ROS1 promoter region serves as a DNAmethylation monitoring sequence (MEMS) that senses DNA meth-ylation and active DNA demethylation activities. Therefore, theROS1 promoter functions like a thermostat (i.e., methylstat) tosense DNA methylation levels and regulates DNA methylation bycontrolling ROS1 expression.

DNA demethylase | ROS1 | methylstat | sensor | rheostat

DNA methylation is a conserved epigenetic mark importantfor development and stress responses in plants and many

animals (1–4). Genome-wide DNA methylation patterns aredynamically regulated by establishment, maintenance, and re-moval activities (4, 5). In plants, de novo DNA methylation iscontrolled by the RNA-directed DNA methylation (RdDM)pathway, in which complementary pairing between long non-coding RNAs and siRNAs mediates cytosine methylation ina sequence-specific manner (2, 4, 6, 7). Best characterized inArabidopsis, RdDM involves a complex array of regulators andmainly targets heterochromatic regions that are enriched withtransposon elements (TEs) and other DNA repeat sequences(8, 9). Once established, Arabidopsis DNA methylation is main-tained via different mechanisms depending on the cytosine contexts[i.e., CG, CHG, CHH (H represents A, T, or C)]. CG and CHGmethylation is maintained by MET1 and CMT3, respectively (2),whereas CHH methylation within pericentromeric long TEs canbe catalyzed by CMT2 and CHH methylation at other loci isestablished de novo by DRM2 during every cell cycle (2, 10). Incontrast to DNA methyltransferases that establish and/or maintaincytosine methylation, plant 5-methylcytosine DNA glycosylasesinitiate a base excision repair pathway that erases DNA methyla-tion, thereby generating, together with the establishment andmaintenance activities, a dynamic landscape of DNA meth-ylation (11, 12).The Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) is

a major DNA demethylase that prunes DNA methylation for

dynamic transcriptional regulation (12). ROS1 counteracts theRdDM pathway to prevent DNA hypermethylation (5, 12–14).Interestingly, ROS1 gene expression is suppressed in mutantsdefective in RdDM (14–17) or MET1 (16), suggesting that DNAmethylation and active demethylation activities are coordinated.However, the mechanism underlying such coordination remainselusive. Here, we report the identification of a regulatory ele-ment at the ROS1 promoter that monitors DNA methylationlevels and accordingly modulates active DNA demethylationof the Arabidopsis genome by controlling ROS1 expression. Ina genetic screen for Arabidopsis mutants that are defective intranscriptional antisilencing of transgenic reporter genes, weisolated several ros1 mutant alleles and many RdDM mutantswith drastically repressed ROS1 gene expression. We found thata helitron TE in the ROS1 gene promoter negatively regulatesROS1 expression, and that such negative regulation is antago-nized by RdDM activities. RdDM of a sequence between ROS15′ UTR and the promoter TE positively correlates with ROS1gene expression. In addition, defective active DNA demethyla-tion resulted in hypermethylation of this sequence, concomitantwith enhanced ROS1 gene expression. ChIP analysis of ROS1occupancy at this region confirmed the existence of coregulationbetween DNA methylation and active demethylation. Theseresults provide important insights into how cells sense the bal-ance between DNA methylation and demethylation, and fine-tune active DNA demethylation accordingly.

Significance

DNA methylation is critical for transposon silencing and generegulation. DNA methylation levels are determined by thecombined activities of DNA methyltransferases and demethy-lases. This study found a 39-bp DNA methylation monitoringsequence (MEMS) in the promoter of the DNA demethylaseREPRESSOR OF SILENCING 1 (ROS1) gene of Arabidopsis plants.DNA methylation of MEMS is responsive to both RNA-directedDNA methylation and ROS1-dependent active demethylation.Thus, MEMS can sense DNA methylation and demethylationactivities, and it regulates genomic DNA methylation by adjustingROS1 expression levels. Our results suggest that the ROS1 pro-moter, with the MEMS and an adjacent helitron transposon ele-ment, functions as a “methylstat” that senses and regulatesgenomic DNA methylation levels.

Author contributions: M.L. and J.-K.Z. designed research; M.L., H.Z., and R.J. performedresearch; M.L., H.Z., K.T., S.X., and J.-K.Z. analyzed data; and M.L., H.Z., and J.-K.Z. wrotethe paper.

Reviewers: J.P., The Sainsbury Laboratory, University of Cambridge; and Y.Q., TsinghuaUniversity.

The authors declare no conflict of interest.

Data deposition: The high-throughput sequencing data generated in this study have beendeposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo(accession nos. GSE53033, GSE64499, and GSE58790). An additional dataset used in thisstudy is GEO accession no. GSE44209.1To whom correspondence should be addressed. Email: jkzhu@purdue.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1502279112/-/DCSupplemental.

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Results and DiscussionMutations in ROS1 and Its Regulator Increased DNA Methylation 1Cause the Silencing of Cauliflower Mosaic Virus 35S Promoter::Sucrose Transporter 2 and 2 × Cauliflower Mosaic Virus 35SPromoter::Hygromycin Phosphotransferase II Transgenes. We pre-viously reported an efficient transgene-based genetic screen forcellular antisilencing factors in Arabidopsis (18, 19). In this sys-tem, a sucrose transporter 2 (SUC2) gene was driven by theconstitutive cauliflower mosaic virus 35S promoter (35S) andhygromycin phosphotransferase II (HPTII) gene was driven bythe 2 × 35S promoter in Arabidopsis (Fig. 1A). When grown onsucrose-containing nutrient media, the transgenic plants (there-after referred as WT) absorb too much sucrose from the me-dium, resulting in root growth inhibition (20). In this geneticscreen, we obtained seven new alleles of ROS1 (Fig. 1B and Fig.S1). Quantitative RT-PCR analysis showed that the transcriptlevels of SUC2 and HPTII were drastically reduced in ros1 mu-tant plants (Fig. 1 C and D). Bisulfite sequencing showed that the35S promoter had higher levels of DNA methylation in ros1 thanin the WT, especially in region A, although the promoter wasalready methylated in the WT (Fig. 1E). We also recovered eightnew alleles of increased DNA methylation 1 (IDM1) (Fig. 1Band Fig. S1), a gene necessary for ROS1 function in active DNAdemethylation, by creating a favorable chromatin environment(21). The results show that active DNA demethylation is re-quired for preventing transcriptional silencing of the reportergenes. The large number of new ros1 and idm1 mutant allelesprovides valuable information regarding structure–function rela-tionships for these two important epigenetic regulators.

RdDM Mutations Cause the Silencing of 35S::SUC2 and 2 × 35S::HPTIITransgenes. Our genetic screen also recovered many RdDMmutants, including six new alleles of nrpd1, 2 nrpe1, 2 nrpd2, 5 rdr2,5 dcl3, 1 dtf1, 1 ago4, 1 drm2, 2 drd1, 4 ktf1, 2 idn1, and 3 idn2 (Fig.2A and Figs. S1 and S2). In the RdDM pathway mutants that weretested, the SUC2 and HPTII transgenes were silenced (Fig. 2 Band C and Fig. S3A). Bisulfite sequencing showed that the 35Spromoter is hypermethylated in the nrpd1-12 mutant comparedwith WT (Fig. 1E). Although the RdDM pathway is known to berequired for epigenetic silencing of some transgenes and numerousendogenous loci, including many transposon repeats (22–25), ourforward genetic screen here identified the RdDM as an anti-silencing pathway. The notion of the RdDM pathway also havingan antisilencing role is consistent with a previous report (17).

RdDM Maintains ROS1 Expression to Prevent the Silencing of 35S::SUC2 and 2 × 35S::HPTII Transgenes. Because the ros1 and RdDMmutants were recovered from the same genetic screen, we hy-pothesized that RdDM may prevent the silencing of the 35S::SUC2and 2 × 35S::HPTII transgenes by maintaining ROS1 expression.We examined ROS1 gene expression, and found that ROS1 wasdramatically repressed in all of the tested RdDM mutants (Fig. 2Dand Fig. S3B), which is consistent with previous reports (14, 15, 17).To test whether the SUC2 and HPTII silencing in the RdDMmutants is caused by down-regulation of ROS1, we tried to restoreROS1 mRNA levels in nrpe1-17 plants by transgenic expression ofROS1 with a 2-kb native promoter. The attempt failed, as revealedby the long root phenotype of the ROS1 promoter (PROS1)::ROS1/nrpe1-17 plants on sucrose-containing medium (Fig. 3A). Quanti-tative RT-PCR results showed that the RNA levels of SUC2,HPTII, and ROS1 in these transgenic plants remained low (Fig. 3B).As a control, the PROS1::ROS1 transgene could rescue the rootphenotype of ros1-12 mutant plants (Fig. S4). These results showthat like the endogenous ROS1, the ROS1 transgene requiresRdDM activity for expression. ROS1 gene promoter containsa helitron TE, which is located 155 bp upstream of the 5′ UTR ofROS1. Deletion of this helitron TE from the ROS1 promoter en-abled transgenic expression of PROS1-ΔTE::ROS1 in RdDM mutants,

and consequently suppressed the silencing of SUC2 and HPTII(Fig. 3 A and B). The results confirmed that the antisilencing roleof RdDM is indeed because it is required for ROS1 expression. Theresults also suggest that the helitron TE negatively regulates ROS1gene expression, and that RdDM-dependent methylation is re-quired for ROS1 expression only when this TE is present in ROS1promoter. We hypothesized that an unknown RdDM target se-quence at the ROS1 locus functions to antagonize the TE to ensureROS1 expression.

RdDM Regulates the DNA Methylation Level of DNA MethylationMonitoring Sequence in the ROS1 Promoter. To investigate howRdDM activity may antagonize the helitron TE in regulatingROS1 gene expression, we examined DNA methylation patterns

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Fig. 1. Mutations in ROS1 resulted in transcriptional silencing of markertransgenes. (A) Schematic diagram of the construct harboring the 35S::SUC2 and2 × 35S::HPTII cassettes that were transformed in the parental line. Black rec-tangles represent exons. (B) Comparison of root growth of Col-0, WT, and sevenros1 mutants. Expression of SUC2 (C) and HPTII (D) transgenes in ros1 mutants.Values are mean ± SD of three biological replicates and are normalized totranscript levels in WT. (E) DNA methylation status at the 35S promoter in WT,ros1-13, and nrpd1-12 mutants. The result from whole-genome bisulfite se-quencing is shown. Vertical bars on each track indicate DNA methylation levels.

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in the ROS1 promoter. We performed individual bisulfite se-quencing to compare DNA methylation levels in the region be-tween the 5′ UTR and the helitron TE in WT, nrpd1-12, andnrpe1-17. NRPD1 and NRPE1 encode the largest subunits of PolIV and Pol V, respectively. Dysfunction of Pol IV and Pol V,both of which are major components of the canonical RdDMpathway (2, 6, 7, 26), substantially reduced DNA methylationlevels at the examined region, especially in CHG and CHHcontexts (Fig. 4A). Similar patterns were observed in nrpd1-3 andnrpe1-11 plants compared with their WT control Columbia-0(Col-0), as revealed in whole-genome bisulfite sequencing(Fig. S5A). In the methylated ROS1 promoter region, Pol IV-dependent and Pol V-dependent 24-nt siRNAs were detected bysmall RNA sequencing (27) (Fig. S5B). Consistently, Pol V couldbind to this region, as shown by published NRPE1 ChIP se-quencing data (28) (Fig. S5C). These results demonstrated thatRdDM directly controls DNA methylation in this particular se-quence of ROS1 promoter. Because the DNA hypomethylationin this particular sequence in the RdDM mutants is concom-itant with ROS1 gene repression, we hypothesized that thissequence may serve as an indicator of general RdDM activitiesto allow a dynamic coordination between DNA methylation

and active DNA demethylation through transcriptional regulationof ROS1. Accordingly, we referred to this sequence as the DNAmethylation monitoring sequence (MEMS) in subsequent analyses.

DNA Methylation Level of MEMS Is also Regulated by Active DNADemethylation. We found that DNA methylation at MEMS isalso regulated by ROS1-dependent active demethylation, asshown by the increased DNA methylation levels when ROS1 isdysfunctional (Fig. 4A). In ros1-12 plants compared with WTplants, the levels of DNA methylation in CG, CHG, and CHHcontexts were increased by 287%, 205%, and 196%, respectively(Fig. 4A). Similarly, DNA hypermethylation was observed inros1-4 in comparison to its Col-0 WT control (Fig. S5A), furthersuggesting that MEMS is targeted by ROS1-dependent DNAdemethylation. To confirm the regulation of MEMS by ROS1-dependent DNA demethylation, chromatin occupancy of ROS1at MEMS was examined by ChIP. We used ros1-4 plants com-plemented with transgenic ROS1 that was driven by its nativepromoter and was epitope-tagged with 3× FLAG. As shown inFig. 4B, anti-FLAG antibody detected ROS1 signals at MEMSand its neighboring regions, consistent with the local DNAhypermethylation in ros1 mutants (Fig. 4A and Fig. S5A).Therefore, DNA methylation at MEMS is controlled by bothRdDM and ROS1-dependent active demethylation.We next asked whether DNA hypermethylation at MEMS might

be accompanied by transcriptional elevation of ROS1. Five point-mutation alleles of ros1 mutants, including ros1-11 to ros1-15, wereexamined. Quantitative RT-PCR results showed that ROS1 gene

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Fig. 2. Dysfunctional RdDM caused transcriptional silencing of marker trans-genes. (A) Comparison of root growth in Col-0, WT, and RdDM mutants, in-cluding nrpd1, nrpe1, nrpd2, rdr2, drd1, and ktf1; ros1 and idm1 were includedas controls. Expression of SUC2 (B) and HPTII (C) transgenes and ROS1 (D) in theRdDMmutants is shown. Values are mean ± SD of three biological replicates andare normalized to transcript levels in WT. ktf1, Kow domain-containing tran-scription factor 1.

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Fig. 3. Antisilencing defect in RdDM mutants is rescued by ROS1 transgenedriven by its promoter with TE deletion. (A) Root phenotype of nrpd1 and nrpe1mutants was rescued by ROS1 gene driven by its promoter with deletion of theTE (PROS1-ΔTE::ROS1). (B) Expression of SUC2 and HPTII transgenes in nrpd1 andnrpe1mutants transformed with PROS1-ΔTE::ROS1. Values are mean ± SD of threebiological replicates and are normalized to transcript levels in WT.

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expression was increased in four of the five ros1 mutant alleles, withthe exception of ros1-12, which contains a C-to-T mutation, and thusa premature stop codon in the fourth exon (Fig. 4C and Fig. S1).RNA decay can be triggered by mutations that cause premature stopcodons (29) and may account for a lack of increased RNA levels ofROS1 in ros1-12. The increased ROS1 expression in all other ex-amined ros1 alleles indicates a positive correlation between MEMSDNA hypermethylation and ROS1 gene up-regulation, furthersupporting a role of MEMS in sensing DNA methylation anddemethylation activities and regulating ROS1 expression.DNA methylation in gene promoters is commonly considered as

transcriptionally repressive. However, DNA methylation of MEMShas a positive role in regulating ROS1 gene expression. Unlikepromoter methylation, gene body methylation is preferentially as-sociated with transcribed genes (8, 30). DNA methylation in theROS1 gene body is unaffected in nrpd1-3 and nrpe1-11mutants (Fig.S6), indicating that RdDM regulation of ROS1 gene expression isindependent of DNA methylation in the ROS1 gene body. Mu-tation of ROS1 causes DNA hypermethylation in nearly 5,000genomic regions, many of which are not RdDM targets (21).Thus, ROS1 down-regulation by defective RdDM may causeDNA hypermethylation in these non–RdDM-targeted regions.

In the nrpd1 mutants, CG hypermethylation was observed at the3′ end of the helitron TE that is adjacent to MEMS (Fig. S5A). It isunlikely that ROS1 gene repression in nrpd1 is a consequence of thishypermethylation, because a greater level of CG hypermethylationin the same region exists in ros1mutants (Fig. S5A), which displayedincreased ROS1 gene expression. This conclusion is also supportedby the observation that ROS1 expression is suppressed in met1mutant plants (16), in which CG methylation is abolished. Recently,it was shown that Pol V can be recruited by preexisting CG meth-ylation at RdDM target loci (31). Therefore, dysfunction of MET1may impair RdDM at MEMS, and thereby decrease methylation-dependent ROS1 gene expression. Indeed, DNA methylation atMEMS is lost in met1-3 mutant plants (32) (Fig. S7).The DNA methylation at MEMS and its adjacent region in the

ROS1 promoter tends to be variable (Figs. S5A and S7), althoughour results show that the methylation at MEMS decreases in nrpd1and nrpe1 mutants and increases in ros1 mutants. The reason forthis high variability is presently unclear but may be a reflection ofthe dynamic regulation of DNA methylation at the ROS1 promoterin plants. It is also unclear how the helitron TE negatively regulatesROS1 gene expression. One possibility is that the TE in ROS1promoter causes a chromatin conformation that is unfavorable fortranscription initiation. DNA methylation at the adjacent MEMSmay help attract a transcriptional activator to overcome the tran-scriptional suppression by the TE.Like a thermostat that senses temperature changes and maintains

a stable temperature, a methylstat may exist in plant cells tomonitor DNA methylation levels and accordingly adjust DNAmethylation and/or active demethylation activities to maintaingenomic DNA methylation patterns. Our results suggest thatthe ROS1 promoter functions as a methylstat that senses bothDNA methylation and demethylation activities and accordinglyfine-tunes ROS1 expression levels. In met1-3 plants, CG hypo-methylation at 5S ribosomal DNA is followed by progressiveincreases in CHH methylation levels in successive generationsas a result of RdDM and lack of ROS1 expression, resulting inre-establishment of transcriptional silencing (16). In humans, thegenome-wide DNA methylation landscape is dynamically fine-tuned, as shown by a strong correlation between biological ageand DNA methylation increases at some loci and decreases atother loci (33). It is possible that a similar methylstat may exist inmammals to regulate DNA methylation.

Materials and MethodsPlant Materials. All mutant and transgenic Arabidopsis plants used in thisstudy were in the Col-0 genetic background. Seeds were surface-sterilized in 20%(vol/vol) bleach, rinsed with sterilized water, and sown on half-strength Murashigeand Skoog (MS) medium plates with 1% (wt/vol) agar. The root phenotype wasobserved when 2% (wt/vol) sucrose was added to the medium. When it wasnecessary to avoid the side effects of sucrose accumulation and to monitor otherphenotypes, sucrose was replaced with 1% glucose. After 2 d of stratification at4 °C, plates were moved to a growth chamber at 22 °C with 100 μmol·m−2·s−1

fluorescent light (16 h of light and 8 h of dark).

Screening for Ethyl Methanesulfonate-Mutagenized Mutant Population. M2seeds from ethyl methanesulfonate-mutagenized plants were grown verticallyon half-strength MS medium with 2% (wt/vol) sucrose for 1 wk. In this con-dition, root growth of WT plants was severely inhibited, whereas putativemutants with long roots were selected and transferred to soil. To map themutated genes, mutants were crossed to Landsberg erecta ecotype WTplants. We selected a mapping population of 96 F2 seedlings that showedlong roots when grown on half-strength MS medium with 2% (wt/vol)sucrose. In this medium, hygromycin (20 μg/L) was added to exclude theseedlings that lack the 35S::SUC2 transgene. After rough mapping withbulk segregant analysis, the mutant genome was sequenced to find themutated genes.

Plasmid Construction and Mutant Complementation. For complementation,ROS1 genomic DNA with an ∼2-kb promoter region was amplified fromCol-0 genomic DNA and cloned into the pENTR/D-TOPO entry vector

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Fig. 4. DNA methylation at MEMS is coregulated by RdDM and activedemethylation. (A) Analysis of DNA methylation levels at the 5′ region ofROS1 gene in nrpd1, nrpe1, and ros1 mutants by genomic bisulfite se-quencing. Methylation levels of boxed regions in each mutant were calcu-lated and are shown in the histograms. Black rectangles represent exons. Theempty box represents the UTR. TSS, transcription start site. (B) ROS1 proteinenrichment at ROS1 promoter. ChIP against ROS1-3×FLAG was performed inCol-0 and two lines of ROS1-3×FLAG/ros1-4. All error bars indicate SD (n = 3).(C) Enhancement of ROS1 transcript levels in ros1 mutants.

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(Invitrogen). To delete the sequence corresponding to the helitron transposon,ROS1 entry clone was amplified using the primers listed in Table S1. After it wassequenced, the genomic DNA was recombined with attR sites into thepGWB16 binary vector (34) using LR Clonase (Invitrogen). Agrobacteriumstrain GV3101 carrying various constructs was used to transform the ros1-13,nrpd1-12, and nrpe1-17 mutants by the standard floral dip method (35).

Bisulfite Sequencing of Individual Loci. Two hundred nanograms of genomicDNA was treated with the BisulFlash DNA Modification Kit (Epigentek) fol-lowing the manufacturer’s protocols. Two microliters of bisulfite-treatedDNA was used for each PCR assay (20-μL final volume) using ExTaq (Takara)with primers specific to the corresponding regions (Table S1). PCR productswere cloned into the pGEM-T easy vector (Promega) following the supplier’sinstructions. For each region in each sample, at least 16 independent top-strand clones were sequenced and analyzed.

Real-Time RT-PCR. For real-time RT-PCR, total RNAs were extracted with theRNeasy Plant Kit (Qiagen). From 1 μg of total RNA, cDNAs were generated usingpoly (T) primer and M-MuLV Reverse Transcriptase (New England Biolabs) ina 20-μL reaction mixture. The resulting cDNAs were used as templates for real-time PCR with iQ SYBR green supermix (Bio-RAD). PCR was performed witha CFX96 real-time PCR detection system (Bio-RAD). TUB8 was used as an internalcontrol. All of the primers used are listed in Table S1.

ChIP Assay. ChIP assays were performed as described by Wierzbicki et al. (28).The antibodies used were anti-FLAG (no. F1804; Sigma). ChIP products werediluted with 80 μL of TE buffer, and 2 μL was used for each quantitative PCRreaction. At least two biological replicates were performed.

ACKNOWLEDGMENTS. This work was supported by NIH Grants R01GM070795and R01GM059138 (to J.-K.Z.) and the Chinese Academy of Sciences.

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