exportin 1a prevents transgene silencing in arabidopsis by ......exportin 1a prevents transgene...

EXPORTIN 1A prevents transgene silencing inArabidopsis by modulating nucleo-cytoplasmicpartitioning of HDA6Guohui Zhu1,2, Yanan Chang3,4, Xuezhong Xu2, Kai Tang1,3, Chunxiang Chen3,4, Mingguang Lei3, Jian-Kang Zhu1,3*

and Cheng-Guo Duan1,3*

1. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA2. College of Life Sciences, South China Agricultural University, Guangzhou 510642, China3. Shanghai Center for Plant Stress Biology and Center of Excellence for Molecular Plant Sciences, the Chinese Academy of Sciences, Shanghai201602, China4. The University of Chinese Academy of Sciences, Beijing 100049, China*Correspondences: Jian-Kang Zhu ([email protected]); Cheng-Guo Duan ([email protected], Dr. Duan is fully responsible for the distributions ofall materials associated with this article)doi: 10.1111/jipb.12787

Abstract In eukaryotic cells, transport of macromole-cules across the nuclear envelope is an essential processthat ensures rapid exchange of cellular components,including protein and RNA molecules. Chromatin regu-lators involved in epigenetic control are among themolecules exported across the nuclear envelope, butthe significance of this nucleo-cytoplasmic trafficking isnot well understood. Here, we use a forward screen toisolate XPO1A (a nuclear export receptor in Arabidopsis)as an anti-silencing factor that protects transgenesfrom transcriptional silencing. Loss-of-function ofXPO1A leads to locus-specific DNA hypermethylation at

transgene promoters and some endogenous loci. Wefound that XPO1A directly interacts with histonedeacetylase HDA6 in vivo and that the xpo1a mutationcauses increased nuclear retention of HDA6 protein andresults in reduced histone acetylation and enhancedtransgene silencing. Our results reveal a new mechanismof epigenetic regulation through the modulation ofXPO1A-dependent nucleo-cytoplasm partitioning of achromatin regulator.

Edited by: Zhizhong Gong, China Agricultural University, ChinaReceived Dec. 29, 2018; Accepted Jan. 25, 2019; Online on Jan. 30,2019

INTRODUCTION

DNA cytosine 5’ methylation is a conserved epigeneticmodification that plays vital roles in many biologicalprocesses including development, oncogenesis andresponses to environmental stimuli (Zhu 2009; Smithand Meissner 2013; Matzke and Mosher 2014; Schubeler2015); patterns of DNA methylation are dynamicallyestablished by both methylation and demethylation(Zhu 2009). In plants, cytosine methylation of DNAoccurs in three different contexts: CG, CHG and CHH(where H represents A, T or C). In Arabidopsis, de novoCHH methylation is catalyzed by DRM1 and DRM2through the RNA-directed DNA Methylation (RdDM)pathway (Matzke et al. 2009; Law and Jacobsen 2010).

CG methylation is maintained by MET1 during DNAreplication (Saze et al. 2003; Li et al. 2017), while CMT3, aplant-specific DNA methyltransferase, maintains CHGmethylation (Lindroth et al. 2001). Active DNA demeth-ylation is achieved by a family of bifunctional DNAglycosylases/lyases: ROS1(DML1), DME, DML2 andDML3(Gong et al. 2002; Zhu 2009). Following removal of themethylated cytosine base and subsequent backbonecleavage, the cytosine gap is filled with an unmethy-lated cytosine through the base excision repair (BER)process, which involves several components, includingZDP, APE1L and LIG1 (Qian et al. 2014; Li et al. 2015a; Liet al. 2015b). Although promoter DNA methylationoften has deleterious effects on downstream genes,recent studies reveal that several Arabidopsis Su(var)3-9

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Article

homologs SUVH1 and SUVH3 bind to methylatedpromoter sequences by interacting with three DNAJdomain-containing homologs (SDJ1, SDJ2 and SDJ3) topromote transcriptional activation (Li et al. 2018; Xiaoet al. 2019; Zhao et al. 2019).

In addition to DNA methylation, histone posttrans-lational modifications (PTMs) and chromatin remodel-ing play vital roles in epigenetic regulation (Liu et al.2010): the DDM1 protein (an SNF2 family nucleosomeremodeler) functions in DNA methylation regulation inall cytosine contexts (Long et al. 2018), and theinterplay between histone modification and DNAmethylation is important for transcriptional regulation(Cedar and Bergman 2009; Saze et al. 2012; Du et al.2015). The coordinated roles of the Su(var)3-9 class ofhistone H3K9 methyltransferase KYP and CMT3methyltransferase is one example of this relationship:KYP binds methylated CHG and recruits the CMT3methyltransferase to enhance CHG methylation(Jackson et al. 2002; Jasencakova et al. 2003). Resultsfrom our recent work show that the IDM histoneacetyltransferase complex functions in target recog-nition of ROS1-dependent active DNA demethylation(Duan et al. 2017). In the IDM complex, two DNA-binding proteins, HDP2 and MBD7, coordinate torecognize hypermethylated regulatory DNA sequen-ces and recruit the IDM1 histone acetyltransferase totarget loci for histone acetylation, believed to provideaccessible chromatin states for the recruitment ofROS1 (Qian et al. 2012; Lang et al. 2015; Duan et al.2017). Malfunction of the IDM complex results inreduced acetylation at H3 lysine 18 (H3K18) and lysine23 (H3K23) sites, as well as DNA hypermethylationand enhanced transcriptional gene silencing (TGS).Histone acetylation and deacetylation are catalyzed byacetyltransferases (HATs) and deacetylases (HDACs),respectively (Hollender and Liu 2008), and acetylatedhistone marks are usually associated with transcrip-tional activation (Hollender and Liu 2008; Lu et al.2008; Yuan et al. 2013; Shen et al. 2015). Arabidopsisencodes 18 histone deacetylases which can beclassified into three groups: RPD3-like, HD-tuins andsirtuin (Hollender and Liu 2008). HDA6 (an RPD3-likehistone deacetylase) participates in the regulation ofgene silencing and locus-specific DNA methylationthrough direct interaction with the DNA methyltrans-ferase MET1 (Probst et al. 2004; To et al. 2011; Liu et al.2012). Reduced gene silencing and decreased DNA

methylation are observed in hda6 mutants (Murfettet al. 2001; Aufsatz et al. 2002; Liu et al. 2012).

The nuclear-cytoplasmic trafficking of macromole-

cules is an essential process in eukaryotic cells, because

the nuclear envelope separates transcription and

translation. Although most epigenetic regulators func-

tion in the nucleus, there is evidence to suggest that

nuclear-cytoplasmic trafficking impacts epigenetic reg-

ulators: in humans, the histone deacetylase HDAC1 is

exported from the nucleus by CHROMOSOMAL REGION

MAINTENANCE 1 (CRM1), and this process is indispens-

able for the onset of axonal damage (Kim et al. 2010); in

Arabidopsis, it has been shown that the siRNA-AGO4

complex is assembled in the cytosol and translocated to

the nucleus to where RdDM reactions occur (Ye et al.

2012). In this study, we isolated the novel anti-silencing

factor XPO1A, which functions to prevent transgene

silencing and promote locus-specific DNA demethyla-

tion. We provide evidence that XPO1A interacts directly

with histone deacetylase HDA6, and mediates its

nuclear export. The xpo1a mutation causes increased

nuclear retention of HDA6 protein, which promotes

transgene silencing and locus-specific DNA hyperme-

thylation. Thus, our study reveals a new mechanism by

which DNA methylation is regulated.

RESULTS

XPO1A is an anti-silencing factor that preventstransgene silencingWe previously reported the results from a forwardgenetic screen in Arabidopsis, which identified cellularfactors required for the prevention of transcriptionaltransgene silencing (Wang et al. 2013; Lei et al. 2015). Inthis system, transgenic Arabidopsis plants overexpress-ing Sucrose transporter 2 (SUC2; driven by the CaMV 35Spromoter) were mutagenized by ethyl methanesulfo-nate (EMS). Besides the SUC2 transgene, the 35S-SUC2transgenic plants also contain two 35S promoter-drivenselectivemarker genes: HPTII and NPTII. When grown onsucrose-containing medium, parental 35S-SUC2 trans-genic plants (wild type; WT) show a short-rootphenotype caused by an over-accumulation of sucrosein roots, whereas transgene-silenced mutants grownormally. Repression of 35S-SUC2 transgene silencingrequires ROS1-dependent DNA demethylation. In addi-tion to ROS1, several anti-silencing factors were

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identified using this system, including ANTI-SILENCING 1(ASI1) (Wang et al. 2013), ASI2/MBD7 (Lang et al. 2015),ASI3/MET18 (Duan et al. 2015), EDM2 (Lei et al. 2014),IDM3 (Lang et al. 2015), HDP1 and HDP2 (Duan et al.2017). The expression of the 35-SUC2 transgene is alsounder the control of the RdDM pathway becauseRdDM is necessary for maintaining ROS1 transcription(Lei et al. 2015).

In the current study, we isolated two recessivemutants, asi4-1 and asi4-2, from a population of EMS-mutagenized 35S-SUC2 transgenic plants. Both mutantsdisplayed a long-root phenotype on 1% sucrose-contain-ing medium due to SUC2 transgene silencing (Figure 1A,B). The expression levels of NPTII and HPTII transgeneswere also significantly reduced in asi4-1 and asi4-2mutants (Figure 1B). As expected, asi4 mutants weresensitive to kanamycin (Figure 1C). We performedchromatin immunoprecipitation (ChIP) assays andobserved a reduction in RNA polymerase II (Pol II)

and an increase in the repressive chromatin markH3K9me2 at the 35S-SUC2 promoter region (Figure 1D),implying that the silencing of transgenes was caused byreduced transcription by Pol II. The above resultssuggest that ASI4 is an anti-silencing factor required forthe prevention of transgene silencing.

Through map-based cloning and whole genomeresequencing, we determined that both the asi4-1 andasi4-2 mutations occur at the AT5G17020 gene locus(Figure S1) which encodes the importin b-like nucleartransport receptor protein, XPO1A � an orthologue ofhuman CRM1 (Fornerod et al. 1997; Fukuda et al. 1997;Ossareh-Nazari et al. 1997). A C-to-T substitution at theseventh exon that creates a premature stop codon, anda G-to-A point mutation at the intron/exon border thatimpairs splicing were detected in the asi4-1 and asi4-2mutants, respectively (Figures 2A, S2). We, therefore,named asi4-1 as xpo1a-4 and asi4-2 as xpo1a-5. To confirmthat the transgene silencing phenotype of asi4 mutant

Figure 1. Characterization of asi4 mutants(A) Root growth phenotype ofwild type (WT) (35S-SUC2), asi4-1 and asi4-1 on 1/2MSmedium containing 1% sucrose or1% glucose. (B) Relative expression levels of SUC2, NPTII and HPTII in WT, asi4-1 and asi4-2 on 1% sucrose mediumsupplemented with DMSO (negative control) or 5mM 5’Aza. (C) Kanamycin (kan)-sensitivity assay. WT, asi4-1 andasi4-2 were cultured on 1/2 MS medium containing 1% glucose (top) or supplemented with 50mg/L of kanamycin(middle) or kanamycin and 5mM5’Aza (bottom). (D) Chromatin immunoprecipitation (ChIP) measurements of Pol IIoccupancy, histone H3K4me3 and H3K9me2 levels at the 35S promoter region of 35S-SUC2 transgene. 35S-1 and 35S-2 represent the upstream and downstream regions of 35S promoter, respectively (Table S1).

XPO1A-mediated nucleo-ctyoplasmic transport of HDA6 3


was caused by XPO1A dysfunction, a genetic comple-mentation assay was performed by transforming nativepromoter-driven genomic DNA of XPO1A fused with a4xMYC tag at the C terminus. A short-root phenotypeon sucrose medium, and restoration of transgeneexpression were observed in pXPO1A::XPO1A-4MYCtransgenic plants (Figure 2B, C), suggesting that thepXPO1A::XPO1A-4MYC transgene complemented thetransgene-silencing phenotype of the asi4 mutant.The expression level of ROS1 was not significantlychanged in xpo1a mutants (Figure S3), suggesting thatASI4/XPO1A may be not a canonical RdDM component.

XPO1A dysfunction results in DNA hypermethylationin transgene promoter regions and specificendogenous lociThe fact that ROS1-dependent DNA demethylation isinvolved in transgene silencing prompted us to

investigate whether the transgene silencing caused

by XPO1A dysfunction is also associated with DNA

methylation. As expected, both the transgene silencing

and kanamycin resistance defects in xpo1a mutants

were rescued by the treatment of DNA methyltransfer-

ase inhibitor 5-aza-2’-deoxycytidine (5-Aza; Figure 1B, C),

suggesting that DNA methylation participates in the

transgene silencing caused by xpo1a mutations. To

assess the effect of XPO1A dysfunction on genome-wide

DNA methylation, whole genome bisulfite sequencing

(WGBS) was performed. We found that the DNA

methylation level in the promoter region of 35S-SUC2

transgene was markedly increased in the xpo1a-4

mutant compared to wild type (WT), especially in the

upstream region (region A, Figures 3A, S4), indicating

that transgene silencing caused by XPO1A dysfunction

may be caused by increased DNA methylation at the

Figure 2. ASI4 encodes the nuclear transport receptor XPO1A(A) Schematic structure of the XPO1A gene and mutation positions of asi4-1 (xpo1a-4) and asi4-2 (xpo1a-5). The asi4-1mutation is a C-to-T mutation in the 7th exon, and the asi4-2mutation is a G-to-A mutation at the 1st intron/2nd exonboundary. Black boxes represent exons, gray boxes represent UTR regions, and lines represent introns. AdditionalT-DNA insertion alleles of XPO1A, xpo1a-1 and xpo1a-3 are also shown. (B) Root phenotypes of the asi4-1 plantscomplementedwith pXPO1A::XPO1A-4MYC grown for 10 d on 1/2MSmedium containing 1% sucrose.Western blottingwas performed to detect transgenic lines using anti-MYC antibody. (C) Relative expression levels of SUC2 and NPTIIin WT, xpo1a-4 and complemented lines grown on 1% sucrose medium. Significant differences were determined byStudent’s t-test (��, P < 0.01).

4 Zhu et al.


transgene promoter region. We next examined DNAmethylation changes in selected endogenous lociknown to be regulated by ROS1-mediated DNAdemethylation (Duan et al. 2017). WGBS analysisindicated an increase in DNA methylation levels at theAT1G26380 and AT1G26410 loci of asi4-1 mutants, but noobvious changes at the AT1G26390 and AT1G26400 loci.To validate these observations, DNA methylation-sensitive PCR was performed in the xpo1a-4 mutant(which is in the 35S-SUC2 background), as well as inxpo1a-1 and xpo1a-3 (two T-DNA insertion mutants in the

Col-0 background characterized in previous studies)

(Blanvillain et al. 2008). Consistent with WGBS analysis,

DNA hypermethylation was observed at the AT1G26380

and AT1G26410 loci in both the 35S-SUC2 transgene-

derived and T-DNA insertion xpo1a mutants compared

to their respective controls (Figure 3B).

The effect of XPO1A dysfunction on genome-wideDNA methylation statusWe next assessed the effect of xpo1a mutations onthe status of genome-wide DNA methylation: WGBS

Figure 3. DNA methylation profile of xpo1a-4 mutant(A) Diagram of the 35S-SUC2 transgene and an IGB snapshot of DNAmethylation levels in the 35S promoter region indifferent cytosine contexts. The positions of the primer pairs (35S-1 and 35S-2) for ChIP-qPCR are as labeled. (B) DNAmethylation-sensitive PCR (Chop-PCR) determines the DNAmethylation level of selected genes in the xpo1amutants.ros1-13 (35S-SUC2background) andT-DNA insertionmutant ros1-4 (Col-0background)wereusedasDNAdemethylationmutant controls, and nrpe1-12 (35S-SUC2 background) was used as DNA methylation mutant control. (C and D) Thenumber (C) and composition (D) of hyper-DMRs and hypo-DMRs in xpo1a-4 compared to WT. TE representstransposable element. (E) DNA methylation levels of three endogenous TE loci calculated from WGBS data (upperpanel), and relative expression levels (normalized to ACTIN2 expression) of these TEs in WT and xpo1a-4. (F) AverageDNA methylation levels in the genes (top) and TEs (bottom) bodies and their flanking sequences (þ/�2 kb).



identified 3,303 hyper-DMRs (differentially methylated

regions) in xpo1a-4mutants, most of which are mapped

to the CHH context and TE regions (Figure 3C, D). We

tested the transcript levels of several selected TE genes

with increased DNA methylation in xpo1a-4. RT-qPCR

analysis showed that the transcript levels of

AT1TE98060, AT1TE38340, and AT5TE42900 were re-

duced in xpo1a-4 compared to WT, indicating that DNA

hypermethylation caused by xpo1a mutation leads to

enhanced silencing of endogenous TEs (Figure 3E).

WGBS analysis further identified 3,096 hypo-DMRs in

xpo1a-4mutants, which also display a preference for the

CHH context and TE regions (Figure 3C, D, F). The

comparable numbers of hyper- and hypo-DMRs in xpo1-

4 suggest that XPO1A may affect DNA methylation

through different pathways.

XPO1A dysfunction results in increased retention ofHDA6 in the nucleusXPO1A is an importin b-like nuclear transport receptorand mediates both export and import of nuclearproteins (Haasen et al. 1999; Conway et al. 2015).Consistent with its function, XPO1A was found to bepresent both in the nucleus and cytosol (Figure S5). Wespeculate that XPO1A may prevent transgene silencingby maintaining proper subcellular localization of anti-silencing factors. In mammals and plants, some histonedeacetylases are known to be subjected to nucleocyto-plasmic trafficking through the CRM1/XPO1-mediatedprotein transport mechanism (Khochbin et al. 2001;Hollender and Liu 2008). HumanHDAC1 is exported fromthe nucleus by CRM1/XPO1 and this process is essentialfor the onset of axonal damage (Kimet al. 2010). HDA6 isa mammalian HDAC1 homolog in Arabidopsis and playsan important role in transcriptional gene silencing(Murfett et al. 2001; Probst et al. 2004). To explorewhether HDA6 may be transported by XPO1A inArabidopsis, we examined the protein interactionbetween HDA6 and XPO1A. Results from yeast twohybrid (Y2H) and split luciferase assays in Nicotianabenthamiana leaves indicate that XPO1A directly inter-acts with HDA6 (Figure 4A, B). To confirm the XPO1A-HDA6 interaction, a co-immunoprecipitation assay wasconducted using F1 plants generated from a crossbetween pXPO1Ap::XPO1A-4MYC and HDA6-GFP.Westernblotting analysis showed that HDA6 could be specificallypulled down by XPO1A (Figure 4C), supporting thehypothesis that XPO1A and HDA6 interact in vivo.

We next examined whether XPO1A may direct thenuclear export of HDA6. To this end, YFP-fused HDA6(HDA6-YFP) was transiently expressed in xpo1a-4 andWT protoplasts. YFP signals were more concentrated inthe nucleus in xpo1a-4 than in WT (Figure 4D), implyingthat XPO1A dysfunction causes increased retention ofHDA6 in the nucleus. We reasoned that the increasednuclear accumulation of HDA6 protein may repress thetranscription of transgenes by increasing histonedeacetylation. To test this, a histone H3ac ChIP assaywas conducted in WT and xpo1a mutants. Thequantitative PCR results indicated that the enrichmentof H3ac mark was significantly reduced in xpo1a-4mutant compared to WT plants. In contrast, nosignificant change was observed at the ACTIN7 locusbetween WT and xpo1a-4 mutant plants. The reductionof H3ac mark at the transgene promoter is consistentwith the higher accumulation of HDA6 in xpo1amutants.This result suggested that transgene silencing in xpo1amutants is at least partially due to enhanced histonedeacetylation resulting from impairment of HDA6export caused by XPO1A dysfunction.

DISCUSSION

Nucleo-cytoplasmic trafficking of macromolecules is anessential process that ensures the rapid exchange ofmaterials and information between the nucleus andcytoplasm. During the nuclear-cytoplasmic trafficking ofmacromolecules, nuclear transport receptors directlybind to cargo proteins and GTP-bound Ran protein(RanGTP) (Merkle 2011). Exportin 1 (XPO1) was firstidentified in the fission yeast as CRM1, which mediatesthe nuclear export of proteins that contain a leucine-rich nuclear export signal (NES) (Fornerod et al. 1997;Fukuda et al. 1997; Ossareh-Nazari et al. 1997).Arabidopsis encodes two XPO1 proteins: XPO1Aencoded by At5g17020, and XPO1B encoded byAt3g03110 (Haasen et al. 1999). While single mutantsof either gene display normal development, xpo1axpo1bdouble mutants are gametophytic lethal, indicating thatXPO1A and XPO1B function redundantly to regulateplant development (Blanvillain et al. 2008). In this work,we discovered that XPO1A promotes proper expressionof transgenes and some endogenous genes throughrepression of DNA hypermethylation. We providedevidence that XPO1A represses DNA hypermethylation

6 Zhu et al.


at least partially by promoting the nuclear export of thesilencing factor HDA6. XPO1A directly interacts withHDA6 (Figure 4A–C), and xpo1a mutations lead toincreased retention of HDA6 in the nucleus, which isexpected to increase DNA methylation and transgenesilencing. Although the methylome of the had6 mutant

is not available in our study, we used public hda6-7methylome data (Stroud et al. 2013) to examine theDNA methylation level in several known XPO1A DNAmethylation target loci, including AT1G26380, AT1G26410and AT1TE98060, and observed a remarkable increase inDNAmethylation in the xpo1amutant (Figure 3B, E). We

Figure 4. XPO1A dysfunction increases the nuclear retention of histone deacetylase HDA6(A–C) XPO1A directly interacts with HDA6. For the Y2H assay (A), XPO1A was fused to the GAL4 binding domain(BD), and HDA6 was fused to the GAL activation domain (AD). The interaction of XPO1A and Ran1 protein wasused as the positive control. The split luciferase complementation assay (B) was conducted in N. benthamianaleaves. XPO1A was fused to the C-terminus of luciferase (cLUC), and HDA6 was fused to nLUC, or vice-versa.Empty vectors were used as negative controls. Co-IP assay (C) was performed in crossed transgenic plants ofMYC-tagged XPO1A and GFP-tagged HDA6 using an anti-MYC antibody. The input and output protein levels weredetected by western blotting using anti-MYC and anti-GFP antibodies. (D) XPO1A dysfunction affects thenucleocytoplasmic partitioning of HDA6. 35S:HDA6-YFP was transiently expressed in Arabidopsis protoplasts ofWT and xpo1a-4, respectively. GFP intensity in the nucleus and protoplasts was calculated by Image J software.Mean� SD values were determined from three replicates (n¼ 60). Significant differences were determined byStudent’s t test (�, P< 0.01). (E) Effect of xpo1amutation on histone H3Ac levels at the 35S promoter. The densityof H3Ac was determined by ChIP-qPCR using an anti-H3Ac antibody. The ChIP signal was quantified relative tohistone H3. Three biological replicates were performed and similar results were obtained. Mean� SD valueswere determined from three technical replicates. Significant differences were determined by Student’s t test(�, P< 0.01).



found the DNA methylation level to be significantlyreduced in the hda6-7mutant compared to its wild-typecontrol (Figure S6), suggesting that these selected lociare commonly targeted by XPO1A and HDA6.

We found that DNA hypermethylation caused byxpo1a mutation is locus-specific and does not affectgenome-wide DNA methylation patterns, althoughthousands of DMRs are identified in xpo1a mutants(Figure 3C). XPO1B may compensate for the lack ofXPO1A in xpo1a mutant plants in mediating HDA6export, considering their high similarity and functionalredundancy in plant development (Haasen et al. 1999;Blanvillain et al. 2008). Indeed, some cytoplasmicaccumulation of HDA6 protein is still observed in thexpo1a mutant (Figure 3D), suggesting that nuclearexport of HDA6 protein is not completely blocked bythe xpo1a mutation. We noticed that XPO1B RNA levelwas increased in xpo1a (Figure S7), perhaps as acompensatory mechanism. More than 700 proteins inyeast and 1,050 proteins in humans have been identifiedas substrates of XPO1/CRM1, and may function in avariety of biological processes (Kirli et al. 2015).Therefore, it is possible that XPO1A also participatesin the nuclear export of other DNA methylation-relatedfactors. The DNA methylation pattern caused by xpo1amutations may reflect the combined effect of failednuclear export of multiple epigenetic factors. Inaddition, XPO1A may affect DNA methylation and

transgene silencing through mechanisms unrelated toits nucleo-cytoplasmic trafficking function.

As one of the 16 Arabidopsis histone deacetylases,HDA6 functions in the maintenance of transcriptionalgene- and TE silencing (Probst et al. 2004). One of thecharacterized mechanisms involves direct interplaybetween HDA6 and MET1 (Liu et al. 2012). Loss-of-function of HDA6 leads to the release of gene silencingand reduced DNA methylation levels (Murfett et al.2001; Aufsatz et al. 2002; Liu et al. 2012). In this study,xpo1a mutations lead to increased retention of HDA6protein in the nucleus which causes DNA hyper-methylation at transgene promoters; a schematicmodel is presented in Figure 5. Although most of theliterature involving HDA6 is focused on its nuclear roles,we cannot rule out the possibility that HDA6 may alsofunction in the cytoplasm. In fact, a previous reportshowed that pathological, condition-induced nuclearexport of HDAC1 mediated by CRM1/XPO1 is vital for theonset of axonal damage in humans (Kim et al. 2010).Moreover, it has been shown that HDAC1 and HDAC3interact with IkappaBalpha in the cytoplasm to mediatetranscriptional regulation in human cells (Viatour et al.2003). The aforementioned evidence suggests that thenucleo-cytoplasmic shuttle plays vital roles in thefunctioning of some epigenetic factors. It will be ofinterest to determine the potential functions of HDA6 inthe cytoplasm in future studies.

Figure 5. Hypothetical model of transgene silencing and DNA hypermethylation caused by XPO1A mutationIn wild-type plants XPO1A interacts with HDA6 and its mediates nuclear export. The xpo1a mutation results inincreased retention of HDA6 protein in the nucleus, thereby reducing histone acetylation and enhancing DNAmethylation in transgene promoters, leading to silencing of the transgene. In both WT and xpo1a mutant plants,XPO1B functions redundantly to mediate the nuclear export of HDA6.

8 Zhu et al.


MATERIALS AND METHODS

Plant materials and growth conditionsAll Arabidopsis plants used in this study were in the

Columbia-0 (Col-0) genetic background. Wild type (WT;

Arabidopsis thaliana L.) refers to transgenic plants

expressing SUC2 driven by CaMV 35S promoter. The

T-DNA insertion mutants xpo1a-1 (SALK_028886) and

xpo1a-3 (SALK_078639) were purchased from ABRC

(http://www.arabidopsis.org). Seeds were stratified at

4°C for 2 d and germinated at 22°C in a standard growth

chamber with a 16 h/8 h light/dark cycle. For root

phenotypic observation, seeds were cultured in 1/2-

strengthMurashige and Skoog (MS)medium containing

1% sucrose. Otherwise, the seeds were grown in 1/2 MS

medium containing 1% glucose. 50mg/L of kanamycin

and 5mM 5’ Aza-2’-deoxycytidine (Sigma) were added

into 1/2 MS medium for kanamycin sensitivity experi-

ment and DNA methylation inhibitor treatment,

respectively.

Screening the anti-silencing mutants and gene cloningAnti-silencing mutants were identified using an EMS

mutagenized population from the 35S-SUC2 background

according to our previous report (Wang et al. 2013).

Briefly, M2 mutants were grown on 1/2 MS medium

containing 1% sucrose and seedlings with roots longer

than WT were selected as potential transgene silencing

mutants. To clone the ASI4 gene, the mutants of asi4-1

and asi4-2were crossed with Landsberg erecta (Ler) and

the F2 generation was planted on 1/2 MS medium

containing 1% sucrose. Seedlings with long and normal

root growth were selected for map-based cloning. The

whole genomic DNA of themutant was re-sequenced to

determine the exact mutated bases in the mapping

region.

Plasmid construction and plant transformationThe complemented plasmids were constructed inGateway-compatible vectors system (Invitrogen). Togenerate the complemented lines of asi4-1, genomicDNA of XPO1A containing the native promoter fragmentwas amplified and cloned into the pENTR-TOPO entryvector (Invitrogen), and then recombined into thepGWB16 vector to form pXPO1A::XPO1A-4MYC plasmid.The pXPO1A::XPO1A-4MYC-containing Agrobacteriumwas then transformed into asi4-1 using the floral dipmethod.

DNA methylation analysis2-week-old xpo1a-4 (asi4-1) seedlings were used for

whole genomic bisulfite sequencing. Genomic DNA was

extracted and sent to BGI (Shenzhen, China) for

bisulfite treatment, library preparation and Illumina

sequencing. Low-quality sequences (q< 20) were

trimmed using “trim” in BRAT-BW (Harris et al. 2012),

and clean reads were mapped to the TAIR10 genome

using BRAT-BW allowing two mismatches. To remove

potential PCR duplicates, the “remove-dupl” command

of BRAT-BW was used. DMRs were identified according

to Stroud et al. with minor modification. In brief, only

cytosines with 4� coverage in all libraries were

considered. 100 base pair bins were divided from the

genome, and methylated versus unmethylated cyto-

sines for each bin were calculated via Fisher’s: absolute

methylation difference in bins corresponding to CG,

CHG, CHH was 0.4, 0.2, 0.1, respectively. Bins with

Benjamini-Hochberg corrected FDR< 0.01, and at least

four cytosines in the corresponding context were

selected. WT data were downloaded from our pub-

lished methylome data (Gene Expression Omnibus

accession number GSE58787, www.ncbi.nlm.nih.gov/

geo; Lang et al. 2015). The whole-genome bisulfite

sequencing data of xpo1a-4 (asi4-1) has been deposited

to NCBI’s Gene Expression Omnibus (GSE119098).For DNA methylation-sensitive PCR experiment,

genomic DNA was extracted from 2-week-old seedlingsusing the DNeasy Plant Minikit (QIAGEN) and digestedwith RNase I. Approximately 50 ng of plant DNA wasincubated with DNA methylation-sensitive restrictionenzymes and then used as template for PCR. Undi-gested genomic DNA was used as a control.

Quantitative RT-PCRTotal RNA was extracted using an RNeasy Plant Kit(Qiagen) and then digested with DNase I to eliminategenomic DNA contamination. One microgram of RNAwas used for reverse transcription using SuperScriptfirst-strand synthesis system (Invitrogen). The transcrip-tion level of the selected gene was measured by iQSYBR green supermix (Bio-Rad). Data were normalizedto amplification of the Arabidopsis ACTIN2 gene.

ChIP assays2-week-old seedlings were used for ChIP assays accord-ing to a previous study (Saleh et al. 2008). Briefly,approximately 1 g of seedlings was crosslinked by



http://www.arabidopsis.org

http://www.ncbi.nlm.nih.gov/geo

http://www.ncbi.nlm.nih.gov/geo

vacuum infiltration of 1% formaldehyde. The nuclei wereisolated and the chromatin was sonicated to a size ofapproximately 250 bp. The antibodies used for Immuno-precipitation were listed as follows: anti-Pol II (Abcam,ab817), anti- H3AC (Abcam, ab8580) and anti-H3K9me2(Wako, 308-32361). The ChIP product was eluted into50mL of TE buffer and diluted 110 folds for qPCR.

Protein interaction analysisFor Y2H constructs, the coding region of HDA6 was

cloned into the pGAD-T7 vector to generate the

AD-HDA6 plasmid, and XPO1A coding sequence was

introduced into pGBK-T7 to generate BD-XPO1A. Trans-

formed Y2H Gold cultures were spotted onto SD plates

lacking Trp and Leu (SD/ �TL) or lacking Trp, Leu, His

and Ade (SD/ �TLHA), and incubated at 30°C for 3 d to

observe yeast growth. For split luciferase assays, the

coding regions of XPO1A and HDA6 were inserted into

the pENTR-TOPO gateway vector (Invitrogen) and then

recombined into amodified pEarleyGate vector carrying

the split luciferase coding sequence (Earley et al. 2006).

Split luciferase constructs were introduced into Agro-

bacterium strain GV3101 and infiltrated into N. ben-

thamiana leaves. After 2 d, the luciferase activity was

monitored using a CCD camera equipped with Winview

software (Princeton instruments). For the Co-IP assay,

pXPO1A::XPO1A-4MYC and HDA-GFP transgenic plants

were crossed, and the F3 homologous plants were used

for Co-IP assay. Anti-MYC agarose beads (Sigma-Aldrich)

were used to pull-down XPO1A, and the complex was

detected by anti-GFP antibodies.

Transient expression and YFP fluorescence assayFor transient expression, the HDA6-YFP fusion protein

driven by 35S promoter plasmid was expressed intoArabidopsis protoplasts extracted fromWT and xpo1a-4.

YFP fluorescence signal was visualized under confocal

microscope (Leica sp5, Germany) after overnight

incubation. YFP intensity in the nucleus and protoplasts

was calculated by Image J software.

ACKNOWLEDGEMENTS

We thank Dr. Keqiang Wu (National Taiwan University)for providing the HDA6-GFP seeds. The work of C.G.D.was supported by the Strategic Priority ResearchProgram of the Chinese Academy of Sciences

(XDB27040203). J.K.Z. was supported by ChineseAcademy of Sciences, and G. Z. was supported by theNationalNatural ScienceFoundationofChina (31570250).

AUTHOR CONTRIBUTIONS

G.Z., C.G.D. and J.K.Z. designed this study. G.Z., Y.C.,

X.X., M.L. and X.X. conducted the experiments. K.T., C.C.

and C.G.D analyzed the data. C.G.D. and J.K.Z. wrote the

manuscript; all authors edited the manuscript and

approved of its content.

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SUPPORTING INFORMATION

Additional Supporting Information may be found onlinein the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.12787/suppinfoFigure S1. Map-based cloning of ASI4 geneThe recombination frequencies of the genetic mappingare shown here. There is a C-to-T mutation in asi4-1andthus produces a stop codon, and a G-to-A mutation inasi4-2 which produces wrong splicing.Figure S2. asi4-2 mutation leads to wrong splicing ofXPO1A transcriptTwo different transcripts were detected in asi4-2compared to WT. Total RNA was extracted from 10-d-old WT and asi4-2 seedlings and then was reversetranscribed as PCR template.Figure S3. Relative expression levels of ROS1 in xpo1amutantsxpo1a mutations do not affect the expression levels ofROS1 mRNA. Data were normalized to amplification ofthe Arabidopsis ACTIN2 gene.Figure S4. xpo1a mutation increases DNA methylationlevels in the promoter region of 35S::SUC2 transgeneDNA methylation levels at region A and region B of35S-SUC2 promoter are calculated from WGBS data.Region A and B are marked in Figure 3.FigureS5.XPO1A localizes tobothnucleus and cytoplasm(A) XPO1A-YFP fusion protein (driven by 35S promoter)was transiently expressed in Arabidopsis protoplasts,and YFP fluorescence was detected after 14 h oftransformation. YFP-NbFib2 (driven by 35S promoter)was co-transformed as a nucleolar localization marker.(B) Detection of GFP fluorescence in the root tip of35S-XPO1A-GFP/xpo1a-3 transgenic plants.Figure S6. hda6 mutant displays reduced DNA methyl-ation level in selected XPO1A target lociA snapshot of published DNA methylome (Stroud et al.2013) showing that DNA methylation levels are reducedin had6-7mutant in comparison with its wild-type in thetested XPO1A target loci (Figure 3C, E).Figure S7. xpo1a mutation increases the expression ofXPO1B geneTotal RNA was extracted from 10-d-old Col-0 and xpo1a-3 (Col-0 background) seedlings and mRNA levels ofXPO1A and XPO1B were determined by quantitativeRT-PCR analysis.Table S1. Primers used in this study

12 Zhu et al.


http://onlinelibrary.wiley.com/doi/10.1111/jipb.12787/suppinfo

http://onlinelibrary.wiley.com/doi/10.1111/jipb.12787/suppinfo

http://onlinelibrary.wiley.com/doi/10.1111/jipb.12787/suppinfo/













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