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The SUMO E3 ligase-like proteins PIAL1 and PIAL2 interact with MOM1 and form a novel complex required for transcriptional silencing

Yong-Feng Han1*, Qiu-Yuan Zhao1,2*, Liang-Liang Dang1*, Yu-Xi Luo1, Shan-Shan Chen1, Chang-Rong

Shao1, Huan-Wei Huang1, Yong-Qiang Li1, Lin Li 1, Tao Cai1, She Chen1, Xin-Jian He1,2

1 National Institute of Biological Sciences, Beijing 102206, China 2 Graduate School of Peking Union Medical College, Beijing 100730, China * These authors contributed equally.

Running title: SUMO ligase-like proteins in transcriptional silencing

Key words: SUMO; PIAL1; PIAL2; MOM1; transcriptional silencing; DNA methylation

Synopsis: Two Arabidopsis SUMO E3 ligase-like proteins interact with MOM1 and form a complex that

mediates transcriptional silencing independently of changes in DNA methylation.

Corresponding author: Xin-Jian He

Tel: 86-10-80707712

Fax: 86-10-80707715

E-mail: [email protected]

Abstract

The mechanism by which MORPHEUS’ MOLECULE1 (MOM1) contributes to transcriptional gene

silencing has remained elusive since the gene was first identified and characterized (Amedeo et al.,

2000). Here, we report that two Arabidopsis thaliana PIAS (PROTEIN INHIBITOR OF ACTIVATED

STAT)-type SUMO E3 ligase-like proteins PIAL1 and PIAL2 function redundantly to mediate

transcriptional silencing at MOM1 target loci. PIAL1 and PIAL2 physically interact with each other and

with MOM1 to form a high-molecular-weight complex. In the absence of either PIAL2 or MOM1, the

formation of the high-molecular-weight complex is disrupted. We identified a previously

uncharacterized IND (interacting domain) in PIAL1 and PIAL2, and demonstrated that IND directly

interacts with MOM1. The CMM2 (conserved MOM1 motif 2) domain of MOM1 was previously

shown to be required for the dimerization of MOM1. We demonstrated that the CMM2 domain is also

required for the interaction of MOM1 with PIAL1 and PIAL2. We found that although PIAL2 has

SUMO E3 ligase activity, the activity is dispensable for PIAL2’s function in transcriptional silencing.

This study suggests that PIAL1 and PIAl2 act as components of the MOM1-containing complex to

mediate transcriptional silencing at heterochromatin regions.

Plant Cell Advance Publication. Published on April 25, 2016, doi:10.1105/tpc.15.00997

©2016 American Society of Plant Biologists. All Rights Reserved.

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INTRODUCTION

In eukaryotic genomes, transposable elements, DNA repeats, and exogenous transgenes are usually

subjected to transcriptional silencing (Slotkin and Martienssen, 2007). In Arabidopsis thaliana,

transcriptional silencing is accompanied by DNA methylation and repressive histone marks (Law and

Jacobsen, 2010; Matzkeand Mosher, 2014; Du et al., 2015). Components involved in DNA methylation

and repressive histone modification were previously identified and demonstrated to be required for

transcriptional silencing (Law and Jacobsen, 2010; Matzkeand Mosher, 2014; Du et al., 2015). The

DNMT1-like DNA methyltransferase MET1 maintains DNA methylation at symmetric CG sites during

DNA replication (Ronemus et al., 1996; Jones et al., 2001). DDM1, a SNF2-type chromatin-remodeling

protein, allows DNA methyltransferases to access H1-containing heterochromatin (Zemach et al., 2013).

The plant-specific DNA methyltransferase CMT3 specifically contributes to DNA methylation at CHG

sites (Lindroth et al., 2001). The histone methyltransferases SUVH4/KYP, SUVH5, and SUVH6

mediate H3K9 dimethylation and act together with CMT3 to facilitate a self-reinforcing loop between

CHG methylation and H3K9 methylation (Jackson et al., 2002; Ebbs and Bender, 2006; Du et al., 2015).

DNA methylation at CHH sites is established and maintained by DRM2, a DNMT3-like DNA

methyltransferase, and CMT2, a plant-specific DNA methyltransferase (Cao and Jacobsen, 2002; Stroud

et al., 2014; Zemach et al., 2013).

To mediate de novo DNA methylation, DRM2 is guided by an RNA-directed DNA methylation

(RdDM) pathway (Law and Jacobsen, 2010; Matzke and Mosher, 2014). In the RdDM pathway, 24-

nucleotide small interfering RNA (siRNA) mediates de novo DNA methylation (Hamilton et al., 2002;

Aufsatz et al., 2002; Matzke and Mosher, 2014). DNA-dependent RNA polymerases IV and V (Pol IV

and Pol V) are two multi-subunit DNA-dependent RNA polymerases that are unique to plants (Ream et

al., 2009; Huang et al., 2009; Law and Jacobsen, 2010). NRPD1 and NRPE1 are the largest subunits of

Pol IV and Pol V, respectively (Herr et al., 2005; Kanno et al., 2005; Pontier et al., 2005; Ream et al.,

2009; Huang et al., 2009). The generation of 24-nt siRNA is dependent on Pol IV, RNA-dependent RNA

polymerase 2 (RDR2), and Dicer-like 3 (DCL3) (Haag and Pikaard, 2011; Xie et al., 2004). The 24-nt

siRNA is loaded into Argonaute 4 (AGO4) and forms an AGO4-siRNA complex that is thought to guide

DRM2 to homologous genomic loci (Pontes et al., 2006; Zhong et al., 2014). Long non-coding RNA

produced by Pol V is believed to base-pair with the AGO4-bound siRNA and is required for the

occupancy of AGO4-siRNA on chromatin (Wierzbicki et al., 2008; Haag and Pikaard, 2011).

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Alteration of DNA methylation is not always required for transcriptional silencing in Arabidopsis

(Amedeo et al., 2000; Vaillant et al., 2006). The plant-specific CHD3-like protein MOM1 was identified

by a forward genetic screen as a unique component in transcriptional silencing (Amedeo et al., 2000). In

the mom1 mutant, transcriptional silencing is released even when DNA methylation is not affected

(Amedeo et al., 2000; Vaillant et al., 2006), suggesting that MOM1 mediates transcriptional silencing

through a mechanism that is different from DNA methylation. Although MOM1 mediates H3K9

dimethylation at a few target loci shared by RdDM and MOM1 (Numa et al., 2010), the involvement of

MOM1 in transcriptional silencing at the whole-genome level is independent of changes not only in

DNA methylation but also in histone H3K9 dimethylation (Probst et al., 2003; Vaillant et al., 2006). To

understand how MOM1 contributes to transcriptional silencing, a series of truncated MOM1 sequences

were transformed into the mom1 mutant for complementation assays (Caikovski et al., 2008). The results

indicated that the conserved C-terminal CMM2 domain is necessary and sufficient for transcriptional

silencing at some MOM1 target loci and that the putative SNF2 and DNA helicase domains in MOM1

are dispensable for transcriptional silencing. The CMM2 domain exists in an anti-parallel coiled-coil

structure and forms a homodimer required for transcriptional silencing (Nishimura et al., 2012). These

results suggest that MOM1 most likely acts as an adaptor component in a multi-subunit complex rather

than as an ATP-dependent chromatin-remodeling enzyme. The molecular mechanism by which MOM1

contributes to transcriptional silencing remains to be elucidated.

Small ubiquitin-related modifier (SUMO) is a conserved posttranscriptional modification in

eukaryotes. SUMO proteins are covalently attached to substrate proteins through the activities of an

enzyme cascade composed of E1 (SUMO activating enzyme, SAE), E2 (SUMO conjugating enzyme,

SCE), and E3 (SUMO ligase) (Gareau and Lima, 2010). In yeast and animals, SUMO modification is

involved in various biological processes including chromatin organization and transcriptional regulation

(Shin et al., 2005; Shiio et al. 2003; Nathan et al., 2006; Cobenas-Potts and Matunis, 2013).

Noncovalent interaction of SUMO proteins with chromatin-associated proteins is also required for

SUMO-dependent transcriptional regulation (Stielow et al., 2008; Ouyang et al., 2009; Cobenas-Potts

and Matunis, 2013). In Arabidopsis, mass spectrometric analysis of purified sumoylated proteins

demonstrated that many SUMO substrates are involved in chromatin structure regulation, transcription,

and RNA metabolism (Budhiraja et al., 2009; Miller et al., 2010). Moreover, several chromatin-

associated proteins were identified by a yeast two-hybrid screen as SUMO-interacting proteins (Elrouby

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et al., 2013). It is unknown, however, whether and how SUMO proteins and SUMO-conjugating

enzymes contribute to transcriptional silencing in plants.

Arabidopsis has three SUMO E1 activating enzymes (SAE1a, SAE1b and SAE2), two SUMO E2

conjugating enzymes (SCE1a and SCE1b), and four SUMO E3 ligases (SIZ1, HPY2, PIAL1, and

PIAL2) (Kurepa et al., 2003; Ishida et al., 2009; Tomanov et al., 2014). Analyses of mutants defective in

these components suggest that they are required not only for growth and development but also for

responses to a variety of stresses (Saracco et al., 2007; Miura and Hasegawa, 2010; Tomanov et al.,

2014). SUMO E3 ligases contain a SP(SIZ-PAS)-RING zinc finger domain that is responsible for

recruiting E2 to substrates (Gareauand Lima, 2010; Miura and Hasegawa, 2010). In this study, we

carried out a forward genetic screen and identified a SUMO E3 ligase-like protein PIAL2 as a regulator

of transcriptional silencing. Our results demonstrate that PIAL2 and its homolog PIAL1 interact with

each other and with MOM1 and form a novel complex that mediates transcriptional silencing

independently of changes in DNA methylation.

RESULTS

Identification of PIAL2 as a Regulator of Transcriptional Silencing

FWA (FLOWERING WAGENINGEN), a flowering repressor gene, is silenced by DNA methylation at

the SINE-type repeats in the FWA transcription start region (Soppeet al., 2000; Kinoshita et al., 2007).

Previous studies demonstrated that the full-length FWA transgene is an efficient target of DNA

methylation and transcriptional silencing (Cao and Jacobsen, 2002; Chan et al., 2004). Here, we

generated a luciferase reporter system under the control of the FWA promoter (pFWA-LUC) to screen for

novel regulators of transcriptional silencing (Supplemental Figure 1, 2). The pFWA-LUC reporter gene

was initially transformed into the nrpe1-11 mutant and the reporter gene was expressed at high levels in

the transgenic lines. A pFWA-LUC transgenic line was selected and then crossed to the wild-type Col-0.

In the F2 generation, the release of silencing in pFWA-LUC co-segregated with the nrpe1-1 mutation

(Supplemental Figure 1, 2), suggesting that the silencing of the pFWA-LUC reporter gene requires

NPRE1, a component of the RNA-directed DNA methylation pathway. A silenced pFWA-LUC

transgenic line in the wild-type background was selected from the F2 generation and its offspring were

subjected to ethyl methane sulfonate (EMS) mutagenesis to create a mutant library (Supplemental

Figure 1, 2). We screened for mutants that released the silencing of the pFWA-LUC transgene by

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luminescence imaging and identified the mutations by map-based cloning. Based on the screening, we

identified several known RdDM components including AGO4, NRPD1, NRPD2, NRPD4, DRD1,

RDR2, and RDM4/DMS4, and various other silencing regulators including MORC6/DMS11, MOM1,

MET1, FPGS1, FAS1, and BRU1 (Supplemental Table 1). We also identified a silencing mutant, #84-3,

in which the silencing of the pFWA-LUC transgene was released (Figure 1A).

We performed RT-qPCR analysis to determine the transcript level of pFWA-LUC and demonstrated

that the silencing of pFWA-LUC was released in #84-3 as well as in nrpd1, drd1, and mom1 (Figure 1B).

Whole-genome bisulfite sequencing analysis was performed to determine the DNA methylation status of

the FWA promoter in the pFWA-LUC transgenic lines in the wild-type, nrpe1, mom1, and #84-3

backgrounds. We found that the DNA methylation level of the FWA promoter was markedly reduced in

nrpe1 but not in mom1 and #84-3 (Figure 1C), suggesting that #84-3 as well as mom1 released the

silencing of pFWA-LUC independently of changes in DNA methylation. By using map-based cloning in

combination with whole-genome sequencing, we identified a G to A mutation at a splice site of

AT5G41580 in the #84-3 mutant (Supplemental Figure 3A-3C). AT5G41580 encodes PROTEIN

INHIBITOR OF ACTIVATED STAT LIKE2 (PIAL2), which is distantly related to PIAS-type SUMO

E3 ligases in animals (Supplemental Figure 4).

To demonstrate that the mutation in PIAL2 is responsible for the defect in transcriptional silencing,

we obtained a homozygous T-DNA pial2 mutant (SALK_043892C, pial2-1) from the Arabidopsis

Biological Resource Center (ABRC). Using RT-qPCR analysis, we found that the silencing of the

endogenous silencing target loci, including solo LTR, SDC, ROMANIAT5, and AT5TE35950 was

released in the pial2-1 mutant (Figure 1D). Furthermore, we generated constructs harboring the full-

length PIAL2 genomic fragment fused with the C-terminal Flag or Myc tag (PIAL2-Flag, PIAL2-Myc)

and transformed the constructs into the pial2-1 mutant for complementation assays. The results indicated

that the PIAL2-Flag and PIAL2-Myc transgenes restored the silencing of these loci in the pial2 mutant

(Figure 1D), demonstrating that PIAL2 is required for transcriptional silencing.

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PIAL2 is Involved in the Silencing of the RD29A-LUC Transgene in the ros1 Mutant

We previously identified silencing regulators using a pRD29A-LUC reporter system in the DNA

demethylation mutant ros1 (He et al., 2009). The pRD29A-LUC reporter gene was silenced when the

DNA demethylation component ROS1 was disrupted (Gong et al., 2002). By a genetic screen for

suppressors of ros1, we identified many RdDM components that mediate the silencing of the pRD29A-

LUC in the ros1 mutant background (He et al., 2009). Here, our further genetic screen identified mom1

as a suppressor of ros1 in the pRD29A-LUC reporter system (Figure 1E). To determine whether PIAL2

contributes to the silencing of the RD29A-LUC reporter gene in the ros1 mutant background, we crossed

pial2-1 to ros1 to obtain a ros1/pial2 double mutant harboring the pRD29A-LUC reporter gene. We

Figure 1. PIAL1 and PIAL2 function redundantly in the silencing of transgenes and endogenous genomic loci. (A) The silencing of the pFWA-LUC transgene in the mom1 and #84-3 mutants as determined by luminescence imaging. Seedlings were grown on MS medium for 10 days and then treated with luciferin for luminescence imaging. (B) The transcript levels of pFWA-LUC in the wild type and the mutants as determined by RT-qPCR. The mutants including nrpd1, drd1, mom1, and #84-3 were identified from the mutant library in the wild-type background harboring the pFWA-LUC transgene. Error bars are s.d. of three biological replicates. (C) The DNA methylation level of the FWA promoter region in the pFWA-LUC transgenic lines in the wild-type, nrpe1, mom1, and #84-3 backgrounds. The DNA methylation level was determined by whole-genome bisulfite sequencing analysis. (D) Silencing of PIAL2 target loci is affected in the pial2 mutant SALK_043892 and is restored by either the Flag- and Myc-tagged PIAL2 transgenes. The transcript levels of PIAL2 target loci were assessed by RT-qPCR. Error bars are s.d. of three biological replicates. (E) The pial2 mutation releases the silencing of the RD29A-LUC transgene in the ros1 mutant background. The pial2 mutation was introduced into the RD29A-LUC reporter system by crossing. The ros1/mom1 mutant identified in the RD29A-LUC system is shown as a control. (F) The transcript levels of PIAL2 target loci are up-regulated synergistically in the pial1pial2 double mutant relative to either of the pial1 and pial2 single mutants. The transcript levels were determined by RT-qPCR. Error bars are s.d. of three biological replicates.

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found that silencing of the pRD29A-LUC reporter gene was released not only in ros1/mom1 but also in

ros1/pial2 (Figure 1E). The result demonstrates that PIAL2 as well as MOM1 are involved in the

silencing of the pRD29A-LUC transgene in the ros1 mutant background.

PIAL1 and PIAL2 Function Redundantly in Transcriptional Silencing

PIAL2 has a homolog, PIAL1, in Arabidopsis and is highly conserved, especially at the N-terminal

regions, in plants (Supplemental Figure 5). It is possible that PIAL1 functions redundantly with PIAL2

to mediate transcriptional silencing. We obtained a homozygous T-DNA pial1 mutant (CS358389) from

ABRC and crossed the pial1 mutant with the T-DNA pial2 mutant to create a pial1 pial2 (pial1/2)

double mutant. As determined by RT-qPCR analysis, the transcript levels of solo LTR, SDC,

ROMANIAT5, and AT5TE35950 were markedly increased in pial2 and were either not increased or

weakly increased in pial1. Moreover, the transcript levels of these loci were significantly higher in the

pial1/2 double mutant than in either of the single mutants (Figure 1F), suggesting that the function of

PIAL1 and PIAL2 in transcriptional silencing is partially redundant. We thereafter analyzed the function

of PIAL1 and PIAL2 together in the pial1/2 double mutant.

PIAL1 and PIAl2 Share Common Targets with MOM1

Several well-known genomic loci were commonly used as MOM1 and/or RdDM targets in previous

reports (Steimer et al., 2000; Yokthongwattana et al., 2010; Numa et al., 2010; He et al., 2009; Blevins et

al., 2014). We examined the transcript levels of these loci in mom1, nrpe1, and pial1/2 by RT-qPCR

analysis. Based on the expression patterns of these loci, we divided these loci into three classes (Figure

2A). Class I loci are markedly up-regulated in mom1 and pial1/2 but are either not up-regulated or are

weakly up-regulated in nrpe1; Class II loci are up-regulated in all three mutants; and Class III loci are

markedly up-regulated in nrpe1 but are either not up-regulated or are weakly up-regulated in mom1 and

pial1/2. The RT-qPCR results indicated that the expression patterns in mom1 and pial1/2 are almost the

same but are different from that in nrpe1 (Figure 2A). Previous studies reported that the transcript level

of ROS1 is decreased in mutants defective in the RdDM pathway (Huettel et al., 2006; He et al. 2009).

Our RT-qPCR result indicated that although the ROS1 transcript level is decreased in the RdDM mutant

nrpe1, it is not decreased in pial1/2 or mom1 (Figure 2A). These results suggest that PIAL1/2 and

MOM1 are not components of the RdDM pathway.

To compare the targets of NRPE1, MOM1, and PIAL1/2 at the whole-genome level, we performed

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RNA deep sequencing to determine the transcriptomes of the wild type, nrpe1, mom1, and pial1/2. A

total of 72, 153, and 105 TEs are significantly up-regulated in nrpe1, mom1, and pial1/2, respectively

(Figure 2B; Supplemental Data Set 1-3). In these mutants, up-regulated TEs are much more abundant

than down-regulated TEs (Supplemental Data Set 1-3), which is consistent with the notion that NRPE1,

MOM1, and PIAL1/2 are involved in TE silencing. Among the 105 TEs up-regulated in pial1/2, 89.5%

(94/105; Class I and II) are up-regulated in mom1 but only 21.9% (23/105) are up-regulated in nrpe1

(Figure 2B). A total of 230, 293, and 205 genes are up-regulated in nrpe1, mom1, and pial1/2,

respectively (Figure 2B; Supplemental Data Set 4-6). Among the 205 genes up-regulated in pial1/2,

77.1% (158/205; Class I and II) are up-regulated in mom1 but only 30.7% (63/205) are up-regulated in

nrpe1 (Figure 2B). Ten identified up-regulated loci were randomly selected for validation by RT-PCR

analysis, and the results demonstrated that the RNA deep sequencing data are reliable (Supplemental

Figure 6). Heat maps indicated that the effects of pial1/2 and mom1 on transcriptomes are highly similar

but are clearly different from that of nrpe1 (Figure 2C). These results demonstrate that PIAL1 and

PIAL2 contribute to transcriptional silencing at most MOM1 target loci.

To determine the genetic relationship between MOM1 and PIAL2, we performed RNA deep

sequencing to analyze the transcript levels of MOM1 and PIAL1/2 common target TEs in the wild type,

mom1, pial2, and mom1pial2. We found that the silencing of MOM1 and PIAL1/2 common target TEs

was markedly released in mom1 and is slightly released in pial2 (Figure 2D). The weak effect of pial2

supports the notion that PIAL2 functions redundantly with PIAL1 in transcriptional silencing. The release

of silencing in MOM1 and PIAL1/2 common target TEs was not significantly enhanced in the mom1 pial2

double mutant relative to the mom1 single mutant (Figure 2D), suggesting that MOM1 and PIAL2 function

in the same pathway.

RdDM components tend to target the euchromatic regions of chromosome arms (Huettel et al,

2006; Cokus et al., 2008; Zemach et al., 2013), whereas MOM1 preferentially targets pericentromeric

heterochromatin regions (Habu et al, 2006; Yokthongwattana et al., 2010). We localized up-regulated

TEs identified in nrpe1, mom1, and pial1/2 on five Arabidopsis chromosomes. The results suggest that

PIAL1/2 and MOM1 tend to target TEs in pericentromeric heterochromatin regions, whereas NRPE1

tends to target TEs in euchromatic regions (Figure 2E). Long TEs are preferentially enriched in

pericentromeric heterochromatin regions, whereas short TEs are mostly present at the intergenic regions

of chromosome arms (Ahmed et al., 2011; Zemach et al., 2013). We found that TEs targeted by PIAL1/2

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and MOM1 are markedly longer than those targeted by NRPE1 (Figure 2F). These results suggest that

PIAL1/2 and MOM1 function together to mediate transcriptional silencing in heterochromatin regions.

The Involvement of PIAL1 and PIAL2 in Transcriptional Silencing is Independent of Changes in

DNA Methylation

We performed whole-genome bisulfite sequencing analysis in the wild type, nrpe1, mom1, and pial1/2.

DNA methylation was separately analyzed at total C, CG, CHG, and CHH sites for differentially

Figure 2. PIAL1 and PIAL2 contribute to the transcriptional silencing of MOM1 target loci as determined by transcriptome analysis. (A) The transcript levels of the indicated loci were determined by RT-qPCR in the wild type, nrpe1, mom1, and pial1 pial2. The transcript level of ACT7 is shown as a control. I, target loci shared by MOM1 and PIAL1/2 but not by NRPE1; II, target loci common to NRPE1, MOM1, and PIAL1/2; III, loci targeted by NRPE1 but not by MOM1 or PIAL1/2. Error bars are s.d. of three biological replicates. (B) Venn diagrams of overlaps between up-regulated TEs or genes in mom1, pial1/2, and nrpe1 relative to the wild type. I, II, and III represent three classes of loci targeted by MOM1, PIAL1/2, and NRPE1 as explained above. (C) Heat maps of differentially expressed TEs and genes in mom1, pial1/2, and nrpe1. Red and blue lines indicate up- and down-regulated TEs or genes, respectively. (D) The transcript levels of common target TEs of MOM1 and PIAL1/2 as determined by RNA deep sequencing analysis. FPKM, fragments per kilo base per million fragments mapped. (E) Distribution of up-regulated TEs throughout the five Arabidopsis chromosomes. Red, blue, and green bars represent up-regulated TEs in nrpe1, pial1/2, and mom1, respectively. (F) Box plots showing the sizes of up-regulated TEs in nrpe1, mom1, and pial1/2 relative to the wild type.

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methylated regions (DMRs). When DNA methylation of total C was analyzed, 5402 hypomethylated-

DMRs (hypo-DMRs) were identified in nrpe1 (Figure 3A). When CG, CHG, and CHH sites were

separately analyzed for DMRs, the number of CHH hypo-DMRs (5261) identified in nrpe1 is much higher

than the numbers of CG hypo-DMRs (313) and CHG hypo-DMRs (874) (Supplemental Figure 7A), which

supports the notion that the RdDM pathway affects DNA methylation especially at CHH sites. MOM1 was

believed to mediate transcriptional silencing without changes in DNA methylation (Amedeo et al., 2000;

Vaillant et al., 2006). When DNA methylation of total C was analyzed, 591 and 509 hypo-DMRs were

identified in mom1 and pial1/2, respectively, which are much lower than number of the hypo-DMRs

identified in npre1 (Figure 3A). When the three different cytosine contexts CG, CHG and CHH were

separately analyzed, the numbers of hypo-DMRs are comparable in mom1 and pial1/2, and are much lower

than in nrpe1 (Supplemental Figure 7A). In the hypo-DMRs identified in nrpe1, DNA methylation is only

slightly reduced in mom1 and pial1/2 (Figure 3B; Supplemental Figure 7B). Heat maps indicated that the

DNA methylation patterns of mom1 and pial1/2 are similar and are comparable to that of the wild type

but are different from that of the RdDM mutant nrpe1 (Figure 3C).

We next determined whether the up-regulation of TEs and genes is correlated with the weak

reduction of DNA methylation in mom1 and pial1/2. Our whole-genome bisulfite sequencing data

suggest that DNA methylation of transcriptionally up-regulated TEs in nrpe1 is preferentially reduced

(Figure 3D, 3E; Supplemental Figure 8, 9; Supplemental Data Set 1), which is consistent with the role of

NRPE1 in RdDM. For most of the transcriptionally up-regulated TEs in mom1 and pial1/2, however,

DNA methylation is not significantly affected (Figure 3D, 3E; Supplemental Figure 8, 9; Supplemental

Data Set 2, 3), demonstrating that release of transcriptional silencing in pial1/2 and mom1 is

independent of changes in DNA methylation. Promoter DNA methylation levels of transcriptionally up-

regulated genes were determined in nrpe1, mom1, and pial1/2 based on the whole-genome bisulfite

sequencing data. When up-regulated genes are methylated (methylated C/total C > 5%), promoter DNA

methylation levels are markedly reduced in nrpe1 but are not reduced or are slightly reduced in mom1

and pial1/2 (Figure 3D, 3E; Supplemental Figure 8, 9; Supplemental Data Set 4-6). These results

suggest that both PAL1/2 and MOM1 contribute to transcriptional silencing independently of changes in

DNA methylation.

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PIAL1, PIAL2, and MOM1 Interact with each other and Form a High-molecular-weight Complex

To determine how PIAL2 functions in transcriptional silencing, we purified PIAL2-interacting proteins

from Flag-PIAL2 transgenic plants using anti-Flag antibody. MOM1 peptides were identified by mass

spectrometric analysis in PIAL2-copurified proteins (Figure 4A; Supplemental Data Set 7). Moreover,

we purified MOM1-interacting proteins from MOM1-Flag transgenic plants and identified PIAL2

Figure 3. The involvement of PIAL1 and PIAL2 in transcriptional silencing is independent of changes in DNA methylation. (A) Venn diagram showing overlaps of hypo-DMRs in nrpe1, mom1, and pial1/2 relative to the wild type. Bins were recognized as DMRs when their DNA methylation change is more than 10% in the mutants relative to the wild type. (B) Box plots showing DNA methylation levels of hypo-DMRs in the wild type, nrpe1, mom1, and pial1/2. The hypo-DMRs in nrpe1 were analyzed. (C) Heat maps showing DNA methylation of total C, CG, CHG, and CHH hypo-DMRs in the wild type, nrpe1, mom1, and pial1/2. The total C, CG, CHG, and CHH hypo-DMRs identified in nrpe1 are separately shown. Black and light yellow indicate high methylation and low methylation, respectively. (D and E) DNA methylation levels of transcriptionally up-regulated TEs or genes are shown by box plots (D) and scatter plots (E) in nrpe1, mom1, and pial1/2 relative to the wild type. *p<0.05 or **p<0.01 was determined by t test. In the box plots, gene promoters were included for DNA methylation analysis only when their DNA methylation levels in the wild type were higher than 5%.

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peptides in MOM1-copurified proteins (Figure 4A; Supplemental Data Set 7). These results suggest that

PIAL2 physically interacts with MOM1 to form a complex in vivo, which is consistent with the co-

expression between PIAL2 and MOM1 (Obayashi et al., 2009). Interestingly, two previously

uncharacterized proteins were also identified by mass spectrometric analysis of MOM1-copurified

proteins (Figure 4A). One (AT4G11560) was named BDT1 (Bromo-adjacent homology (BAH) domain-

containing transcriptional regulator 1); the other (AT1G43770) was named PHD1 (PHD domain-

containing protein 1). BDT1 was identified not only in MOM1-copurified proteins but also in PIAL2-

copurified proteins (Figure 4A). Both the BAH domain and the PHD domain typically interact with

histones or other chromatin-related proteins. It is possible that BDT1 and PHD1 act as components of

the MOM1-containing complex on chromatin.

Although PIAL1 and PIAL2 function redundantly in transcriptional silencing, the mass

spectrometric analysis identified PIAL2 but not PIAL1 in MOM1-copurified proteins. We performed co-

immunoprecipitation (co-IP) analysis to determine whether MOM1 interacts with PIAL1 as well as

PIAL2. Transgenic plants harboring the PIAL1 or PIAL2 genomic sequence fused to a Myc tag were

generated and crossed to the transgenic plants harboring the MOM1-Flag fusion transgene. Plants from

the F1 generation were used to determine the interaction between MOM1 and PIAL1 or PAL2 by co-IP.

The results indicated that MOM1-Flag copurified with PIAL1-Myc as well as PIAL2-Myc (Figure 4B),

demonstrating that both PIAL1 and PIAL2 interact with MOM1 in vivo. MOM1 was previously

demonstrated to contain a conserved MOM1 motif (CMM2), which is sufficient for the function of

MOM1 in transcriptional silencing at a subset of MOM1 target loci (Caikovski et al., 2008; Nishimura et

al., 2012). The bacterially expressed CMM2 domain folds into an unusual hendecad-based coiled-coil

structure and forms a homodimer as determined by X-ray crystallography (Nishimura et al., 2012).

However, it is unknown whether the full-length MOM1 forms a homodimer in Arabidopsis. Transgenic

lines carrying the MOM1-Myc fusion transgene were generated and crossed to the MOM1-Flag

transgenic line. F1 plants harboring both the MOM1-Myc and MOM1-Flag transgenes were used to

assess dimerization of MOM1 by co-IP. We found that MOM1-Myc co-precipitated with MOM1-Flag

(Figure 4B), demonstrating that MOM1 forms a homodimer in vivo. To determine whether PIAL2

interacts with PIAL2 and PIAL1, we generated Flag-PIAL2 transgenic plants and crossed them to

PIAL2-Myc or PIAL1-Myc transgenic plants to obtain F1 plants carrying both Flag-PIAL2 and PIAL2-

Myc or PIAL1-Myc. Co-IP assay indicated that both PIAL2-Myc and PIAL1-Myc co-precipitated with

13

Flag-PIAL2 (Figure 4B), demonstrating that PIAL2 interacts not only with PIAL2 but also with PIAL1

in vivo.

The interaction between PIAL1, PIAL2, and MOM1 suggests that these proteins form a complex in

vivo. We examined whether PIAL1, PIAL2 and MOM1 exist in a complex by gel filtration. Proteins

were extracted from epitope-tagged transgenic plants and were separated on a Superose 6 (10/300 GL)

column; the eluted fractions were detected by immunoblotting. We found that PIAL1-Myc and PIAL2-

Myc were predominantly eluted in fractions of ~220 KDa, whereas MOM1 was absent in these fractions

(Figure 4C). The ~220 KDa faction is much higher than the PIAL1 or PIAL2 monomer and corresponds

to the PIAL1 and/or PIAL2 dimer. Given the interaction of PIAl2 with PIAL1 and PIAL2 as determined

by co-IP (Figure 4B), PIAL1 and/or PIAl2 may form a homodimer or a heterodimer in vivo. Moreover,

the elution of PIAL1 and PIAL2 was extended to fractions of > 669 KDa, whereas MOM1 was present

in the fractions (Figure 4C), suggesting that PIAL1/2 and MOM1 form a high-molecular-weight

complex in the fractions. To determine whether MOM1 is required for PIAL2 to form a complex, we

expressed the PIAL2-Myc transgene in the mom1 mutant. The gel filtration result indicated that the

elution of PIAL2 disappeared in the high-molecular-weight fractions in the mom1 mutant (Figure 4C),

suggesting that MOM1 is required for the formation of the high-molecular-weight PIAL2-containing

complex. Furthermore, we expressed the MOM1-Myc transgene in the pial1/2 double mutant to

determine whether the pial1/2 mutation affects the MOM1 elution pattern in the gel filtration assay. Our

result indicated that although MOM1 forms a complex in the pial1/2 mutant, the complex is clearly

smaller than that in the wild type (Figure 4C), suggesting that PIAL1 and PIAL2 are required for the

formation of the high-molecular-weight MOM1-containing complex. Our co-IP result demonstrated that

MOM1 forms a homodimer (Figure 4B). The remaining MOM1-containing complex in the pial1/2

mutant may at least contain a MOM1 homodimer. Together, these results suggest that PIAL1, PIAL2,

and MOM1 interact with each other and form a high-molecular-weight complex in vivo.

14

The IND Domains of PIAL1 and PIAL2 Interact with each other and with the CMM2 Domain of

MOM1

The interaction of MOM1 with PIAL1 or PIAl2 was confirmed by yeast two-hybrid analysis (Figure 5A,

5B). To determine the interaction domain of MOM1, we performed yeast two-hybrid analysis with

truncated versions of MOM1. MOM1 contains a domain that is distantly related to a part of the catalytic

Figure 4. PIAL1, PIAL2, and MOM1 form a high-molecular-weight complex in vivo. (A) PIAL2- and MOM1-copurified proteins identified by mass spectrometric analysis. Proteins were extracted from Flag-PIAL2 or MOM1-Flag transgenic plants and were subjected to affinity purification with anti-Flag antibody. (B) The interaction between PIAL1, PIAL2, and MOM1. By crossing, PIAL1-Myc, PIAL2-Myc, and MOM1-Myc, transgenes were separately introduced into MOM1-Flag transgenic plants, and PIAL1-Myc and PIAL2-Myc transgene were separately introduced into Flag-PIAL2 transgenic plants. F1 generation plants were subjected to protein extraction and co-immunoprecipitation. (C) Gel filtration analyses of PIAL1, PIAL2 and MOM1. Proteins were extracted from PIAL1-Myc, PIAL2-Myc, or MOM1-Myc transgenic plants in the wild-type background, from PIAL2-Myc transgenic plants in the mom1 mutant background, and from MOM1-Myc transgenic plants in the pial1/2 double mutant background. The proteins were eluted on a Superose 6 (10/300 GL) column. The epitope-tagged proteins in different fractions were detected by antibodies against the Myc tag. Arrows indicate the fractions that correspond to the standard proteins of 67 KDa, 220 KDa, and 669 KDa.

15

SNF2 domain in chromatin remodeling proteins (Amedeo et al., 2000). Additionally, MOM1 contains

three conserved MOM1 motifs (CMM1, CMM2, and CMM3), which are specifically present in

homologs of MOM1 in plants (Caikovski et al., 2008). A series of truncated versions of MOM1 were

used in the yeast two-hybrid analysis. The results indicated that both PIAL1 and PIAL2 interact with

MOM1-P2, MOM1-P4 and MOM1-P5 but not with MOM1-P1 or MOM1-P3 (Figure 5A, 5B),

suggesting that the conserved CMM2 is necessary and sufficient for the interaction of MOM1 with

PIAL1 and PIAL2.

We further carried out yeast two-hybrid analysis to identify the domain of PIAL2 that is required

for interaction with the CMM2 domain of MOM1. A series of truncated versions of PIAL2 were

analyzed in the yeast two-hybrid analysis and the results indicated that the CMM2 domain of MOM1

interacts with PIAL2-P1 and PIAL2-P5 but not with PIAL2-P2, PIAL2-P3, or PIAL2-P4 (Figure 5C,

5D). The results suggest that a previously uncharacterized domain in PIAL2-P5 (143-301aa) is

necessary and sufficient for the interaction with the CMM2 domain of MOM1. This previously

uncharacterized domain of PIAL2 was thereafter named IND (Interacting domain).

To determine whether the CMM2 domain of MOM1 directly interacts with the IND domain of

PIAL2, we examined the interaction by GST pull-down analysis. The CMM2 and IND domains were

fused to the GST and HIS tags, respectively, and were co-expressed in E. coli. GST pull-down analysis

indicated that HIS-IND co-precipitated with GST-CMM2 (Figure 5E), demonstrating that the CMM2

domain of MOM1 directly interacts with the IND domain of PIAL2. Moreover, as determined by GST

pull-down analysis, HIS-IND also co-precipitated with GST-IND (Figure 5E), suggesting that the IND

domain is required for the dimerization of PIAL2. Because of the sequence similarity between PIAL1

and PIAL2, we carried out GST pull-down to determine whether the PIAL1 fragment (PIAL1-IND, 113-

271 aa) corresponding to the IND domain of PIAL2 is also required for dimerization and MOM1

interaction. The results indicated that PIAL1-IND interacts with PIAL1-IND, PIAL2-IND, and MOM1-

CMM2 (Figure 5E), suggesting that the IND domains of PIAL1 and PIAL2 interact with each other and

with the CMM2 domain of MOM1.

16

SUMO Ligase Activity is not Required for the Function of PIAL2 in Transcriptional Silencing

PIAL2 contains a RING zinc finger motif (RING) and a SUMO interaction motif (SIM), which are

necessary for the SUMO ligase activity of PIAL2; mutations in RING and SIM impair the SUMO ligase

activity of PIAL2 (Tomanov et al., 2014). We introduced mutations into the RING and SIM domains

(RING-M: C329A/H331A; SIM-M: VFDL425AAAA) and performed a sumoylation assay to determine

the effect of the RING and SIM mutations on the SUMO ligase activity of PIAL2 (Figure 6A). The

Figure 5. The IND domain of PIAL1 and PIAL2 interacts with the CMM2 domain of MOM1. (A) Truncated versions of MOM1 used in yeast two-hybrid analysis. P1, 1-832 aa; P2, 798-2001 aa; P3, 798-1662 aa; P4, 1660-2001 aa; P5, 1660-1860 aa. (B) The interaction of a series of truncated forms of MOM1 with PIAL1 or PIAL2 as determined by yeast two-hybrid assays. The full-length MOM1 and truncated forms of MOM1 were fused with GAL4-BD; the full-length PIAL1 and PIAL2 were fused with GAL4-AD. Yeast strains carrying the indicated combinations of fusion proteins were grown for yeast two-hybrid analysis. “Vec” represents the empty GAL4-AD or GAL4-BD vector. (C) Truncated versions of PIAL2 used in yeast two-hybrid assays. P1, 1-301 aa; P2, 276-386 aa; P3, 358-760 aa; P4, 1-142 aa; P5, 143-301 aa. (D) The interaction of the CMM2 domain with truncated forms of PIAL2 as determined by yeast two-hybrid assays. The full-length PIAL2 and truncated forms of PIAL2 were fused with GAL4-AD, and the CMM2 domain of MOM1 was fused with GAL4-BD. (E) The IND domains of PIAL1 and PIAL2 interact with each other and with the CMM2 domain of MOM1 as determined by GST pull-down assays. PIAL1-IND, 143-301 aa; PIAL2-IND, 113-271 aa; MOM1-CMM2, 1700-1824 aa. GST- and HIS-tagged proteins were co-expressed in E. coli, and were purified with anti-GST antibody.

17

result indicated that the SUMO ligase activity of PIAL2 was abolished by the mutation in the RING

domain (C329A/H331A) and was partially affected by the mutation in the SIM domain

(VFDL425AAAA) (Figure 6A). To determine whether the SUMO ligase activity is required for

transcriptional silencing, we introduced the RING and SIM mutations into the full-length PIAL2

construct and carried out complementation assays in the pial2 mutant. The expression levels of the wild-

type and mutated PIAL2 transgenes were determined by immunoblot analysis in randomly selected

transgenic lines. We found that the expression levels of the mutated transgenes PIAL2-RING-M and

PIAL2-RING-SIM-M were markedly lower than that of the wild-type PIAL2 transgene, whereas the

expression level of the mutated transgene PIAL2-SIM-M was only slightly lower than that of the wild-

type PIAL2 transgene (Figure 6B; Supplemental Figure 10). RT-qPCR indicated that the wild-type

PIAL2 transgene restored the silencing of the PIAL2 target loci (solo LTR, ROMANIAT5, SDC, and

AT5TE35950) in the pial2 mutant (Figure 6C). The mutated transgene PIAL2-RING-M restored the

silencing of solo LTR, ROMANIAT5, and AT5TE35950 to the same level of silencing as observed in the

wild-type PIAL2 transgene, whereas it restored the silencing of SDC to a lesser degree (Figure 6C). The

mutated transgene PIAL2-SIM-M restored the silencing of all of the PIAL2 target loci to an equal degree

as the wild-type PIAL2 transgene (Figure 6C). The mutated transgene PIAL2-RING-SIM-M also restored

the silencing of all of the PIAL2 target loci even though the efficiency was slightly lower than that of the

wild-type PIAL2 transgene (Figure 6C). The reduced expression levels of the mutated PIAL2 transgenes

(PIAL2-RING-M and PIAL2-RING-SIM-M) may explain the reduced efficiency in the complementation

assays. These results indicate that the mutations in RING and SIM do not markedly affect the silencing

of PIAL2 target loci, suggesting that the SUMO ligase activity is not required for the function of PIAL2

in transcriptional silencing.

The interaction between PIAL2 and MOM1 suggests that MOM1 is probably a substrate of

sumoylation. Prediction of sumoylated lysine sites using the SUMOylation prediction website

(http://sumosp.biocuckoo.org/) indicated that the lysine sites in MOM1 most likely to be sumoylated are

K674, K1204, and K1762. To determine whether the sumoylation of MOM1 is required for

transcriptional silencing, we mutated the three lysine sites to arginine to block the possible sumoylation

of these sites, and we then transformed each of the mutated MOM1 sequences into the mom1 mutant for

complementation testing. RT-PCR analysis indicated that the silencing of the MOM1 target loci solo

LTR and PDR10 was restored by the mutated MOM1 sequences as well as by the wild-type MOM1

18

sequence (Figure 6D), suggesting that even if MOM1 is sumoylated at these lysine sites, sumoylation is

not required for the function of MOM1 in transcriptional silencing. To determine whether MOM1 is

sumoylated, we precipitated the MOM1-Flag protein from the MOM1-Flag transgenic plants with anti-

Flag antibody and determined whether a sumoylated MOM1 signal was present. No sumoylated MOM1

signal was detected by immunoblot analysis (Figure 6E). These results suggest that MOM1 is not a

substrate of sumoylation even though MOM1 interacts with the SUMO E3 ligase-like proteins PIAL1

and PIAL2.

Figure 6. The SUMO E3 ligase activity of PIAL2 is not required for transcriptional silencing. (A) The mutations in the RING and SIM domains of PIAL2 affect the SUMO E3 ligase activity. The SUMO activating enzyme SAE1, the SUMO conjugating enzyme E2, and SUMO1 were used for the SUMO conjugation assay in the presence of either the full-length wild-type PIAL2 (PIAL2-WT) or the full-length mutated PIAL2 (RING-M, SIM-M, and RING-SIM-M). The SUMO chain signals were visualized by immunoblotting. The wild-type and mutated PIAL2 proteins in the reaction are shown by Coomassie blue staining. (B) The expression levels of the wild-type (PIAL2-WT) and mutated PIAL2-Flag transgenes (RING-M, SIM-M, and RING-SIM-M) harboring the mutations in the RING and SIM domains in the pial2 mutant background. The transgenic lines were selected from the lines shown in Supplemental Figure 10 and used for RT-qPCR analysis. (C) Effect of the mutations in the RING and SIM domains on transcriptional silencing. The wild-type and mutated PIAL2-Flag transgenes were separately expressed in the pial2 mutant for complementation assays. The transcript levels of the indicated PIAL2 target loci were determined by RT-qPCR. ACT7 was used as an internal control. Two biological replicates were performed and similar results were obtained. The results of a set of representative experiments are shown. Error bars indicate s.d. of three technical replicates. (D) Mutation of putative sumoylated sites in MOM1 does not affect MOM1-mediated transcriptional silencing. The wild-type MOM1-Flag or the mutated MOM1-Flag carrying the K674R, K1204R, or K1762R mutation was expressed in the mom1 mutant for complementation assays. The transcript levels of the MOM1 target loci solo LTR and PDR10 were determined by RT-PCR. ACT7 was used as a control. For the RT-PCR experiments, at least two biological replicates were performed. (E) MOM1 is not sumoylated in vivo. The MOM1-Flag fusion protein was precipitated by anti-Flag antibody from the proteins of MOM1-Flag transgenic plants. Anti-SUMO1/2 antibody was used to detect possible sumoylated MOM1 signals. Free SUMO1 and SUMO2 signals were detected by anti-SUMO1/2 antibody in input protein.

19

DISCUSSION

PIAS proteins were initially identified as protein inhibitors of activated STAT in animals (Chung et al.,

1997; Liu et al., 1998). Later, these proteins were found to form a SUMO E3 ligase family defined by a

RING zinc finger motif (Johnson et al., 2001). PIAS proteins function in transcriptional regulation

through various mechanisms in animals (Cobenas-Potts and Matunis, 2013). In plants, it is unknown

whether and how PIAS homologs are involved in transcriptional regulation. In this study, we found that

the Arabidopsis SUMO E3 ligase-like proteins PIAL1 and PIAL2 interact with the plant-specific protein

MOM1 and thus mediate transcriptional silencing, suggesting that the molecular mechanism underlying

the function of PIAL1 and PIAL2 in plants is different from that of PIAS proteins in animals. This study

revealed a novel mechanism by which SUMO E3 ligase-like proteins contribute to transcriptional

silencing in plants.

SUMO E3 ligases interact with substrates and SUMO proteins, thereby facilitating sumoylation of

substrates in animals (Gareau and Lima, 2010). PIAL1 and PIAL2 were shown to have SUMO ligase

activity, which is required for SUMO chain formation (Tomanov et al., 2014). Although the RING and

SIM domains are necessary for the SUMO ligase activity of PIAL2, they are dispensable for

transcriptional silencing (Figure 6A-6C). Thus, the SUMO ligase activity of PIAL2 is not required for

the function of PIAL2 in transcriptional silencing. This result is consistent with the finding that MOM1

is not sumoylated in vivo (Figure 6E). Together, these results suggest that PIAL2 serves as an interaction

partner of MOM1 rather than as a SUMO ligase for MOM1.

MOM1 contains a domain that is distantly related to the catalytic SNF2 domain in chromatin-

remodeling proteins (Amedeo et al., 2000). The full SNF2 domain is composed of seven conserved

DNA helicase motifs and is required for the function of chromatin-remodeling proteins (Thoma et al.,

2005). However, the SNF2-related domain in MOM1 is dispensable for the function of MOM1 in

transcriptional silencing (Caikovski et al., 2008). Instead, CMM2, a conserved MOM1 motif, is not only

necessary but is also sufficient for transcriptional silencing at a subset of MOM1 target loci (Caikovski

et al., 2008; Nishimura et al., 2012). The CMM2 domain forms a homodimer as determined by X-ray

crystallography (Nishimura et al., 2012). Our results demonstrate that the CMM2 domain is required not

only for forming a homodimer but also for interacting with PIAL1 and PIAL2 (Figure 5A-5E). The dual

functions of the CMM2 domain are consistent with the finding that this domain plays a critical role in

transcriptional silencing (Caikovski et al., 2008; Nishimura et al., 2012). Our results suggest that PIAL1

20

and PIAL2 interact with each other and with MOM1 to form a novel PIAL1/2- and MOM1-containing

complex in vivo. Considering that PIAL1 and PIAL2 are necessary for transcriptional silencing at most

MOM1 target loci, we predict that the integrity of the complex is required for transcriptional silencing.

The involvement of MOM1 in transcriptional silencing is independent of changes in DNA

methylation in heterochromatin regions and remains a poorly understood silencing mechanism in plants

(Habu et al, 2006; Vaillant et al., 2006; Yokthongwattana et al., 2010). Previous reports showed that

SUMO proteins and SUMO-conjugating enzymes mediate transcriptional repression at heterochromatin

regions in yeast and animals (Shin et al., 2005; Shiio et al. 2003; Nathan et al., 2006), but it is unknown

whether and how these proteins function at heterochromatin regions in plants. Our results demonstrate

that the SUMO ligase-like proteins PIAL1 and PIAL2 interact with each other and with MOM1 to form

a high-molecular-weight complex (Figure 4A-4C). Although we demonstrate that the SUMO ligase

activity is not required for the function of PIAL2 in transcriptional silencing (Figure 6A, 6C), we can not

exclude the possibility that the function of the PIAL1/2-MOM1 complex in transcriptional silencing is

related to sumoylation. A yeast two-hybrid screen previously demonstrated that MOM1 physically

interacts with SUMO1 (Elrouby et al., 2013). Several silencing regulators, including the RdDM

components SUVR2, KTF1, and IDN2, have been demonstrated to be covalently sumoylated in vivo

(Budhiraja et al., 2009; Miller et al., 2010; Miller et al., 2013). Sumoylation is probably necessary for

these silencing regulators to function in transcriptional silencing. MOM1 was previously shown to be

functionally linked to the RdDM pathway (Numa et al., 2010; Yokthongwattana et al., 2010). It is

possible that the PIAL1/2-MOM1 complex cooperates with these sumoylated silencing regulators and

thereby facilitates transcriptional silencing on chromatin.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana seedlings were grown on MS (Murashige and Skoog) medium plates under

standard long-day conditions (16 h/light, 8 h/dark, 22°C). The T-DNA insertion mutants pial2

(SALK_043892), pial1 (CS358389), and mom1-3 (SALK_141293) were in the Col-0 background and

obtained from the Arabidopsis Biological Resource Center. The ros1 mutant in the C24 background

harboring both pRD29A-LUC and p35S-NPTII transgenes was previously reported (Gong et al., 2002).

The ros1/pial2 mutant was generated by crossing the ros1and pial2 single mutants. For luminescence

21

imaging, seedlings were grown on MS medium for 10 days and then kept at 4°C for 2 days to activate

the stress-responsive pRD29A-LUC transgene. Following the cold treatment, the seedlings were sprayed

with luciferin and subjected to luminescence imaging.

The full -length of PIAL1, PIAL2 or MOM1 driven by the corresponding native promoter was

introduced into the modified pCAMBIA1305 or pRI909 vector to express the C-terminal 5xMyc- or

3xFlag-tagged proteins. The constructs were introduced into the Agrobacterium strain GV3101 and

transformed into Arabidopsis by the flower-dipping method (Clough and Bent, 1998). Site-directed

mutagenesis was performed to introduce point mutations into the epitope-tagged PIAL2 and MOM1

constructs, and the function of the mutated PIAL2 and MOM1 proteins was then determined by

complementation testing. In the N-terminal Flag-tagged PIAL2 construct, the PIAL2 native promoter

was placed in front of the Flag-tag sequence in the pRI909 vector. All constructs were sequenced for

verification, and the primers used for the construction are listed in Supplemental Data Set 8.

RT-PCR, RT-qPCR, and RNA deep sequencing

Total RNA was extracted with Trizol reagent (Invitrogen) from 10-day-old Arabidopsis seedlings or

from rosette leaves. The cDNA was obtained using a Reverse Transcription Kit (TaKaRa, RR012A). RT-

qPCR was performed with SYBR Green Master Mix (TaKaRa) according to the manufacturer’s

instruction. The actin gene ACT7 was used as an internal control. The primers used for RT-PCR are

listed in Supplemental Data Set 8. Total RNA was extracted from two-week-old Arabidopsis seedlings

and then used to produce RNA libraries for deep sequencing (HiSeq 2000, Illumina). To analyze the

data, the Arabidopsis genome sequences and annotated gene models were downloaded from TAIR10

(http://www.arabidopsis.org/). After the adaptor sequences were removed, 49-bp reads were mapped to

the TAIR10 Arabidopsis genome using Tophat (v2.0.12, http://ccb.jhu.edu/software/tophat/), allowing

up to two mismatches. Cufflinks (v2.2.1, http://cole-trapnell-lab.github.io/cufflinks/) was performed to

assemble transcripts and calculate transcript abundances. Gene expression differences were evaluated

using a combination of Fisher’s exact test (P < 0.01) and the fold change of the normalized reads

(Log2(fold change) > 1). The gplots package in R was used to draw the heat maps of the differentially

expressed genes and TEs. The quantsmooth package in Bioconductor was modified to draw the

distribution of up-regulated TEs throughout the five Arabidopsis chromosomes. The raw RNA deep

sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (accession no.

22

GSE80303).

Whole-genome DNA methylation analysis

For whole-genome DNA methylation analysis, genomic DNA was extracted from the two-week-old

Arabidopsis seedlings with hexadecyltrimethyl ammonium bromide (CTAB) and subjected to bisulfite

treatment that converted unmethylated cytosine to uracil. The converted genomic DNA was amplified

and then used for whole-genome bisulfite sequencing with HiSeq 2000 (Illumina). Bisulfite sequencing

reads were mapped to the TAIR10 reference genome using Bismark, allowing two mismatches (Krueger

and Andrews, 2011). Reads that were mapped to more than one position were removed to retain only

reads that were uniquely mapped. The methylation level of each cytosine site was represented by the

percentage of the number of reads reporting a C relative to the total number of reads reporting a C or T.

Only sites with at least five-fold coverage were included in the results. Gene and TE annotations were

downloaded from TAIR. The methylation levels of genes and TEs were estimated by pooling the read

counts that show at least five-fold coverage. The method for identifying differentially methylated

regions (DMRs) was previously described (Stroud et al., 2013). Bins of 200 base pair were analyzed and

the DNA methylation levels of total cytosine, CG, CHG, and CHH sites were separately evaluated. Bins

were considered DMRs when the absolute DNA methylation change was more than 10%, 40%, 20%,

and 10% for total C, CG, CHG, and CHH, respectively. We compared the DNA methylation levels

between each mutant and three different wild-type controls to obtain three batches of DMRs. Only those

DMRs identified in the mutant relative to all three controls were selected for further analysis. Whole-

genome DNA methylation of Col-0 was determined by bisulfite sequencing analysis two times and the

data were used for two wild-type controls; another wild-type control was from a previous report (Stroud

et al., 2013). To evaluate the correlation between expression and DNA methylation, we determined the

DNA methylation levels of different classes of differentially expressed loci. The raw bisulfite

sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (accession no.

GSE80303).

Affinity purification, mass spectrometric analysis, co-immunoprecipitation, and gel filtration

For affinity purification, 6 g of flower tissue was ground to a fine powder and then suspended in lysis

buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 0.5 mM

DTT, 1 mM PMSF, and 1 protease inhibitor cocktail tablet/50 ml [Roche]). The supernatant obtained by

23

centrifugation was incubated with anti-Flag antibody-conjugated beads (Sigma, A2220) for 2.5 h at 4°C.

After the beads were vigorously washed several times, the proteins was eluted with 3xFLAG peptides

(Sigma, F4799), and separated on a 7.5% SDS-PAGE gel. The gel was stained with the ProteoSilver

Silver Stain Kit (Sigma PROT-SIL1), and the bands were then subjected to mass spectrometric analysis.

For co-immunoprecipitation, the protein extracted from flower tissue or from seedlings was incubated

with anti-Flag M1 agarose (Sigma, A2220) for affinity purification. After the sample was washed, it was

boiled and subjected to SDS-PAGE and then to immunoblotting. For gel filtration, 0.5 g of seedlings

was ground to a powder and suspended in 2 ml of lysis buffer. After centrifugation, the supernatant was

passed through a 0.22-micron filter, and 500 μl of the filtrate was loaded onto a Superose 6 (10/300 GL)

column (GE Healthcare, 17-5172-01). The eluate was collected in a series of fractions (500 μl/fraction)

and run on SDS-PAGE gel for immunoblotting.

Yeast two-hybrid assay

The cDNA sequences and the different truncated forms of MOM1, PIAL1, and PIAL2 were cloned into

pGADT7 and pGBKT7 vectors in frame to the 3’-termini of GAL4-AD and GAL4-BD using the One-step

Cloning Kit (Vazyme biotech), respectively. The DNA primers used for cloning are listed in

Supplemental Data Set 8. The yeast strains AH109 and Y187 were transformed with pGADT7 and

pGBKT7 constructs and grown on synthetic dropout medium lacking Leu and Trp, respectively. The

positive clones from the synthetic dropout medium minus Leu were mated with the positive clones from

the synthetic dropout medium minus Trp in YPD medium for 16-20 h. The mixture was spotted on

synthetic dropout medium minus Leu and Trp. The positive yeast colonies were spotted on both the

synthetic dropout medium minus Trp and Leu, and the synthetic dropout medium minus Trp, Leu, and

His. Growth of transformed, positive yeast strains on SD-TLH indicates the interaction between the

GAL-AD fusion protein and the GAL4-BD fusion protein in corresponding yeast strains. A 5 mM

solution of 3-AT was used to inhibit the background growth of transformed strains on the synthetic

dropout medium minus Trp, Leu, and His.

Protein induction and pull-down assays in bacteria

PIAL1-IND, PIAL2-IND, and MOM1-CMM2 were fused with 5’-terminal GST or 6xHIS in the

constructs pGEX-6P-1 and pET28a+, respectively. The primers used for the construction are listed in

Supplemental Data Set 8. The constructs expressing both GST- and HIS-tagged proteins were co-

24

transformed into the E. coli expression strain Transetta (DE3). After selection on solid LB medium

containing both kanamycin and ampicillin (Amresco, both at 50 mg/L), a single colony was transferred

to 5 ml of LB liquid medium containing antibiotics (50 mg/L kanamycin, 50 mg/L ampicilin, and 34

mg/L chloromycetin) and grown at 37°C with rapid shaking (>200 rpm) overnight. A 1-ml volume of the

LB was then added to 100 ml of LB for growth under the same conditions. When the OD600 of the

culture increased to 0.4-0.5, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and then

the culture was shaken continuously at 18°C for another 24 h for protein induction.

The bacteria in the culture were collected by centrifugation, and 4 ml of the bacterial culture was

suspended in 1.5 ml of protein extraction buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10%

glycerol, 0.5% Tween-20, 15 mM β-mercaptoethanol, 1 mM PMSF, and 1 protease inhibitor cocktail

tablet/50 ml [Roche]). The sample was sonicated four times (10 s on and 59 s off), and then centrifuged

at 13000 rpm for 10 min at 4°C. A 1.4-ml volume of the supernatant was transferred to a 1.5-ml

Eppendorf tube. Of this supernatant, 200 μl was used as input, and 1.2 ml was incubated with 50 μl of

GST-beads for a pull-down assay. After incubation for 1.5 h with gentle rotation at 4°C, the sample was

centrifuged at 2000 rpm for 1 min at 4°C. The supernatant was discarded and the GST-beads were

washed six times (5 min per time) with 1.5 ml of protein extraction buffer at 4°C. After the last wash,

200 μl of protein extraction buffer and 50 μl of 5xSDS sample buffer were added to the tube. The

samples were boiled for 6 min at 100°C and separated on 15% SDS-PAGE gels for immunoblotting with

GST antibody (Abmart, 12G8) and HIS antibody (Abmart, 10E2).

In vitro sumoylation assay

All proteins used in the in vitro sumoylation assay were expressed and purified in E. coli using Rosetta

(DE3). SUMO1 was expressed in pET28a vector with a C-terminal His tag, and was purified with Ni2+

beads, followed by removal of His tag with Ulp1 enzyme. SAE1b was expressed in pETDuet-1 vector

with no tags, while SAE2 was expressed in a modified pET28a vector with an N-terminal His tag followed

by a PreScission Site. The two subunits of E1 enzyme (SAE1b and SAE2) were co-expressed in Rosetta

(DE3) and purified with Ni2+ beads followed by His tag removal using PreScission Protease. The E2

enzyme SCE1 was expressed in a modified pET28a vector with a His tag followed by PreScission Site in

the N terminal, and was purified with Ni2+ beads followed by His tag removal. PIAL2 and its mutants

were tagged with hexa-His in the N terminal and were purified with Ni2+ beads.

25

The in vitro sumoylation reaction buffer contained 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 15%

glycerol, 5 mM ATP, and 5 mM MgCl2. For the assay, 8 µg SUMO1 was incubated with 0.5 µg E1 enzyme,

4 µg E2 enzyme, and 1 µg PIAL2 or its mutant for 2 h at 37°C. The total volume of the reaction was 50

µl. The samples were then separated by SDS-PAGE and analyzed by immunoblotting for detection of

sumoylated bands.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL

databases under the following accession numbers: PIAL1 (AT1G08910), PIAL2 (AT5G41580), MOM1

(AT1G08060), NRPD1 (AT1G63020), NRPE1 (AT2G40030), DRD1 (AT2G16390), BDT1

(AT4G11560), PHD1 (AT1G43770), FWA (AT4G25530), SDC (AT2G17690), ROS1 (AT2G36490),

MORC6 (AT1G19100), AGO4 (AT2G27040), RDR2 (AT4G11130), NRPD4 (AT4G15950), RDM4

(AT2G30280), NRPD2 (AT3G23780), MET1 (AT5G49160), FPGS1 (AT5G05980), FAS1

(AT1G65470), BRU1 (AT3G18730).

SUPPLEMENTAL DATA

Supplemental Figure 1. The FWA-LUC reporter system.

Supplemental Figure 2. Luminescence imaging of pFWA-LUC transgenic plants.

Supplemental Figure 3. Map-based cloning and characterization of PIAL2.

Supplemental Figure 4. Alignment of PIAL1 and PIAL2 in Arabidopsis, PIAS2 in human, and

SU(VAR)2-10 in Drosophila.

Supplemental Figure 5. Alignment of Arabidopsis PIAL1 and PIAL2 and their homologs in other

plants including Glycine max, Brassica napus, Vitis vinifera, Populus trichocarpa, Oryza sativa, and

Sorghum bicolor.

Supplemental Figure 6. Validation of the transcript levels of the MOM1, PIAL1/2, and/or NPRE1

target loci in the wild type, nrpe1, mom1, and pial1/2.

Supplemental Figure 7. Analyses of hypo-DMRs in nrpe1, mom1, and pial1/2.

Supplemental Figure 8. Box plots showing CG, CHG, and CHH methylation of transcriptionally up-

regulated TEs and genes in nrpe1, mom1, and pial1/2 relative to the wild.

Supplemental Figure 9. Scatter plots showing CG, CHG, and CHH methylation of transcriptionally up-

regulated TEs or genes in nrpe1, mom1, and pial1/2 relative to the wild type.

http://www.arabidopsis.org/servlets/TairObject?id=30854&type=locus

















26

Supplemental Figure 10. The expression levels of the wild-type and mutated PIAL2 transgenes in the

pial2 mutant.

Supplemental Table 1. Full list of mutants identified by map-based cloning in this study.

Supplemental Data Set 1. RNA transcript and DNA methylation levels of differentially expressed TEs

in nrpe1.


in mom1.


in pial1/2.

Supplemental Data Set 4. RNA transcript and DNA methylation levels of differentially expressed

genes in nrpe1.


genes in mom1.


genes in pial1/2.

Supplemental Data Set 7. Full list of proteins identified by mass spectrometric analyses in affinity

purification of MOM1 and PIAL2.

Supplemental Data Set 8. DNA oligonucleotides used in this study.

ACKNOWLEDGMENTS

This work was supported by the National Basic Research Program of China (973 Program)

(2012CB910900) and the 973 Program (2011CB812600) from the Chinese Ministry of Science and

Technology.

AUTHOR CONTRIBUTIONS

YFH, QYZ, LLD, and XJH conceived and designed the experiments; YFH, QYZ, LLD, YXL, SSC,

CRS, LL, and SC performed the experiments; YFH, QYZ, LLD, HWH, YQL, TC, and XJH analyzed

the data; YFH, QYZ, and XJH wrote the manuscript.

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Aufsatz, W., Mette, M.F., van der Winden, J., Matzke, A.J., and Matzke, M. (2002). RNA-directed DNA methylation in Arabidopsis.Proc Natl Acad Sci U S A 99 Suppl 4, 16499-16506.


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Cao, X., and Jacobsen, S.E. (2002). Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and genesilencing. Curr Biol 12, 1138-1144.


Chan, S.W., Zilberman, D., Xie, Z., Johansen, L.K., Carrington, J.C., and Jacobsen, S.E. (2004). RNA silencing genes control de novoDNA methylation. Science 303, 1336.


Chung, C.D., Liao, J., Liu, B., Rao, X., Jay, P., Berta, P., and Shuai, K. (1997). Specific inhibition of Stat3 signal transduction byPIAS3. Science 278, 1803-1805.


Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsisthaliana. Plant J 16, 735-743.


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Elrouby, N., Bonequi, M.V., Porri, A., and Coupland, G. (2013). Identification of Arabidopsis SUMO-interacting proteins that regulatechromatin activity and developmental transitions. Proc Natl Acad Sci U S A 110, 19956-19961.


Gareau, J.R., and Lima, C.D. (2010). The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition.Nat Rev Mol Cell Biol 11, 861-871.


Gong, Z., Morales-Ruiz, T., Ariza, R.R., Roldan-Arjona, T., David, L., and Zhu, J.K. (2002). ROS1, a repressor of transcriptional genesilencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803-814.


Haag, J.R., and Pikaard, C.S. (2011). Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant genesilencing. Nat Rev Mol Cell Biol 12, 483-492.


Habu, Y., Mathieu, O., Tariq, M., Probst, A.V., Smathajitt, C., Zhu, T., and Paszkowski, J. (2006). Epigenetic regulation oftranscription in intermediate heterochromatin. EMBO Rep 7, 1279-1284.


Hamilton, A., Voinnet, O., Chappell, L., and Baulcombe, D. (2002). Two classes of short interfering RNA in RNA silencing. EMBO J21, 4671-4679.


He, X.J., Hsu, Y.F., Pontes, O., Zhu, J., Lu, J., Bressan, R.A., Pikaard, C., Wang, C.S., and Zhu, J.K. (2009). NRPD4, a protein relatedto the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNAmethylation. Genes Dev 23, 318-330.


Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005). RNA polymerase IV directs silencing of endogenous DNA.Science 308, 118-120.


Huang, L., Jones, A.M., Searle, I., Patel, K., Vogler, H., Hubner, N.C., and Baulcombe, D.C. (2009). An atypical RNA polymeraseinvolved in RNA silencing shares small subunits with RNA polymerase II. Nat Struct Mol Biol 16, 91-93.


Huettel, B., Kanno, T., Daxinger, L., Aufsatz, W., Matzke, A.J., and Matzke, M. (2006). Endogenous targets of RNA-directed DNAmethylation and Pol IV in Arabidopsis. EMBO J 25, 2828-2836.


Ishida, T., Fujiwara, S., Miura, K., Stacey, N., Yoshimura, M., Schneider, K., Adachi, S., Minamisawa, K., Umeda, M., and Sugimoto, K.(2009). SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis. Plant Cell 21, 2284-2297.


Jackson, J.P., Lindroth, A.M., Cao, X., and Jacobsen, S.E. (2002). Control of CpNpG DNA methylation by the KRYPTONITE histoneH3 methyltransferase. Nature 416, 556-560.


http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 18%5BVolume%5D AND 1166%5BPagination%5D AND Ebbs%2C M%2EL%2E%2C and Bender%2C J%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ebbs,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

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http://search.crossref.org/?page=1&rows=2&q=Elrouby, N., Bonequi, M.V., Porri, A., and Coupland, G. (2013). Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions. Proc Natl Acad Sci U S A 110, 19956-19961.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Elrouby,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Elrouby,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nat Rev Mol Cell Biol%5BTitle%5D%29 AND 11%5BVolume%5D AND 861%5BPagination%5D AND Gareau%2C J%2ER%2E%2C and Lima%2C C%2ED%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gareau, J.R., and Lima, C.D. (2010). The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11, 861-871.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gareau,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gareau,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28ROS%5BTitle%5D%29 AND 1%5BVolume%5D AND a repressor of transcriptional gene silencing in Arabidopsis%2C encodes a DNA glycosylase%2Flyase%2E Cell 111%2C 803%5BPagination%5D AND Gong%2C Z%2E%2C Morales%2DRuiz%2C T%2E%2C Ariza%2C R%2ER%2E%2C Roldan%2DArjona%2C T%2E%2C David%2C L%2E%2C and Zhu%2C J%2EK%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gong, Z., Morales-Ruiz, T., Ariza, R.R., Roldan-Arjona, T., David, L., and Zhu, J.K. (2002). ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803-814.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gong,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gong,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nat Rev Mol Cell Biol%5BTitle%5D%29 AND 12%5BVolume%5D AND 483%5BPagination%5D AND Haag%2C J%2ER%2E%2C and Pikaard%2C C%2ES%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Haag, J.R., and Pikaard, C.S. (2011). Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nat Rev Mol Cell Biol 12, 483-492.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Haag,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Haag,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28EMBO Rep%5BTitle%5D%29 AND 7%5BVolume%5D AND 1279%5BPagination%5D AND Habu%2C Y%2E%2C Mathieu%2C O%2E%2C Tariq%2C M%2E%2C Probst%2C A%2EV%2E%2C Smathajitt%2C C%2E%2C Zhu%2C T%2E%2C and Paszkowski%2C J%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Habu, Y., Mathieu, O., Tariq, M., Probst, A.V., Smathajitt, C., Zhu, T., and Paszkowski, J. (2006). Epigenetic regulation of transcription in intermediate heterochromatin. EMBO Rep 7, 1279-1284.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Habu,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Epigenetic regulation of transcription in intermediate heterochromatin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Epigenetic regulation of transcription in intermediate heterochromatin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Habu,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28EMBO J%5BTitle%5D%29 AND 21%5BVolume%5D AND 4671%5BPagination%5D AND Hamilton%2C A%2E%2C Voinnet%2C O%2E%2C Chappell%2C L%2E%2C and Baulcombe%2C D%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hamilton, A., Voinnet, O., Chappell, L., and Baulcombe, D. (2002). Two classes of short interfering RNA in RNA silencing. EMBO J 21, 4671-4679.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hamilton,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Two classes of short interfering RNA in RNA silencing&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Two classes of short interfering RNA in RNA silencing&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hamilton,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28NRPD%5BTitle%5D%29 AND 4%5BVolume%5D AND a protein related to the RPB4 subunit of RNA polymerase II%2C is a component of RNA polymerases IV and V and is required for RNA%5BPagination%5D AND He%2C X%2EJ%2E%2C Hsu%2C Y%2EF%2E%2C Pontes%2C O%2E%2C Zhu%2C J%2E%2C Lu%2C J%2E%2C Bressan%2C R%2EA%2E%2C Pikaard%2C C%2E%2C Wang%2C C%2ES%2E%2C and Zhu%2C J%2EK%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=He, X.J., Hsu, Y.F., Pontes, O., Zhu, J., Lu, J., Bressan, R.A., Pikaard, C., Wang, C.S., and Zhu, J.K. (2009). NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes Dev 23, 318-330.

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http://search.crossref.org/?page=1&rows=2&q=Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005). RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118-120.

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http://scholar.google.com/scholar?as_q=RNA polymerase IV directs silencing of endogenous DNA&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=RNA polymerase IV directs silencing of endogenous DNA&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Herr,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nat Struct Mol Biol%5BTitle%5D%29 AND 16%5BVolume%5D AND 91%5BPagination%5D AND Huang%2C L%2E%2C Jones%2C A%2EM%2E%2C Searle%2C I%2E%2C Patel%2C K%2E%2C Vogler%2C H%2E%2C Hubner%2C N%2EC%2E%2C and Baulcombe%2C D%2EC%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Huang, L., Jones, A.M., Searle, I., Patel, K., Vogler, H., Hubner, N.C., and Baulcombe, D.C. (2009). An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II. Nat Struct Mol Biol 16, 91-93.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Huang,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huang,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28EMBO J%5BTitle%5D%29 AND 25%5BVolume%5D AND 2828%5BPagination%5D AND Huettel%2C B%2E%2C Kanno%2C T%2E%2C Daxinger%2C L%2E%2C Aufsatz%2C W%2E%2C Matzke%2C A%2EJ%2E%2C and Matzke%2C M%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huettel,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 21%5BVolume%5D AND 2284%5BPagination%5D AND Ishida%2C T%2E%2C Fujiwara%2C S%2E%2C Miura%2C K%2E%2C Stacey%2C N%2E%2C Yoshimura%2C M%2E%2C Schneider%2C K%2E%2C Adachi%2C S%2E%2C Minamisawa%2C K%2E%2C Umeda%2C M%2E%2C and Sugimoto%2C K%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ishida,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nature%5BTitle%5D%29 AND 416%5BVolume%5D AND 556%5BPagination%5D AND Jackson%2C J%2EP%2E%2C Lindroth%2C A%2EM%2E%2C Cao%2C X%2E%2C and Jacobsen%2C S%2EE%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jackson,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Direct binding of CoREST1 to SUMO-2/3 contributes to gene-specific repression by the LSD1/CoREST1/HDAC complex&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ouyang,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pontes,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pontier,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Cell%5BTitle%5D%29 AND 33%5BVolume%5D AND 192%5BPagination%5D AND Ream%2C T%2ES%2E%2C Haag%2C J%2ER%2E%2C Wierzbicki%2C A%2ET%2E%2C Nicora%2C C%2ED%2E%2C Norbeck%2C A%2ED%2E%2C Zhu%2C J%2EK%2E%2C Hagen%2C G%2E%2C Guilfoyle%2C T%2EJ%2E%2C Pasa%2DTolic%2C L%2E%2C and Pikaard%2C C%2ES%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ream,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Demethylation-induced developmental pleiotropy in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ronemus,&as_ylo=1996&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Genetic analysis of SUMOylation in Arabidopsis: conjugation of SUMO1 and SUMO2 to nuclear proteins is essential&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Saracco,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci U S A%5BTitle%5D%29 AND 100%5BVolume%5D AND 13225%5BPagination%5D AND Shiio%2C Y%2E%2C and Eisenman%2C R%2EN%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Histone sumoylation is associated with transcriptional repression&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shiio,&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Soppe,&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 12%5BVolume%5D AND 1165%5BPagination%5D AND Steimer%2C A%2E%2C Amedeo%2C P%2E%2C Afsar%2C K%2E%2C Fransz%2C P%2E%2C Mittelsten Scheid%2C O%2E%2C and Paszkowski%2C J%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Identification of SUMO-dependent chromatin-associated transcriptional repression components by a genome-wide RNAi screen&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Stielow,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nat Struct Mol Biol%5BTitle%5D%29 AND 12%5BVolume%5D AND 350%5BPagination%5D AND Thoma%2C N%2EH%2E%2C Czyzewski%2C B%2EK%2E%2C Alexeev%2C A%2EA%2E%2C Mazin%2C A%2EV%2E%2C Kowalczykowski%2C S%2EC%2E%2C and Pavletich%2C N%2EP%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Thoma,&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Thoma,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 26%5BVolume%5D AND 4547%5BPagination%5D AND Tomanov%2C K%2E%2C Zeschmann%2C A%2E%2C Hermkes%2C R%2E%2C Eifler%2C K%2E%2C Ziba%2C I%2E%2C Grieco%2C M%2E%2C Novatchkova%2C M%2E%2C Hofmann%2C K%2E%2C Hesse%2C H%2E%2C and Bachmair%2C A%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tomanov,&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Arabidopsis PIAL1 and 2 promote SUMO chain formation as E4-type SUMO ligases and are involved in stress responses and sulfur metabolism&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tomanov,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28EMBO Rep%5BTitle%5D%29 AND 7%5BVolume%5D AND 1273%5BPagination%5D AND Vaillant%2C I%2E%2C Schubert%2C I%2E%2C Tourmente%2C S%2E%2C and Mathieu%2C O%2E%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vaillant, I., Schubert, I., Tourmente, S., and Mathieu, O. (2006). MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis. EMBO Rep 7, 1273-1278.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vaillant,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vaillant,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Cell%5BTitle%5D%29 AND 135%5BVolume%5D AND 635%5BPagination%5D AND Wierzbicki%2C A%2ET%2E%2C Haag%2C J%2ER%2E%2C and Pikaard%2C C%2ES%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wierzbicki,&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wierzbicki,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28PLoS Biol%5BTitle%5D%29 AND 2%5BVolume%5D AND 104%5BPagination%5D AND Xie%2C Z%2E%2C Johansen%2C L%2EK%2E%2C Gustafson%2C A%2EM%2E%2C Kasschau%2C K%2ED%2E%2C Lellis%2C A%2ED%2E%2C Zilberman%2C D%2E%2C Jacobsen%2C S%2EE%2E%2C and Carrington%2C J%2EC%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xie,&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Genetic and functional diversification of small RNA pathways in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xie,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28EMBO J%5BTitle%5D%29 AND 29%5BVolume%5D AND 340%5BPagination%5D AND Yokthongwattana%2C C%2E%2C Bucher%2C E%2E%2C Caikovski%2C M%2E%2C Vaillant%2C I%2E%2C Nicolet%2C J%2E%2C Mittelsten Scheid%2C O%2E%2C and Paszkowski%2C J%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yokthongwattana,&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Cell%5BTitle%5D%29 AND 153%5BVolume%5D AND 193%5BPagination%5D AND Zemach%2C A%2E%2C Kim%2C M%2EY%2E%2C Hsieh%2C P%2EH%2E%2C Coleman%2DDerr%2C D%2E%2C Eshed%2DWilliams%2C L%2E%2C Thao%2C K%2E%2C Harmer%2C S%2EL%2E%2C and Zilberman%2C D%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Molecular mechanism of action of plant DRM de novo DNA methyltransferases&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhong,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

Supplemental Figure 1

Supplemental Figure 1. The FWA-LUC reporter system. (A) Schematic representation of

the pFWA-LUC construct. The primers used to generate the FWA promoter sequence are

shown. The recognition sites of BamHI and SalI are shown in red in the primer sequences.

(B) The flow diagram used for mutant library construction and map-based cloning.

B

A

pFWA-LUC in nrpd1b-11 (LUC: bright) X Col

F1 (LUC: dark)

U

F2: pFWA-LUC in nrpd1b mutant (bright)

EMS mutagenesis

U

F2, pFWA-LUC in wild type (dark)

U

F3: pFWA-LUC in nrpd1b mutant (bright)

Mutant library screening

mom1, dms11, pial2, ago4, nrpd1a, rdr2…

selfing

selfing selfing

(LUC: bright)

pFWA-LUC construct

transformation (GV3101 strain)

F3, pFWA-LUC in wild type (dark)

Tnos pFWA

-1616 bp

BamHI

ATG

SalI SacI

+1653 bp HindIII

LUC

SINE repeats

RB LB HYG p35S

pFWA-BamHI-F: CGGGATCCCGCCTTTCTCTTCCTCATCTGC

pFWA-SalI-R: ACGCGTCGACCATTTTCCCTCAATGCAATAACCTG

Supplemental Data. Han et al. (2016). Plant Cell 10.1105/tpc.15.00997

1

pFWA-LUC in nrpe1 X Col-0 F2

pFWA-LUC in nrpe1-11


Supplemental Figure 2. Luminescence imaging of pFWA-LUC transgenic plants. (A)

Luminescence imaging of pFWA-LUC transgenic plants in the nrpe1-11 background. pFWA-

LUC was transformed into the nrpe1-11 background and a transgenic line highly expressed

the pFWA reporter gene. (B) The expressed pFWA-LUC transgenic line was crossed to the

wild-type Col-0 and the pFWA-LUC reporter gene was silenced in the F1 plants. (C)

Luminescence imaging of the F2 plants from the cross of pFWA-LUC in nrpe1 and the wild-

type Col-0. The pFWA-LUC transgenic plants in the nrpe1-11 background are shown as a

control.

C

pFWA-LUC in nrpe1-11 pFWA-LUC in nrpe1-11

X Col-0 F1 A B


2

A

B


Chr. 5 TFL1 TT4 NGA139 PHYC LFY3 M555

PIAL2, G2769 to A

Marker coordinates:

Recombination:

15461301

8/153 2/153

16576701

1/153

16764801

4/153

17033501

16626337 - 16630783 bp

+1 +4097 pial2-3

G2769 to A

PIAL2

pial2-1

SALK_043892

C

760 aa

RING

298-375 aa

PIAL2

Supplemental Figure 3. Map-based cloning and characterization of PIAL2. (A)

Diagram for map-based cloning of PIAL2. A ~188-kb region on chromosome 5 was

determined by map-based cloning. A mutation was identified by whole-genome DNA

deep sequencing at a splice site of PIAL2 in the ~188-kb region. A black bar with a

blank circle represents chromosome 5. The centromere is indicated by the blank circle.

Positions of indicated genes and markers are shown on the top of the black bar. (B)

Schematic representation of the PIAL1 gene and the mutations in the pial2-1 and

pial2-3 mutants. Boxes and lines represent exons and introns, respectively. 5’ and 3’ UTRs are indicated by blank boxes, whereas coding regions are indicated by filled

boxes. (C) The PIAL2 protein contains a RING-type zinc finger motif that is conserved

in SUMO E3 ligases. The blank bar represents the full-length PIAL2 proteins. The

position and size of the conserved RING-type zinc finger motif are shown.


3

Arabidopsis PIAL1 1 MVIPATSR------------------FGFR-------------AEFNTKEFQASCISLANEIDAAIGRNEVPGNIQELAL

Arabidopsis PIAL2 1 MSTAAAARPVAGTGLREKTAASLVNSFRLASVTQRLRYHIQDGAKVDPKEFQICCISFAKGIDFAIANNDIPKKVEEFPW

Human PIAS2 1 --------------------------------------------------------------------------------

Drosophila SU(VAR)2-10 1 ---------------------------------------------------MRKTRSQTARTQAENAATSSSPGHQSTSS

Arabidopsis PIAL1 50 ILNNVCRRKCDDYQTRAVVMALMISVKSACQLGWFPERETQELLAIIDLMWNGFSCPENVTSCVNSPVTLISQVIERFYP

Arabidopsis PIAL2 81 LLKQLCRHGTDVY-TKTALMVLMISVKHACHLGWFSDSESQELIALADEIRTCFGSSGSTSPGIKSPGSTFSQIMERFYP

Human PIAS2 1 ---------MADFEELRNMVSSFRVSELQVLLGFAGRNKSGRKHDLLMRALHLLKSG--------CSPAVQIKIRELYRR

Drosophila SU(VAR)2-10 30 APIAVNPFDSTKYKECEQMVQMLRVVELQKILSFLNISFAGRKTDLQSRILSFLRT---------NLELLAPKVQEVYAQ

Arabidopsis PIAL1 130 CVKLGHILVSFEAKPESKMMMKDFHISKKMPHSPKQKVGLFVVRTEDISRSNCIVHPQGVSFLLNGKGIDKRVN--ISME

Arabidopsis PIAL2 160 FVKLGHVLVSFEVKAGYTMLAHDFYISKNMPHSLQEKIRLFVAQTDNIDTSACISNPPEVSFLLNGKGVEKRVN--IAMD

Human PIAS2 64 RYPRTLEGLSDLSTIKSSVFSLDGGSSPVEPDLA--VAGIHSLPSTSVTPHSPSSPVGSVLLQDTKPTFEMQQPSPPIPP

Drosophila SU(VAR)2-10 101 SVQEQNATLQYIDPTR--MYSHIQLPPTVQPNP----VGLVGSGQGVQVPGGQMNVVGGAPFLHTH-SINSQLP------

Arabidopsis PIAL1 208 SGPQLPTNVTALLNLGANLLQAIGCFGGSYLIAIAFMDVIPLPNKPLLKDYVHPEVVGSNSDCDIIEGPSRISLSCPISR

Arabidopsis PIAL2 238 TGPQLPTNVTAQLKYGTNLLQVMGNFKGNYIIIIAFTGLVVPPEKPVLKDYLQSGVIEASPDSDIIEGPSRVSLSCPISR

Human PIAS2 142 VHPDVQLKNLPFYDVLDVLIKPTSLVQSSIQRFQEKFFIFALTPQQVREICISRDFLPGGRRDYTVQVQLRLCLAETSCP

Drosophila SU(VAR)2-10 168 IHPDVRLKKLAFYDVLGTLIKPSTLVPRNTQRVQEVPFYFTLTPQQATEIASNRDIRNSSKVEHAIQVQLRFCLVETSCD

Arabidopsis PIAL1 288 TRIKLPVKGHVCKHLQCFDFWNYVNMNTR------------------RHHGAARIILEEVGRNAADVVISADGTWMVETE

Arabidopsis PIAL2 318 KRIKLPVKGQLCKHLQCFDFSNYVHINMRNPTWRCPHCNQPVCYPDIRLDQNMAKILKDVEHNAADVIIDAGGTWKVTKN

Human PIAS2 222 QEDNYPNSLCIKVNGKLFPLPGYAPPPKNG---------------------------IEQKRPGRPLNITSLVRLSSAVP

Drosophila SU(VAR)2-10 248 QEDCFPPNVNVKVNNKLCQLPNVIPTNRPN---------------------------VEPKRPPRPVNVTSNVKLSPTVT

Arabidopsis PIAL1 350 NDEDVELVPETTHDHGDPNSFIN--LGPTVKNPARD---ENEMETSTQVEEHNPCLSEIQGPSND----THRPASDYTML

Arabidopsis PIAL2 398 TGETPEPVREIIHDLEDPMSLLN--SGPVVFDLTGDDDAELEVFGDNKVEDRKPCMSDAQGQSNNNNTNKHPSNDDYSSI

Human PIAS2 275 NQISISWASEIGKNYSMSVYLVRQLTSAMLLQRLKMKGIRNPDHSRALIKEKLTADPDSEIATTSLRVSLMCPLGKMRLT

Drosophila SU(VAR)2-10 301 NTITVQWCPDYTRSYCLAVYLVKKLTSTQLLQRMKTKGVKPADYTRGLIKEKLTEDADCEIATTMLKVSLNCPLGKMKML

Arabidopsis PIAL1 421 NQS-----------HTSTNTLPQLPRTLNAFDGQQFVNLPQVINTRDSPASQALPMTFSPTPSPQDILATNAANFGTSMP

Arabidopsis PIAL2 476 FDISDVIALDPEILSALGNTAPQPHQASNTGTGQQYSNLSQIPMSIDP---MPVPVPFSQTPSPRDRPATTSTVFTIPNP

Human PIAS2 355 IPCR-----------------AVTCTHLQCFDAALYLQMNEKKPTWICP--------VCDKKAAYESLILDGLFMEILND

Drosophila SU(VAR)2-10 381 LPCR-----------------ASTCSHLQCFDASLYLQMNERKPTWNCP--------VCDKPAIYDNLVIDGYFQEVLGS

Arabidopsis PIAL1 490 AAQSSQFQGSHVTSLGNCEGRTSDLMARWNHIYGRVQTQFPPAPLSHHHYSMQNQSPSPAQQRPVPSYIAHPQTFHVNYG

Arabidopsis PIAL2 553 SPQYSQVHASPVTPTGTYLGRTTS--PRWNQTY---QSQAP--PMTTPYTSRKVSVPVTSQS------------------

Human PIAS2 409 ---CSDVDEIKFQEDG-----------SWCP--MRPKKEAMKVSSQPCTKIESSS-------------------------

Drosophila SU(VAR)2-10 436 SLLKSDDTEIQLHQDG-----------SWSTPGLRSETQILDTPSKPAQKVEVISDDIELIS------------------

Arabidopsis PIAL1 570 ENADQRWMPSSIAHPQTLPVNYGGNTNQRPIPSSIAHPQTLPVNYRGNTDHRSTPYSITHLQTLLNYGGNADQRPMPSSI

Arabidopsis PIAL2 607 ------------------PANVSSFVQSQHVPRVLSQPN--------------------------NYG------------

Human PIAS2 448 -----------------VLSKPCSVTVASEASKKKVDVIDLT--------------------------------------

Drosophila SU(VAR)2-10 486 ------------DDAKPVKRDLSPAQDEQPTSTSNSETVDLT--------------------------------------

Arabidopsis PIAL1 650 TNLQTLPATYGGYAHQRPMSSSITHPRTSPVNYGGTPDQRPMPSSITHPQTLPVSYGGTTDQILNPGGAMGQFSSREFMN

Arabidopsis PIAL2 631 --VRGLTSSHASTSRQHPSGPTVQS--------------------------------------------VSRLSDLVDVD

Human PIAS2 473 --IESSSD----EEEDPPAKRKCIF--------------------------------------------MSETQS-----

Drosophila SU(VAR)2-10 516 --LSDSDDDMPLAKRRPPAKQAVAS--------------------------------------------STSNGSGGGQR

Arabidopsis PIAL1 730 LTPANTENWRPQSRMRGSVAPG---TGYDHMIIHPTRPVHPQAQT-PPAPLSTSYDGADEIQAFIGHPSYPVSNNETQAG

Arabidopsis PIAL2 666 LTVPDTSNWRP--RMRGSLVPGSHSTALDHMIIRPSQQSQTSTRLNSSQPVQTPSVQTSQAQSPFTTAAYRT---ETVLG

Human PIAS2 498 ------SPTKGVLMYQPSSVRVPSVTSVDPAAIPPSLTDY--SVPFHHTPISSMSSDLPGEQRRNDINNELKLGTSSDTV

Drosophila SU(VAR)2-10 551 AYTPAQQPQQSESPEQQASRQSPEKQTVSEQQLQQQQHEQPATAAVHASLLESLAAAVADQKHFQLLDLAAVAAAAAATA

Arabidopsis PIAL1 806 TSSLPVAEGLGYSGSFWSMPPETW

Arabidopsis PIAL2 741 NRNHPVPAPP---GIVRPTGPTS-

Human PIAS2 571 QQ----------------------

Drosophila SU(VAR)2-10 631 SSGQSQNAGP--------------


Supplemental Figure 4. Alignment of PIAL1 and PIAL2 in Arabidopsis, PIAS2 in human,

and SU(VAR)2-10 in Drosophila. Black and gray boxes indicate that the residues are highly

and weakly conserved, respectively.


4

Aravidopsis thaliana 1 ---------------------------------------MVIPATSRFGFR---------AEFNTKEFQASCISLANEID

Arabidopsis thaliana 1 ------------MSTAAAARPVAGTGLREKTAASLVNSFRLASVTQRLRYHIQDG-----AKVDPKEFQICCISFAKGID

Glycine max 1 -----------------MNNSLPALT--SDTSPSVVNLFRINKVADRLSWIAQPG-----NRGEPYEFYNLCLSLSRGID

Brassica napus 1 -----------MSSASTAVRPVAGTGLPEKAAAALVNSFRLASVTQRLAFHIQTG-----AKSDVKEFQSCCISLAKGID

Vitis vinifera 1 ------MTGATISLPTSVSNIAGVETTSTSYSASLANSFRLNAVLERLAMHVRSGHRILDGQRSTEEFHNLCLSLARGID

Populus trichocarpa 1 ---MVGMMAGTMMPPPQVAEGVTIRAAGQQMSASLANSFRVHAVAERLSLHIQPG---FDS--NPTEFFGLCLSLARGID

Oryza sativa 1 MASAAPLP--PTPPPSQQQQPQPQGKEQQQQMAVAMNARRLVMIGDRLRTHFRGGG---GTVLEPPDLAHLVYAFARGID

Sorghum bicolor 1 MATAAATAPHPTPPTPLQQQQHQQDKDQKQQKALSMNAIRLKAIGDRLKAHLRG-----MNVPPIAEFAHLVYAFARGID

Aravidopsis thaliana 33 AAIGRNEVPGNIQELALILNNVCRRKCDDYQTRAVVMALMISVKSACQLGWFPERETQELLAIIDLMWNGFSCPENVTSC

Arabidopsis thaliana 64 FAIANNDIPKKVEEFPWLLKQLCR-HGTDVYTKTALMVLMISVKHACHLGWFSDSESQELIALADEIRTCFGSSGSTSPG

Glycine max 57 YALANGETPPKAHELPLLVKQICQ-LKNDECSQAAMMVLMISIKNACEIGWFQTKESEELVTIADEIRKVYSSLGTINVG

Brassica napus 65 FAIANNEIPKKVEDLPSLLKQVCR-HRDDVYTKTAVLVLMISIKHACKLGWFSDSEAQELIALADQMKNGFGNPENTIPV

Vitis vinifera 75 YSLANGEVPARVQDLPLLLKQICQ-RRNDLFLLGGIMVLMVSVKNACKVGWFTEKDTEELLTLVNEIGSNFCNLGDNNTE

Populus trichocarpa 73 FAVANNETLLKAQELPFLLKQVCQ-RKNDVFLQAAIMVLMASVKNACMVGWFQEKETQELVTLATEIGKVFCTPGDINAG

Oryza sativa 76 FALSSGDVPTVASEIPSILKKVYL-VGKDQFLQSSVMVLMISCKNACSEKWFQPTDCTEILRMANELSGKFCTPVSQPDN

Sorghum bicolor 76 FAVSAGDIPQMAGDIPDILRKVYD-LRREMFIQSSLMVLVISCKNACSNNWFQPADSRDIFRMANELSGNFCTSTGQAAS

Aravidopsis thaliana 113 VNSPVTLISQVIERFYPCVKLGHILVSFEAKPESKMMMKDFHISKKMPHSPKQKVGLFVVRTEDISRSNCIVHPQGVSFL

Arabidopsis thaliana 143 IKSPGSTFSQIMERFYPFVKLGHVLVSFEVKAGYTMLAHDFYISKNMPHSLQEKIRLFVAQTDNIDTSACISNPPEVSFL

Glycine max 136 PRSCSTAISTIMQKFYPKFKLGPILASIEAQPGYGASVVDFHITKS--EVLKDKIFLLVAQTDNIETSACLINPQQVNFL

Brassica napus 144 IQSPGGTLSQIMERFYPLVKLGHVLVSLEVKSGYTMLAHDFHISKNMPHSPQEKIRLFVVQTENIDTSACIINPPEVSFI

Vitis vinifera 154 PISFHPTISKIMTRFYPRMEMGQILASREVKPGYGTFLVDFHISKSTKFSSQEKIRLFVAQTDNIETSSCIITPPQVNFL

Populus trichocarpa 152 TTDSLSIIPTIMSRFYPLMKMGQIIVSLEVKPGFGAHVIDFHISKTTRNSTEDKI-------------------------

Oryza sativa 155 DSTVIQIISTIMPRYYPQLKFERLVTSLEAKVGYDVLMADFFIHKN--VPREEKINLIVVQKEDLNASSCIANPPHVSFL

Sorghum bicolor 155 DSTVLEIISQIMPRYYPRLKFERLITSIEAKVGYDILMADFFIERN--LSRDEKIRLIVVQKENLDASSCVSSPPHVSFL

Aravidopsis thaliana 193 LNGKGIDKRVNISMESGPQLPTNVTALLNLGANLLQAIGCFGGSYLIAIAFMDVIPLPNKPLLKDYVHPEVVGSNSDCDI

Arabidopsis thaliana 223 LNGKGVEKRVNIAMDTGPQLPTNVTAQLKYGTNLLQVMGNFKGNYIIIIAFTGLVVPPEKPVLKDYLQSGVIEASPDSDI

Glycine max 214 LNGKGVLNRTNVQMDPGPQVPTNVTGMLKFGTNLLQAVGQFNGRYVVLVAYMSFTPFLEDPVLQDYLQPAVTSVDSDSDI

Brassica napus 224 LNGKVVEKRVNISMDSGPQLPTNVTAILKYGTNLLQVMGNSNGHYIIVIAFTGLAQLPEKPVLKEYVQSGVVEASPDSDI

Vitis vinifera 234 LNGKGVERRTNVFMDSGPQIPTNVTPMLKYGTNLLQAVGQFNGHYILAIAFMAVISSPDNPVLQDYVQPAVSMLHSDNEI

Populus trichocarpa 206 --------------DTGPQMPTNVTGMLKYGTNLLQAVGQFK-----AVAFMSVEPKPETPVLQDYVHPCAAELDP----

Oryza sativa 233 VNGKGVDKRTNVSMETGPQFPTDITRMLKYGANIIQAIGYFNANYIIAVAFLNKLESFDAPNLNDYAQP-VAADPPDSDL

Sorghum bicolor 233 VNGRGVDRRTNVSMEPGPQFPTDITKMLKYGANIIQAVGYFNANYIIAVAFVNNLTSFSAPKLDDYAQP-ITVYPADSDV

Aravidopsis thaliana 273 IEGPSRISLSCPISRTRIKLPVKGHVCKHLQCFDFWNYVNMNTR------------------RHHGAARIILEEVGRNAA

Arabidopsis thaliana 303 IEGPSRVSLSCPISRKRIKLPVKGQLCKHLQCFDFSNYVHINMRNPTWRCPHCNQPVCYPDIRLDQNMAKILKDVEHNAA

Glycine max 294 IEGASQISLNCPISFTRIKTPVKGHSCKHFQCFDFDNFINMNSKRPSWRCPHCIQNVCYADIRLDRNMVEVLKNVGENIT

Brassica napus 304 IEGPSRVSLRCPISRSRIKLPVKGQLCKHLQCFDFWNYVNINMRNPSWRCPHCNQPVSYPEIRLDQNM--ILKDAGRNAA

Vitis vinifera 314 VEGPSRISLNCPISRTRIKVPVKGHSCKHLQCFDFGNFVEINSRRPSWRCPHCNQYVCYTDIRIDQNMVKVLKEVGENVA

Populus trichocarpa 263 -------------------------ATHESELLSKGIHADIFS---------------MSATQIFELIKTWLRQV---VG

Oryza sativa 312 LEGPSRVSLKCPISFRRIKTPIKGRLCKHYQCFDYDNYMELNLRKPTWRCPFCNTPSNFTDLRIDQKMVKILQETGEDTI

Sorghum bicolor 312 LEGPSRVSLNCPISFDVELIFSLPSLVAVLGCFDYDNYMDMNSRKPNWRCPYCNTSSSFTDLRIDQKMMKILEETGDDVT

Aravidopsis thaliana 335 DVVISADGTWMVETENDEDVELVP-ETTHDHGDPNSFIN------LGPTVKNPARDE------NEMETSTQVEEHNPCL-

Arabidopsis thaliana 383 DVIIDAGGTWKVTKNTGETPEPVR-EIIHDLEDPMSLLN------SGPVVFDLTGDD--DAELEVFG-DNKVEDRKPCM-

Glycine max 374 EVIVLANGSWKAVLEKDHDVDKMQKKARNCEKEQTQPQESTCP----PGTVDLTKDD----DGLDTVGSCDIVERKPTP-

Brassica napus 382 DVVIHAGGTWKVAMENNGNEEPVRDAIIHDLEDPNSLLN------AGPVVLDLTGDDEDDADIELFGSTSKAVDQKPHL-

Vitis vinifera 394 DVIISADGSWKAILESNDHVDQPRVGTLNSQQKGPDLQGSTSFSNASPNVWDLTEGD----DEMNAFDACEIEDRKPFQ-

Populus trichocarpa 301 DVIISADGSLKAISESDNKVDQTQERTLHCEKGMPEQVESMTSTRALPMVMDLTVDD----DEINGEDNIDAEDRKPFL-

Oryza sativa 392 DVLVFADGSWKAISTNDERSDRHSSDVIQQSRDTMDTDA------TADDVIDLINED--NDGDVPMSFTSASEDVKPFLN

Sorghum bicolor 392 DVLVFADGSWKAAPAQDEKSDRHRGDAIQQTGDSTETDS------PSSDVIDLINGN--DDGDMQMDWVSAPEDTKPLLN

Aravidopsis thaliana 400 ---SEIQGPSND----THRPASDYTMLNQSHTSTNTLPQLPRTL--------------------NAFDGQQFVNLPQVIN

Arabidopsis thaliana 451 ---SDAQGQSNNNNTNKHPSNDDYSSIFDISDVIALDPEILSALG---------NTAPQPHQASNTGTGQQYSNLSQIPM

Glycine max 444 ---ASIHSQFVTPN----STSLGMNSTGVNQN-VATQIDDFWPG----------------VCFVRSRSDTPTVGNSELPV

Brassica napus 454 ---SDAQGQSNNNNANKDASVDDYCSMFNFSDVISLDEVMLDHL--------------------NTGTGQDYSNLSQVPM

Vitis vinifera 468 ---SNIQGHSITTK--QTMAPELNNATEVNQNAVSRVQDGFCSGILLSTYGSSTHSARSDAQFIGGTSQPSPANFLLPPV

Populus trichocarpa 375 ---ATLQNHPVDTNPIPTMPSQLINANAPSRN-FSTLADEFWS----SPYWS---SSASDAQMVNGFSEPSTTTFMTSPV

Oryza sativa 464 CQDLSVADYLSDLPMNTVSQAEDLYAGGASRGNNERGNATSTSGQ---------------NSSLPSTGGLGSSSFGTLES

Sorghum bicolor 464 SQDLSVSDYLTDLPM--AVQTGDLYRG---DGNNGGSNMAFTSRQ---------------NLLLPPTSGLGSSSFGTLES

Aravidopsis thaliana 454 TRDSPASQALPMTFSPTPSPQDILATNAANFGTSMPAAQSSQFQGSHVTSLGNCEGRTSDLMARWNHIYGRVQTQFPPAP



5

Arabidopsis thaliana 622 -------------------------------------------------------VLS-QPNNYG----VRGLTS-----

Glycine max 657 NTSAPTQPQNQ---YRANAFSEFRNLHLQQALNLRPPPPRSSNAQWPRIQQGVPQSGNFQAAARG-ASVAAGQGSSHARN

Brassica napus 598 -------------------------------------------------------VLAPQPNSYF----ARSLNS-----

Vitis vinifera 686 AGHPTTSQRPGPGAYRTSSGLPTEPQTLQQQ---QSPQARTHSNLLRSSAHHHSRSQVQQGGAQGRATHAVGTGISQNAQ

Populus trichocarpa 598 VTLPSSSQLAG--AHRASS-----PNLLYQQP-LRVPQSRSHSPNVVRSSLPLAPAQTQQGAAQVGVGNSAGATNSQQSR

Oryza sativa 666 QQEIRATQNYH----------------QGQFIGLTAPQNFMGTRPPPGVPGQAIGANAHGAPPAQQSHHVHRLVSNLMN-

Sorghum bicolor 655 -------------------------------------------------------AASFPAPAASDSL------------

Aravidopsis thaliana 676 RTS----------------PVNYGGTPDQRPMPSSITHPQTLPVSYGG-------TTDQILNPGGAMGQFSSREFMNLTP

Arabidopsis thaliana 637 ---------------------SHASTSRQHPS-----GPTVQSVSR-----------------------LSDLVDVDLTV

Glycine max 732 ----V-PTSGATTHSHQARGMVANQPAR--PSVLVQNQSTVAGTPFHG-------LTTEQRGNTA---QSVSRPEELFSS

Brassica napus 614 ---------------------NHLTTQTQRPS-----SPPVLSVSR-----------------------TSDLMDVDSAT

Vitis vinifera 762 ----P-MVA----------AQRAAQMTRMP--LPVQNQTSRTGSAFPVNANGGRSTAGEQRGNIEGMVQAVSRPESLVDL

Populus trichocarpa 669 ----L-MVA----------AQLAAQRARQPPSVPVQIQTSGAGASYLTSADGIRAPATEQRGNAGGALPAVSGTEGLVDL

Oryza sativa 728 ---------------------QLGQATVAQPSTAPQVLPSQPGGTSAVNPQIRGHLFPAQQRSQAMRPQAVPRPTISQAP

Sorghum bicolor 668 --------------------------------------------------------------------------------

Aravidopsis thaliana 733 ANTENWRPQSRMRGSVAPG---TGYDHMIIHPTRPVHPQAQTPPAPLSTSYDGADEIQAFIGHPSYPVSNNETQAGTSSL

Arabidopsis thaliana 669 PDTSNWRP--RMRGSLVPGSHSTALDHMIIRPSQ----QSQTSTRLNSSQPVQTPSVQTSQAQSPFTTAAYRTETVLGNR

Glycine max 796 QSEQNWAPTGRMRGSLDLSQLNDESIAQRIITPTQG-QNSRPPGPQPIRRTGIS-SLQPATT-QQDVLIANNRNANAHNR

Brassica napus 646 PDTSNWRP--RMRGSITPGSYSPALDHMIIRPTQ----QSQ--TRLQVSQPGQTPPVQTSQALPPFST------------

Vitis vinifera 826 ASEQNWRPTGLMRGSLVG-RAYNSALNQLVIQPTQPTQSTRPPTPITSPPPGFPPHLQALLTNIRTPLVPQAPNYPMTQP

Populus trichocarpa 735 ASEQNWRPTGRMRGSLSG-RAYSAALKEFMVQPTQQTQTPRPPPNLPPSQSSMPPHLQFLFA--RNAQVPQAQSSPVTGS

Oryza sativa 788 PRAQSPFLPATARPPSTPPPIGTSDDLQELPVDESWRPTGQMRGSLTGEAYSVAIGRYNPSVNIAGQQTSHVTSQARPAG

Sorghum bicolor 668 --------------------------------------------------------------------------------

Aravidopsis thaliana 810 PVAEGLGYSGSFWSMPPETW--

Arabidopsis thaliana 743 NHPVPAPPGIVRPTGPTS----

Glycine max 873 SSSR------------------

Brassica napus 705 -----APPTFTRPSGPTAPWGI

Vitis vinifera 905 ASTTGGSGILPERSLGLH----

Populus trichocarpa 812 AISNGSSSILP-----------

Oryza sativa 868 PDARR-----------------

Sorghum bicolor 668 ----------------------

Sorghum bicolor 464 SQDLSVSDYLTDLPM--AVQTGDLYRG---DGNNGGSNMAFTSRQ---------------NLLLPPTSGLGSSSFGTLES

Aravidopsis thaliana 454 TRDSPASQALPMTFSPTPSPQDILATNAANFGTSMPAAQSSQFQGSHVTSLGNCEGRTSDLMARWNHIYGRVQTQFPPAP

Arabidopsis thaliana 520 SIDP---MPVPVPFSQTPSPRDRPATTSTVFTIPNPSPQYSQVHASPVTPTGTYLGRTTSPRWNQTYQS---QAPPMTTP

Glycine max 501 LPDTVSPTFSQESAGHDNNP-VVNSAMHNQFLGPNNLQ-MQMNHMNSVN--EYGRSSSAPRHIHRTPVAVQALPVQSQAL

Brassica napus 512 PRDP---AHVPAPFSQAPSPRERPAATSTVFPS-------SRVHASPVTPAGTYLSRTSS------------QMSSLTTS

Vitis vinifera 544 LTDAISPALNRGTEDIRGNTHLTTSTLHDQLPIPDSLQLQQAQFGHSIVSNEYGRFPTIPRHITRTPIAVQALPAQTQTS

Populus trichocarpa 445 ITDSVSPALNCDVGGYGNTT--TSSVMHNQLSASIYLQSLQQNFVNSVANGEYGTLPPIY-HVDRSPIAVQALPARPQTP

Oryza sativa 529 ILPHNILHPVITDAVSPSLDTSNSVVLRQHVAQGTRSDIVPSQPRIDPQLRLEIARPPIPRNVAREPTGIQALPVQPQ--

Sorghum bicolor 524 ILPQNVLRPVITDAVSPSLETSTSTSGMQHVSQETHCGTVQLQAQIGPVHGSELRRLPIPRNPRREPVGVQALAVPPQNP

Aravidopsis thaliana 534 LSHHHYSMQN------------------QSPSPAQQRPVPSYIAHPQTFHVNYGENADQRWMPSSIAHPQTLPVNYGGNT

Arabidopsis thaliana 594 YTSRKVSVPV------------------TSQSPANV---------------------------SSFVQSQHVPR------

Glycine max 577 GPQQNSITNLNSSLLPSNSSATPHISLSNPTSVDTLNAILSDTERQQHFSRTPMNLPQVSGVNSPAFQHHTATQNRGPLI

Brassica napus 570 SQSRVHPVQV------------------TSQSLGNG---------------------------SSLAQSPRIPR------

Vitis vinifera 624 GPHHRSRTT----------------LISMVP--NGPNTVGSDMERPQQFSRSIFNPVQISDISASALQHHSMSQNWNQQV

Populus trichocarpa 522 APQQRSRTPN--PAISSGASLSSHGTLPEAA--NGLSPVSGNMDRQQQFARSLNTNSSSSQNWNMQDHPFMHGQSAQQQA

Oryza sativa 606 ----RVRPNIYN----------------CPPPFPQSSP-ASAYQVHQVTNADSVITAMSTGIGSLSRAPDAAPLLQHQST

Sorghum bicolor 604 GSSTRLQPNILN----------------CPPPIPLSSPSSSTYQTHQVTNPDSVIAPMNSGSGPLPRT------------

Aravidopsis thaliana 596 NQRPIPSSIAHPQTLPVNYRGNTDHRSTPYSITHLQTLLNYGGNADQRPMPSSITNLQTLPATYGGYAHQRPMSSSITHP

Supplemental Figure 5. Alignment of Arabidopsis PIAL1 and PIAL2 and their homologs in

other plants including Glycine max, Brassica napus, Vitis vinifera, Populus trichocarpa,

Oryza sativa, and Sorghum bicolor. Black and gray boxes indicate that the residues are

highly and weakly conserved, respectively.


6

ACT7

AT5TE40510

AT4TE18715

AT2TE82000

AT3TE47360

AT3TE54005

AT4TE04415

AT2G13770

AT2G10975

AT2TE17230

AT2TE18240

(II)

(III)

(I)


Supplemental Figure 6. Validation of the transcript levels of the MOM1, PIAL1/2, and/or

NPRE1 target loci in the wild type, nrpe1, mom1, and pial1/2. Indicated loci were randomly

selected from up-regulated loci identified by the RNA deep sequencing analyses. The

transcript levels of the indicated loci were determined by RT-PCR. The loci that are

transcriptionally up-regulated in nrpe1, mom1, and/or pial1/2 were placed into three classes

based on the transcript patterns of the loci. I, loci up-regulated in mom1 and pial1/2 but not

in nrpe1; II, loci up-regulated in nrpe1, mom1, and pial1/2; III, loci up-regulated in nrpe1 but

not in mom1 or pial/2. The actin gene ACT7 was used as an internal control. The RT-PCR

experiment was biologically repeated for at least two times and the result of a representative

experiment is shown.


7


nrpe1 (5261) mom1 (177)

4972

26 92

55

142 25

4

pial1/2

(226)

CHH hypo-DMRs

nrpe1 (313) mom1 (173)

263

118 31

14

5 30

10

pial1/2

(59)

CG hypo-DMRs

nrpe1 (874) mom1 (38)

861 25 9

1 3 5

3

pial1/2

(12)

CHG hypo-DMRs A

B CHH hypo-DMRs CG hypo-DMRs CHG hypo-DMRs

Supplemental Figure 7. Analyses of hypo-DMRs in nrpe1, mom1, and pial1/2. (A) Overlaps of

hypo-DMRs in nrpe1, mom1, and pial1/2 relative to the wild type at CG, CHG, and CHH sites.

Hypo-DMRs were separately defined based on the DNA methylation levels of CG. CHG, and

CHH sites in the mutants relative to the wild type. Bins were recognized as DMRs when their

absolute DNA methylation change is more than 40%, 20%, and 10% for CG, CHG, and CHH,

respectively. (B) DNA methylation levels of hypo-DMRs in the wild type, nrpe1, mom1, and

pial1/2. CG, CHG, and CHH hypo-DMRs in nrpe1 were separately analyzed.


8

Up-regulated

in pial1/2

WT mom1 nrpe1 WT

Up-regulated

in nrpe1

Up-regulated

in mom1

pial1/2 WT

Up-regulated

in pial1/2

WT mom1 nrpe1 WT

Up-regulated

in nrpe1

Up-regulated

in mom1

pial1/2 WT

TEs Genes

CG

me

thyla

tio

n

CH

H m

eth

yla

tion

CH

G m

eth

yla

tio

n


**

**

**

**

** * 9.63E-03

6.67E-05

4.33E-09

1.31E-02

1.23E-11

1.35E-14

* 1.40E-02

Supplemental Figure 8. Box plots showing CG, CHG, and CHH methylation of

transcriptionally up-regulated TEs and genes in nrpe1, mom1, and pial1/2 relative to the

wild type. *p<0.05 or **p<0.01 was determined by t test. In the box plots, gene promoters

were included for DNA methylation analysis only when their DNA methylation levels in the

wild type were higher than 5%.


9

0%

10%

20%

30%

40%

50%

0%

10%

20%

30%

40%

50%

0%

10%

20%

30%

40%

50%

0% 10% 20% 30% 40% 50% 0% 10% 20% 30% 40% 50%

0%

20%

40%

60%

80%

0%

20%

40%

60%

80%

0%

20%

40%

60%

80%

0% 20% 40% 60% 80%0% 20% 40% 60% 80%

0%

20%

40%

60%

80%

100%

0%

20%

40%

60%

80%

100%

0%

20%

40%

60%

80%

100%

0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%

Up in nrpe1 Up in nrpe1

Up in mom1 Up in mom1

Up in pial1/2 Up in pial1/2

Genes TEs

Meth

yla

tio

n

in n

rpe

1

Meth

ylation

in p

ial1

/2

Meth

yla

tio

n

in m

om

1

Up in nrpe1 Up in nrpe1



Genes TEs Up in nrpe1 Up in nrpe1



Genes TEs CG methylation CHH methylation CHG methylation


Supplemental Figure 9. Scatter plots showing CG, CHG, and CHH methylation of

transcriptionally up-regulated TEs or genes in nrpe1, mom1, and pial1/2 relative to the wild type.

The DNA methylation levels of TEs and genes that are transcriptionally up-regulated in the

mutants were analyzed. Diagonal red lines indicate that the DNA methylation levels of mutants are

the same as those of the wild-type.

Methylation in WT


10

PIAL2-WT PIAL2-RING-M

PIAL2-Flag + pial2

PIAL2-SIM-M

PIAL2-RING-

SIM-M

1 2 1 2 3 4 5 6 1 2 3 1 2

PIAL2-Flag

Ponceau S.

stained


Supplemental Figure 10. The expression levels of the wild-type and mutated PIAL2

transgenes in the pial2 mutant. The expression levels were determined by immunoblot

assays with anti-Flag antibody. Rubisco proteins stained by Ponceau S. are shown as a

loading control. Boxed transgenic lines were used for complementation assays by

quantitative RT-PCR and the results are shown in Figure 6C. The expression of PIAL2-Flag

in the boxed transgenic lines is also shown in Figure 6B.


11

Mutant No. Mutant gene Mutation position

related to ATG Mutation Code change Mutation type

84-3 PIAL2 2769 C→T splice site

4-1 MORC6 548 G→A TGG→TGA premature stop codon

22-1-1 MORC6 3049 G→T GAG→TAG premature stop codon

59-1 MORC6 3031 C→T CAG→TAG premature stop codon

138-2 MORC6 4123 C→T CAA→TAA premature stop codon

215-3 MORC6 2904 G→A splice site

249-1 MORC6 3170 G→A GGA→GAA G→E

250-1 MORC6 3169 G→A GGA→AGA G→R

200-1 MORC6 315 G→A splice site

294-1 MORC6 1800 C→T GCT→GTT A→V

31-2 MOM1 4782 G→A splice site

38-1 MOM1 400 C→T CGA→TGA premature stop codon

270-2 MOM1 1036 C→T CAA→TAA premature stop codon

310-9 MOM1 2350 G deletion

13-2 MOM1 6181 G→A splice site

241-1 MOM1 3007 G→A GAA→AAA E→K

78-2 AGO4 2457 C→T splice site

91-1 AGO4 4213 C→T splice site

139-3 AGO4 469 C→T CAA→TAA premature stop codon

136-3 AGO4 3256 G→A splice site

62-1 AGO4 2555 G→A GGA→GAA G→E

227-2 AGO4 3893 G→A TGG→TGA premature stop codon

310-17 AGO4 3370 G→A TGG→TGA premature stop codon

190-1 AGO4 3785 G→A splice site

24-1 AGO4 199 C→T CAA→TAA premature stop codon

144-1 AGO4 3148 G→A GAG→AAG E→K

178-1 AGO4 1789 G→A TGC→TAC C→Y

324-2 AGO4 3199 G→A GGG→AGG G→R

131-1 NRPD1 2839 C→T CAG→TAG premature stop codon

45-1 NRPD1 3412 C→T splice site

120-3 NRPD1 3169 C→T CAG→TAG premature stop codon

200-2 NRPD1 2752 C→T CAA→TAA premature stop codon

268-2 NRPD1 225 G→A GGA→AGA G→R

95-1 NRPD1 1828 C→T CCT→TCT P→W

40-1 DRD1 2014 C→T CCA→TCA P→S

226-1 DRD1 3127 G→A GAA→AAA E→K

231-1 DRD1 3209 G→A GGG→GAG G→E

272-5 RDR2 2548 C→T CAG→TAG premature stop codon

33-1 RDR2 3120 C→T CTT→TTT L→F

218-2 NRPD4 199,200 CC→GG splice site

139-2 RDM4 49 C→T CCT→TCT P→S

17-4 NRPD2 4022 G→A GGG→AGG G→R

25-1 MET1 4596 C→T CCT→CTT P→L

218-1 MET1 3016 G→A GAG→AAG E→K

28-6 MET1 4557 G→A CGA→CAA R→Q

68-2 FPGS1 3803 G→A TGG→TGA premature stop codon

25-9 FPGS1 3403 G→A TGG→TGA premature stop codon

104-1 FAS1 455 G→A TGG→TAG premature stop codon

112-2 BRU1 5091 G→A splice site

Supplemental Table 1. Full list of mutants identified by map-based cloning in this study.


12

DOI 10.1105/tpc.15.00997; originally published online April 25, 2016;Plant Cell

Huan-Wei Huang, Yong-Qiang Li, Lin Li, Tao Cai, She Chen and Xin-Jian HeYong-Feng Han, Qiu-Yuan Zhao, Liang-Liang Dang, Yu-Xi Luo, Shan-Shan Chen, Chang-Rong Shao,

complex required for transcriptional silencingThe SUMO E3 ligase-like proteins PIAL1 and PIAL2 interact with MOM1 and form a novel

This information is current as of April 25, 2016

Supplemental Data http://www.plantcell.org/content/suppl/2016/04/25/tpc.15.00997.DC1.html

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