RESEARCH ARTICLE 1 2

HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition 3 from Seed Maturation to Vegetative Growth in Arabidopsis 4

5 Naichong Chen1,2, Vijaykumar Veerappan1,3, Haggag Abdelmageed1,4, Miyoung Kang1,2, 6 Randy D. Allen1,2* 7

8 1Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, OK 73401, USA 9 2 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, 10 OK, USA 11 3Department of Biology, Eastern Connecticut State University, Willimantic, CT 06226, USA 12 4Department of Agricultural Botany, Faculty of Agriculture, Cairo University, Giza 12613, Egypt 13 *Corresponding author: randy.allen@okstate.edu14

15 Short title: AGL15 is a regulatory target of HSI2/VAL1 16

17 One-sentence summary: AGL15 is a direct regulatory target of the transcriptional silencing 18 factor HSI2/VAL1, which is involved in the developmental transition from seed maturation to 19 germination and growth. 20

21 The author responsible for distribution of materials integral to the findings presented in this 22 article in accordance with the policy described in the Instructions for Authors 23 (www.plantcell.org) is: Randy D. Allen (randy.allen@okstate.edu). 24

25 ABSTRACT 26 Gene expression during seed development in Arabidopsis thaliana is controlled by transcription 27 factors including LEAFY COTYLEDON 1 and 2 (LEC1 and LEC2), ABA INSENSITIVE 3 28 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE 15 (AGL15). The 29 transition from seed maturation to germination and seedling growth requires the transcriptional 30 silencing of these seed maturation-specific factors leading to down-regulation of structural genes 31 including those that encode seed storage proteins, oleosins, and dehydrins. During seed 32 germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the 33 transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis 34 indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, 35 ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain 36 interacts with two RY elements upstream of the AGL15 coding region and at least one of them is 37 required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed 38 maturation regulatory genes LEC1, ABI3, FUS3 and LEC2 in different genetic backgrounds 39 demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory 40 program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This 41 hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the 42 Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 43 locus. 44

45

INTRODUCTION 46

Plant Cell Advance Publication. Published on February 23, 2018, doi:10.1105/tpc.17.00655

©2018 American Society of Plant Biologists. All Rights Reserved

2

Seed development in plants involves two major phases: morphogenesis and maturation. 47

During morphogenesis, specification of various developmental domains of the embryo is 48

followed by the differentiation of tissues and organ systems. The subsequent seed 49

maturation phase is characterized by accumulation of storage carbohydrates, proteins, and 50

oils followed by acquisition of desiccation tolerance and dormancy. Establishment of the 51

seed maturation program requires at least four master regulators, collectively known as 52

the LAFL group of transcription factors (reviewed by Jia et al., 2014). This group 53

includes the CAAT-box family protein LEAFY COTYLEDON 1 (LEC1) (Lotan et al. 54

1998), and the B3-family proteins ABSCISIC ACID INSENSITIVE 3 (ABI3), FUSCA3 55

(FUS3) and LEC2 (Luerben et al., 1998; Stone et al., 2001; Finkelstein et al., 2008). 56

These LAFL regulators form a complex genetic network and act in concert to promote 57

the seed maturation program by activating the expression of downstream genes related to 58

storage product accumulation and desiccation tolerance (Holdsworth et al., 2008; Suzuki 59

and McCarty 2008; Jia et al. 2013). 60

During embryogenesis, AGAMOUS-Like15 (AGL15), a MADS domain transcriptional 61

regulator, is preferentially expressed and primarily accumulates during early stages of 62

seed development (Heck et al., 1995; Rounsley et al., 1995). In Brassica, maize, and 63

Arabidopsis, AGL15 transcription remains high during embryo morphogenesis until the 64

seeds start to dry (Perry et al., 1996). Constitutive expression of AGL15 promotes seed 65

development and somatic embryogenesis in Arabidopsis and loss-of-function mutation in 66

both AGL15 and the closely related MADS domain gene AGL18 leads to decreased 67

somatic embryo tissue production (Harding et al., 2003; Thakare et al., 2008). AGL15 68

has been found to physically bind to the chromatin of LEC2, ABI3 and FUS3 and 69

upregulate the expression of these genes during embryogenesis (Zheng et al, 2009). In 70

previous reports, we showed that expression of AGL15 is strongly upregulated in both 71

hsi2-2 knockout Arabidopsis plants and in hsi2-4 plants that carry a C to Y substitution in 72

the HSI2 PHD motif (Veerappan et al., 2012). This change in expression is associated 73

with a dramatic loss of histone 3 lysine 27 tri-methylation (H3K27me3) at the AGL15 74

locus (Veerappan et al., 2014). 75

3

Under favorable conditions, seed storage reserves are remobilized and degraded to 76

provide energy for subsequent germination and seedling growth. The seed maturation 77

program is inhibited in germinated seeds by two transcriptional repressors, HIGH-78

LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE 2 (HSI2) and HSI2-LIKE 1 79

(HSL1), to ensure the developmental transition from seeds to seedlings (Tsukagoshi et al. 80

2005, Tsukagoshi et al. 2007). HSI2 and HSL1 are also named as VP1/ABI3-LIKE1 81

(VAL1) and VAL2, respectively (Suzuki et al.; 2007). HSI2 and HSL1 share high 82

sequence similarity and, like LEC2, ABI3 and FUS3, belong to the B3 DNA-binding 83

domain-containing family of transcription factors. Upon germination, expression of HSI2 84

and HSL1 is induced and reaches to peak at 5 days after germination (Tsukagoshi et al. 85

2005, Tsukagoshi et al. 2007). HSI2 and HSL1 show partial functional redundancy. 86

Although morphologically normal, hsi2 or hsl1 mutants show increased expression of 87

seed maturation genes during seedling growth. However, double hsi2 hsl1 mutants show 88

much higher expression of seed maturation genes, leading to inviable seedlings with 89

embryonic characteristics (Suzuki et al. 2007, Tsukagoshi et al. 2007; Holdsworth et al. 90

2008, Suzuki and McCarty 2008). 91

The B3 domain is a plant-specific DNA binding domain that specifically interacts with 92

RY DNA motifs (CATGCA) in target genes (Suzuki et al. 1997; Reidt et al. 2000; Monke 93

et al. 2004; Braybrook et al. 2006; Jia et al. 2013; Qüesta et al., 2016; Yuan et al., 2016). 94

The majority of genes up-regulated by more than four-fold in hsi2hsl1 double mutant 95

seedlings contain one or more RY motifs in the upstream region or the first intron 96

(Suzuki et al., 2007). Recently, electrophoretic mobility shift and yeast-one hybrid assays 97

indicate that the HSI2-B3 domain binds to RY elements of FLOWERING LOCUS C 98

(FLC) and represses its expression (Qüesta et al., 2016; Yuan et al., 2016). In addition, 99

HSI2 contains other conserved motifs, including a plant homeodomain (PHD), cysteine 100

and tryptophan residue-containing domain (CW), and an ethylene-responsive element 101

binding factor-associated amphiphilic repression (EAR) domain. The PHD finger is a 102

zinc finger domain that is reported to recognize histone 3 tri-methylated at lysine 4 103

(H3K4me3) and di- or tri-methylated lysine 27 (H3K27me2/3) (Chakravarty et al., 2009; 104

Zeng et al., 2010; Yuan et al., 2016). Genetic disruption of the HSI2-PHD domain in 105

Arabidopsis results in decreased H3K27me2/3 and increased H3K4me3 enrichment at 106

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certain seed-related genes, correlating with ectopic expression of those genes in seedlings 107

(Veerappan et al. 2012; Veerappan et al. 2014; Yuan et al., 2016). The CW domain also 108

recognizes H3K4me2 and H3K4me3 in vitro (Hoppmann et al. 2011) and the HSL1-CW 109

domain interacts with HISTONE DEACETYLASE 19 (HDA19) in vivo to down-regulate 110

a subset of seed maturation genes by alteration of histone acetylation at the target gene 111

loci during seed germination (Zhou et al. 2013). The EAR domain is a repressive motif 112

that recruits co-repressors, such as SWI-independent 3 (SIN3) and TOPLESS (TPL) that 113

subsequently recruit a histone deacetylase complex (HDAC) to target gene loci 114

(Tsukagoshi et al. 2005; Ohta et al. 2001; Kazan 2006; Kagale et al. 2011), and 115

disruption of the EAR motif of HSI2 reduces its ability to repress the expression of a 116

luciferase reporter gene driven by the sweet potato Sporamin A1 gene promoter in 117

Arabidopsis protoplasts (Tsukagoshi et al., 2005). However, the functions of these HSI2 118

functional domains and their interactions in the repression of seed maturation genes in 119

seedlings remain unclear. 120

Polycomb group (PcG) proteins regulate cell fate and identity in higher eukaryotes by 121

chromatin-mediated gene repression. In Arabidopsis, two groups of PcGs, polycomb 122

repressive complex 1 (PRC1) and polycomb repressive complex 2 (PRC2) exist in 123

multiple variants due to gene family expansion and functional diversification 124

(Derkacheva and Hennig, 2014). During vegetative development in Arabidopsis, PRC2 125

consists of CURLY LEAF (CLF) or SWINGER (SWN), EMBRYONIC FLOWER 2 126

(EMF2), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), and MULTICOPY 127

SUPPRESSOR of IRA 1 (MSI1). CLF and SWN have methyltransferase activity and 128

specifically catalyze tri-methylation of lysine 27 of histone H3 (H3K27me3) at target 129

gene loci to establish a repressive chromatin state (Chanvivattana, et al., 2004; Yoshida, 130

et al., 2001; Jiang, et al., 2008; Kinoshita, et al., 2001). MSI1 is a WD40 repeat protein 131

that is an indispensable component of PRC2 at all developmental stages (Kohler et al., 132

2003; Schonrock et al., 2006). MSI1 is required for seed development and loss of 133

function causes female sterility and seed abortion (Hennig et al., 2003; Kohler et al., 134

2003). Transgenic MSI1 co-suppression lines (msi1-cs), in which MSI1 expression is 135

reduced to 5% of that in wild-type plants, lose repression of some EMF2-target genes and 136

show strong defects in vegetative and reproductive development leading to sterility 137

5

(Hennig et al., 2003; Kohler et al., 2003). Recently, Mehdi et al. (2016) reported that 138

MSI1 interacts with HDA19 to form a histone deacetylase complex involved in the 139

suppression of ABA receptor gene expression, which is thought to attenuate ABA 140

signaling in Arabidopsis. Thus, MSI1 may have functions outside of the classical PRC2 141

context. 142

Here we report that two RY elements located upstream of AGL15 are direct targets of the 143

B3 domain of HSI2 and at least one of these elements is required for HSI2-dependent 144

silencing of the AGL15 promoter. Furthermore, the PRC2 protein MSI1 interacts with 145

HSI2 and is required for full transcriptional silencing of the AGL15 promoter. We 146

propose a regulatory model in which the LAFL genes LEC1, ABI3, FUS3, and LEC2 are 147

positively regulated by AGL15. HSI2-dependent silencing of AGL15 expression in 148

Arabidopsis seedlings results in down-regulation of these genes and repression of the 149

seed maturation developmental pathway. 150

RESULTS 151

HSI2 is enriched at the AGL15 promoter region 152

Since expression of AGL15 is strongly upregulated and H3K27me3 enrichment is 153

reduced in hsi2 mutants (Veerappan et al., 2012; Veerappan et al., 2014), we hypothesize 154

that the AGL15 locus is a direct regulatory target of HSI2. To determine if HSI2 interacts 155

with AGL15 in vivo, an epitope-tagged HSI2-rescued Arabidopsis line was generated by 156

the introduction of HSI2-HA coding sequence under control of the native HSI2 promoter 157

(HSI2pro:HSI2-HA) into hsi2-2 knockout plants that contain the GSTF8pro:LUC (hsi2-158

2LUC) reporter gene (Veerappan et al., 2012; Veerappan et al., 2014). Expression of the 159

luciferase reporter gene and selected seed-specific major transcriptional regulator genes 160

in plants of the rescued line was reduced from the high levels seen in hsi2-2LUC plants to 161

levels similar to that in wild-type plants, indicating that HSI2-HA is functional (Figure 162

1A-C). Expression of HSI2-HA in the rescued line was confirmed by immunoblot 163

analysis using anti-HA (Figure 1D). Chromatin Immunoprecipitation (ChIP) assays were 164

conducted on these plants to investigate the enrichment of HSI2 on chromatin of seed 165

maturation regulatory genes including LEC1, ABI3, FUS3, LEC2, and AGL15. Different 166

6

primer sets were used to query promoter (P) and the coding regions (C) of candidate 167

target genes by ChIP-qPCR. Significant HSI2 enrichment was detected on the proximal 168

promoter regions (P1 and P2) and 5ʹ coding area (C1) of AGL15, with strongest 169

enrichment at P1. Significant HSI2 enrichment was not detected at the distal upstream 170

region (P3) or the far downstream coding region C2 (Figure 2). Significant HSI2 171

accumulation was not observed in chromatin of the promoter or coding sequences of 172

LEC1, ABI3, FUS3, or LEC2 loci (Figure 2). Thus, among these master transcription 173

factor genes, only AGL15 appears to be enriched for HSI2 under the test conditions used. 174

RY elements are required for HSI2-dependent repression of AGL15 175

Sequence analysis indicated the presence of two canonical RY elements in the region 176

upstream of the transcriptional start site (TSS) of AGL15. This region corresponds to P1, 177

which showed significant HSI2 enrichment in ChIP-qPCR assays (Figure 2). To examine 178

the role of these RY elements in AGL15 repression by HSI2, a luciferase reporter gene 179

controlled by the AGL15 promoter (AGL15pro:LUC) or a mutated AGL15 promoter in 180

which both RY elements were disrupted (AGL15mpro:LUC) was tested by transient 181

expression in Nicotiana benthamiana leaves by agroinfiltration (Figure 3A). Substantial 182

levels of luminescence, indicating luciferase activity, and LUC mRNA were seen in 183

leaves agroinfiltrated with either the AGL15pro:LUC or the AGL15mpro:LUC construct 184

alone (Figure 3B). However, expression from the AGL15pro:LUC reporter construct was 185

strongly reduced when co-infiltrated with an HSI2-expressing effector construct, while 186

expression from the AGL15mp:LUC construct was unaffected by HSI2 co-expression 187

(Figure 3B). Furthermore, luminescence and luciferase expression assays of seedlings of 188

stable transgenic Arabidopsis lines that contained the AGL15pro:LUC or 189

AGL15mpro:LUC constructs in a wild-type genetic background showed that loss of the 190

RY elements in this promoter led to de-repression of the AGL15 promoter, resulting in 191

increased expression of the luciferase reporter gene (Figure 3C, D). 192

To investigate whether both RY elements are required for HSI2-dependent AGL15 193

transcriptional repression, luciferase reporter genes driven by mutated AGL15 promoters 194

in which one or the other RY element was disrupted (AGL15mpro1:LUC and 195

AGL15mpro2:LUC) were developed (Figure 3A) and agroinfiltrated, with or without the 196

7

HSI2-expressing effector construct, into N. benthamiana leaves for transient expression 197

assays. High levels of luciferase activity and LUC mRNA were detected when either the 198

AGL15mpro1:LUC or the AGL15mpro2:LUC construct was transiently expressed in N. 199

benthamiana leaves. However, reporter gene expression was significantly reduced when 200

either of these constructs was co-expressed with HSI2 (Figure 3E). These results 201

demonstrate that, at least in agroinfiltrated N. benthamiana leaves, a single RY element is 202

necessary and sufficient for HSI2-dependent repression of transcription by the AGL15 203

promoter. 204

B3 domain is required for HSI2 to bind to RY elements and repress AGL15 205

The B3 domain of HSI2 recognizes and binds to RY motifs in the FLC silencing 206

elements and is required to repress FLC expression (Qüesta et al., 2016; Yuan et al., 207

2016). Since, as shown in Figure 3, an RY element is required for the HSI2-dependent 208

repression of the AGL15 promoter, the HSI2 B3 domain may also play a role in AGL15 209

repression. To test this hypothesis, an effector construct that encodes a B3 domain-210

deleted HSI2 (HSI2-ΔB3) was co-infiltrated with the AGL15pro:LUC reporter construct 211

into N. benthamiana leaves (Figure 4A). Expression of the AGL15pro:LUC reporter gene 212

was strongly repressed by co-expression with HSI2 but, when co-expressed with HSI2-213

ΔB3, reporter gene expression remained high (Figure 4B). Previously reported soluble 214

structure analysis indicated that two lysine residues, which correspond to K297 and K314 215

of HSI2, are located on the DNA binding surfaces of ABI3 and RAV1 and these residues 216

were predicted to make direct contact with DNA (Yamasaki et al., 2004). Furthermore, it 217

was demonstrated that the DNA binding activity of maize VIVIPOROUS1 (zmVP1), a 218

member of the ABI3 family of B3 domain proteins, was lost almost completely when 219

K519, which corresponds with K297 of HSI2, was replaced by arginine (Suzuki et al., 220

2014). Therefore, we predicted that these two conserved lysines are required for the 221

function of HSI2. To test this hypothesis, K297 and K314 were replaced by arginine 222

(K297R, K314R) by site directed mutagenesis (Figure 4C). The HSI2pro:HSI2mB3 223

construct was transformed into hsi2-2LUC Arabidopsis to measure the ability of HSI2mB3 224

to repress the expression of the AGL15 genes in planta. Expression of HSI2mB3-GFP 225

was confirmed in the HSI2mB3 transgenic line by the GFP expression (Figure 4D). 226

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Relative expression of AGL15 in HSI2mB3 seedlings was significantly higher than in 227

WTLUC plants and similar to the levels in hsi2-2LUC (Figure 4D; Supplemental Figure 1). 228

Taken together, these results demonstrate that the HSI2 B3 domain is required for HSI2-229

dependent repression of the AGL15 promoter. 230

To test whether the HSI2 B3 domain can bind directly to the RY elements in the AGL15 231

promoter in vitro, His-Trx-tagged HSI2-B3 fusion protein was expressed in Escherichia 232

coli and purified using Dynabeads. For DNA pull-down experiments, this recombinant 233

protein was incubated independently with DNA probes containing two intact RY 234

elements or probes in which the 5ʹ (RY1) or 3ʹ (RY2), or both RY elements were mutated 235

(Figure 5A). Following purification of the protein–DNA complexes, the probe was 236

quantified using qPCR. The HSI2 B3 domain-containing fusion protein bound strongly 237

with DNA fragments containing tandem RY elements, while little binding activity was 238

detected when incubated with DNA fragments in which both RY elements were mutated 239

(Figure 5A). Binding of probes that contain single intact RY elements in the HSI2 B3 240

domain was reduced by more than 50% compared to the probe with two RY elements. To 241

confirm that single RY elements can bind the HIS B3 domain in vitro, electrophoretic 242

mobility shift assays (EMSAs) were also performed. RY1- and RY2-containing DNA 243

probes from the AGL15 proximal promoter were labeled with biotin at the 3ʹ end, while 244

the unlabeled probes were used as competitor. EMSA results showed that HSI2-B3 was 245

able to bind to both RY1 and RY2-containing probes (Figure 5B). Probes in which either 246

RY1 or RY2 sequences were mutated failed to bind HSI2-B3, and an HSI2-B3 247

polypeptide with a disrupted B3 domain failed to bind intact RY1 and RY2 probes. These 248

results indicate that the HSI2-B3 domain can specifically binds to the both AGL15 RY1 249

and RY2 elements. 250

To investigate if disruption of the HSI2 B3 domain affects HSI2 enrichment at the 251

AGL15 locus in plant cells, we performed ChIP-qPCR assays using hsi2-2 Arabidopsis 252

leaf protoplasts to express HSI2-GFP and HSI2mB3-GFP under control of the HSI2 253

native promoter, as described in the Methods. Our analysis revealed that, compared to 254

HSI2, accumulation of HSI2mB3 at P1, P2 and C1 areas decreased significantly (Figure 255

5C). However, compared to control protoplasts that express GFP only, enrichment of 256

9

HSI2mB3 at P1 and P2 sites was somewhat greater. This suggests that other motifs, in 257

addition to the B3 domain, could be involved in the recruitment of HSI2 to its target loci. 258

PHD domain is required for HSI2 to repress AGL15 expression 259

HSI2-dependent transcriptional repression of GSTF8pro:LUC reporter gene, along with 260

other HSI2-repressed genes, including AGL15, is compromised in hsi2-4 plants that carry 261

a C-to-Y substitution at position 65 in the PHD domain (Veerappan et al. 2012). To test 262

the hypothesis that the HSI2 PHD domain is required for HSI2 transcriptional repression 263

of AGL15, expression of AGL15pro:LUC reporter gene when co-expressed with intact 264

HSI2 or a mutant form of HSI2 in which the eight conserved amino acids of the PHD 265

domain are substituted (C39S, C42S, C65S, C68S, H73L, C76S, C93S, C96S) was 266

analyzed by transient expression in N. benthamiana leaves (Figure 6A, B). As expected, 267

both luciferase activity and mRNA expression were significantly reduced when 268

AGL15pro:LUC was co-expressed with intact HSI2 but co-expression with HSI2mPHD 269

had no significant effect on reporter gene expression (Figure 6C). The transcriptional 270

repressor activity of HSI2mPHD was also assayed in stable transgenic Arabidopsis plants 271

that express the HSI2pro:HSI2mPHD-HA construct in the hsi2-2LUC background. 272

Expression of HSI2mPHD-HA was confirmed in these plants by immunoblot analysis 273

using anti-HA (Figure 6D). Relative expression of the native AGL15 gene in HSI2mPHD 274

seedlings was significantly upregulated compared to WTLUC, and the upregulation was 275

similar to that in hsi2-2LUC seedlings (Figure 6E; Supplemental Figure 1). These results 276

confirm that the HSI2 PHD domain is required for transcriptional repression of the 277

AGL15 promoter. 278

The HSI2 PHD domain has been reported to recognize and bind to H3K27me3 in vitro 279

(Yuan et al., 2016) and disruption of HSI2 PHD domain leads to decreased H3K27me3 280

chromatin marks at the AGL15 locus (Veerappan et al., 2014), resulting in de-repression 281

of AGL15 expression. One possible role for the HSI2 PHD domain could be that it is 282

involved in targeting HSI2 to specific loci. To test this hypothesis, ChIP-qPCR assays 283

were performed to measure the enrichment of PHD-mutated HSI2 at the AGL15 locus in 284

HSI2pro:HSI2mPHD-HA and HSI2pro:HSI2-HA transgenic Arabidopsis seedlings 285

(Figure 6F). Compared to HSI2-HA, the accumulation of HSI2mPHD-HA was 286

10

significantly lower at the AGL15 locus, showing levels similar to WT seedlings. Thus, an 287

intact PHD domain is required for HSI2 enrichment at the AGL15 locus, indicating that it 288

is necessary for HSI2 binding in vivo (Figure 6F). Using pull-down assays, we confirmed 289

that a fusion protein that contained the HSI2 PHD domain could interact with H3K27me3 290

peptides as reported by Yuan et al. (2016) and showed that it could also interact with 291

H3K4me3 peptides (Figure 6G). These interactions were lost, for the most part, when a 292

fusion protein that contained a mutated PHD domain (C65Y) was used in the pull-down 293

assay (Figure 6G). 294

EAR motif is required for full HSI2 activity 295

The HSI2 EAR motif was shown to be necessary for full HSI2-dependent repression of a 296

luciferase reporter gene under control of the sugar-responsive Sporamin promoter from 297

sweet potato (Spomin) in Arabidopsis protoplasts (Tsukagoshi et al., 2005). Therefore, we 298

tested whether the EAR motif contributes to HSI2-dependent transcriptional repression of 299

the AGL15 promoter. Co-infiltration of a construct encoding EAR motif-deleted HSI2 300

(HSI2-ΔEAR) with the AGL15pro:LUC reporter gene construct into N. benthamiana 301

leaves resulted in reduced luciferase expression compared to infiltration with the reporter 302

gene alone but repression was only about half that seen with co-expression of intact HSI2 303

(Figure 7A, B). These results show that expression of HSI2 that lacks the EAR motif 304

leads to partial de-repression of AGL15, indicating that this motif contributes to, but is 305

not essential for the transcriptional repressor activity of HSI2 in this assay. 306

Protein sequence comparison indicates that the EAR motif includes a core of three 307

conserved amino acids that are predicted to be critical for EAR function (Tsukagoshi et 308

al., 2005; Zhang et al., 2013). Site directed mutagenesis was used to replace the highly 309

conserved leucine at position 730 within the EAR motif with alanine (L730A) (Figure 310

7C). The transcriptional repressor activity of HSI2mEAR was assayed in planta by stable 311

transformation of the HSI2pro:HSI2mEAR-HA construct into hsi2-2LUC Arabidopsis. 312

Expression of HSI2mEAR-HA was confirmed in the HSI2mEAR transgenic line by 313

immunoblot analysis using anti-HA (Figure 7D). Luminescence and LUC mRNA 314

expression in HSI2mEAR seedlings was much stronger than in WTLUC plants, which 315

express intact HSI2, but was significantly weaker than hsi2-2LUC knockout plants (Figure 316

11

7E). Relative expression of the native AGL15 gene in HSI2mEAR seedlings was also de-317

repressed significantly compared to WTLUC but, like the reporter gene, failed to reach to 318

the expression level seen in hsi2-2LUC seedlings (Figure 7F; Supplemental Figure 1). 319

These results confirm that the HSI2 EAR motif contributes to HSI2-mediated 320

transcriptional repression of the native AGL15 gene. 321

CW domain does not contribute to HSI2-dependent repression of AGL15 322

Previous research showed that the CW domain of HSL1, but not of HSI2, physically 323

interacts with HDA19, leading to histone de-acetylation of chromatin at its target loci and 324

thus plays a role in their repression (Zhou et al., 2013). Therefore, we examined whether 325

the CW domain of HSI2 contributes to the repression of AGL15. Based on protein 326

sequence alignment between HSI2 and other CW domain-containing proteins in 327

Arabidopsis, two tryptophan and four cysteine residues were predicted to be critical for 328

CW domain function (Figure 8A). These four conserved cysteines at positions 547, 550, 329

568 and 580 were replaced by serine (C547S, C550S, C568S, C580S) through site-330

directed mutagenesis (Figure 8A). The HSI2pro:HSI2mCW construct was transformed 331

into hsi2-2LUC Arabidopsis to measure the ability of HSI2mCW to repress the expression 332

of the GSTF8pro:LUC reporter and AGL15 genes in planta. Expression of HSI2mCW-333

HA was confirmed in the HSI2mCW transgenic line by immunoblot analysis using anti-334

HA (Figure 8B). Luminescence from the GSTF8pro:LUC reporter and relative 335

expression of both LUC mRNA and the native AGL15 gene in HSI2mCW seedlings was 336

similar to the expression levels seen in WTLUC, but was strongly repressed compared to 337

hsi2-2LUC seedlings (Figure 8C, D; Supplemental Figure 1). Since the ability of HSI2 to 338

repress transcription of either the GSTF8pro:LUC transgene or native AGL15 was not 339

deleteriously affected by multiple substitution mutations at critical positions in the CW 340

domain, it appears that the CW domain is not required for the HSI2-mediated 341

transcriptional repression in this system. 342

343

HSI2 represses key regulators of seed maturation through downregulation of 344

AGL15 345

12

Based on the results reported here, we hypothesize that HSI2 directly targets AGL15 but 346

not LEC1, ABI3, FUS3 and LEC2, leading to repression of the seed maturation program. 347

Based on this hypothesis, we predict that HSI2 is epistatic to AGL15 and represses key 348

regulators of seed maturation via the downregulation of AGL15. To test this prediction, 349

the relative expression of LEC1, ABI3, FUS3 and LEC2 was examined in seedlings of 350

Arabidopsis hsi2-2 and agl15-4 T-DNA knockout lines and an AGL15-overexpressing 351

line (Figure 9A). Changes in the expression of AGL15 in these lines was confirmed by 352

RT-qPCR analysis (Figure 9B). Since AGL15 expression is repressed in WT seedlings, 353

we predicted that agl15 loss-of-function mutations should have little effect on the 354

expression of downstream target genes and this proved to be the case, as expression of 355

LEC1, ABI3, FUS3 and LEC2 in agl15-4 seedlings was not significantly reduced relative 356

to WT (Figure 9A). However, expression of LEC1, ABI3, FUS3 and LEC2 was 357

significantly elevated in AGL15-overexpressing plants, consistent with AGL15 358

functioning to activate the expression of these genes (Zheng et al, 2009), yet the increase 359

in LAFL gene expression in these lines was not equivalent to the levels seen in hsi2-2 360

seedlings. Interestingly, relative to hsi2-2, hsi2-2 agl15-4 double knockout seedlings 361

showed significantly reduced expression of LEC1, ABI3, FUS3 and LEC2 (Figure 9A), 362

yet the levels of expression remained significantly greater than in WT plants. We 363

interpret these results to indicate that HSI2-dependent silencing of these LAFL seed 364

maturation regulatory genes in Arabidopsis seedlings is indirect and mediated, in large 365

part, by direct transcriptional silencing of AGL15. However, since the increase in LAFL 366

gene expression in OE-AGL15 lines is weaker than in hsi2-2 plants and the rescue of the 367

hsi2-2 gene expression phenotype by agl15-4 is incomplete, it is likely that other HSI2 368

target genes may also be involved in suppressing the seed maturation developmental 369

program in seedlings. 370

371

MSI1 interacts with HSI2 and regulates AGL15 372

Since enrichment of H3K27me3 at the AGL15 locus is dependent on HSI2 (Veerappan et 373

al., 2014), we hypothesized that HSI2 represses AGL15 expression by recruitment of 374

PRC2. To test this hypothesis, yeast two-hybrid assays were carried out to screen for 375

13

protein–protein interactions between HSI2 and PRC2 component proteins including CLF, 376

EMF2, FIE and MSI1. Among these PRC2-specific bait proteins, HSI2 was found to 377

interact only with MSI1 in yeast (Figure 10A), and the protein–protein interaction 378

between HSI2 and MSI1 was confirmed by in vivo co-immunoprecipitation assays using 379

protein extracts from transgenic plants that co-express MSI1-myc and HSI2-HA (Figure 380

10B). Additional yeast two-hybrid analyses were carried out using HSI2 subdomains, and 381

HSI2 with a B3 domain deletion or mutated B3 domain as baits (Figure 10C). The 382

results showed that interaction between HSI2 and MSI1 was limited to a peptide region 383

that contained an intact B3 domain. To determine if MSI1 is required for HSI2-dependent 384

repression, the expression of AGL15 was examined in an Arabidopsis msi1 co-385

suppression (msi1-cs) line (Hennig et al., 2003). Relative to WT, AGL15 expression in 386

the msi1-cs line was significantly elevated (about 2-fold) but not to the same level as in 387

hsi2-2 plants (Figure 10D), indicating that MSI1 is required for full repression of AGL15 388

expression. It should be noted that the msi1-cs plants are not null and retain 5% MSI1 389

expression relative to WT plants. To test if, like HSI2, MSI1 is enriched at the AGL15 390

locus, protoplasts isolated from WT and hsi2-2 Arabidopsis plants were transformed with 391

a MSI1-myc expressing construct (35Spro:MSI1-myc) and analyzed by ChIP-qPCR 392

(Figure 10E). The results showed significantly higher MSI1 signal at the proximal 393

promoter and 5ʹ coding region of the AGL15 locus in WT Arabidopsis protoplasts than in 394

hsi2-2 protoplasts, indicating that accumulation of MSI1 at the AGL15 locus is HSI2 395

dependent. Since enrichment of MS1 at the 5ʹ coding region is stronger than at the 396

proximal promoter, it is possible that HSI2 may recruit MSI1 to form a PRC2 nucleation 397

site at the proximal promoter region of AGL15, which then expands into the 5ʹ coding 398

region. We interpret these results to indicate that HSI2-mediated repression of AGL15 is 399

dependent on the PRC2 component MSI1. 400

401

DISCUSSION 402

The major regulatory genes of seed maturation, LEC1, ABI3, FUS3, and LEC2 constitute 403

a genetic pathway to activate the seed maturation program (Lotan et al. 1998; Luerben et 404

al., 1998; Stone et al., 2001; Finkelstein et al., 2008; Holdsworth et al., 2008; Suzuki and 405

14

McCarty 2008). AGL15 is reported to act upstream of these key regulatory genes to 406

promote their expression during seed development (Zheng et al, 2009). The regulatory 407

model shown in Figure 11 summarizes our results and those presented by Chhun et al. 408

(2016) regarding the role of HSI2 in the repression of the seed maturation developmental 409

program in seedlings after germination. Accordingly, expression of several key 410

transcriptional regulators of the seed maturation gene expression program, including 411

LEC1, ABI3, FUS3, LEC2, and AGL15 is upregulated in hsi2-2 loss-of-function 412

Arabidopsis seedlings (Figure 1C, Suzuki et al. 2007, Tsukagoshi et al. 2007; Holdsworth 413

et al. 2008, Suzuki and McCarty 2008; Veerappan et al., 2012). Increased expression of 414

ABI3 and LEC1 was accompanied by relatively minor decreases in H3K27me3 415

deposition at these loci and no change was seen at the FUS3 and LEC2 loci (Veerappan et 416

al., 2014). On the other hand, H3K27me3 was strongly decreased at the AGL15 locus in 417

both hsi2 knockout and PHD domain mutants (hsi2-2 and hsi2-4, respectively; Veerappan 418

et al., 2014). Results of ChIP analyses indicate that that HA-HSI2 is substantially 419

enriched in the proximal promoter region of the AGL15 locus, which contains two 420

putative B3 domain-binding RY elements, but no detectable enrichment of HA-HSI2 was 421

observed at the LEC1, ABI3, FUS3 or LEC2 loci in our assays (Figure 2). Based on these 422

results, we predict that the expression of AGL15 in Arabidopsis seedlings is directly 423

repressed by HSI2, while down-regulation of LEC1, ABI3, FUS3, and LEC2 expression is 424

likely to be indirect, and possibly a result of the down-regulation of AGL15. These 425

findings conflict with those reported by Chhun et al. (2016), who found enrichment of 426

HSI2 at 5ʹ regions of LEC1, LEC2, ABI3, and FUS3 loci, leading them to propose these 427

genes as direct targets of HSI2 transcriptional repression. However, the plants used for 428

their ChIP experiments expressed HA-tagged HSI2 under control of the strongly 429

constitutive CaMV 35S promoter instead of the native HSI2 promoter and had transcript 430

levels 30-fold higher than WT. One explanation for these conflicting results could be that 431

HSI2 has low affinity for the RY sites upstream of these LAFL genes and binds only 432

when intracellular concentrations of HSI2 are quite high, as in the HSI2-HA over-433

expressing plants used by Chhun et al. (2016). Whether interaction under these 434

conditions is relevant to WT plants has yet to be elucidated. Thus, our results indicate 435

that, when expressed under control of its native promoter, HSI2-HA accumulates at the 436

15

AGL15 locus but not at LAFL gene loci in seven-day-old Arabidopsis seedlings. It 437

remains possible that HSI2 could interact with LAFL genes at other developmental stages 438

or under different physiological conditions. 439

Analysis of the direct transcriptional silencing of the AGL15 promoter by transient co-440

expression of luciferase reporter genes controlled by either the intact AGL15 promoter or 441

AGL15 promoters in which the RY elements were disrupted indicate that at least one RY 442

element is required for HSI2-dependent repression of the AGL15 promoter (Figure 3). 443

Protein–DNA binding assays confirmed that the HSI2-B3 domain is able to bind to each 444

of these RY elements individually and binding increased additively when two intact RY 445

elements were present, while binding was abrogated with the disruption of both RY 446

elements (Figure 5A). Furthermore, while the RY elements found in the AGL15 promoter 447

include the core consensus sequence 5ʹ-CATGC-3ʹ, they vary somewhat from the 448

elements targeted by LEC2, FUS3 and ABI3, in which G is preferred immediately 449

upstream of the core sequence (Baud et al., 2016). So, it is possible that HSI2 may have 450

stronger affinity to a subset of RY elements that differ somewhat from those recognized 451

most strongly by other B3-domain proteins, but this is yet to be tested directly. 452

The B3 domain deletion effector construct (HSI2-ΔB3) failed to repress AGL15 promoter 453

activity when co-infiltrated into N. benthamiana leaves with the AGL15pro:LUC 454

reporter, and a HSI2 B3 mutant with K279R K314R substitutions failed to rescue the 455

transcriptional repression of AGL15 in transgenic hsi2-2 plants (Figure 4). These data 456

indicate that the B3 domain is required for HSI2-mediated transcriptional repression in 457

this system. Furthermore, the HSI2 PHD domain is also required for AGL15 repression 458

activity. This outcome was predicted by the de-repression of AGL15 expression and 459

associated loss of H3K27me3 chromatin marks in the hsi2-4 mutant line (Veerappan et 460

al., 2012, 2014) and was confirmed here by co-expression of the HSI2mPHD effector 461

construct with the AGL15pro:LUC reporter in N. benthamiana leaves. The PHD domain 462

was reported to be a histone code reader that recognizes H3K4me3 and H3K27me2/3, 463

and we confirmed the interaction between HSI2 and these methylated H3 peptides in 464

vitro (Figure 6G, Li et al., 2006, Chakravarty et al., 2009; Zeng et al., 2010; Yuan et al., 465

2016). Therefore, it is possible that targeting of HSI2 to the AGL15 promoter requires 466

16

both DNA binding activity of the B3 domain and interaction of the PHD domain with 467

histone methylation marks associated with chromatin at the AGL15 locus. Our ChIP 468

results confirmed that a mutant form of HSI2 with a disrupted PHD domain fails to 469

accumulate at the 5ʹ region of the AGL15 locus in vivo (Figure 6). Through RNA 470

sequencing analysis, Schneider et al. (2016) identified two HSI2 splice variants: a full-471

length form and a truncated form that lacks coding sequences for the PHD domain. 472

Translation of the truncated HSI2 transcript would produce a protein variant, similar to 473

HSL2 that contains intact B3, CW, and EAR motifs but lacks the PHD domain (Jia et al., 474

2014). These authors suggest that this putative protein variant could still silence target 475

genes by binding directly to RY motifs through the B3 domain. However, results reported 476

here indicate that this truncated form of HSI2 is unlikely to be an effective repressor of 477

AGL15. 478

Disruption of the HSI2 EAR motif reduced its ability to repress the expression of a 479

luciferase reporter gene controlled by the sweet potato Sporamin A1 promoter in 480

Arabidopsis protoplasts (Tsukagoshi et al., 2005). Likewise, co-expression of the 481

HSI2-ΔEAR construct with the AGL15pro:LUC reporter gene in N. benthamiana leaves 482

or stable expression of an HSI2 construct with a point mutation within the EAR domain 483

in Arabidopsis plants resulted in compromised transcriptional repressor activity (Figure 484

7). However, the activity remained substantially higher than in hsi2-2, B3, or PHD 485

domain mutants. Thus, while the EAR motif is necessary for full activity, it appears to 486

play a reduced role in HSI2-dependent AGL15 transcriptional repression compared to the 487

B3 or PHD domains. Furthermore, expression of a HSI2mCW construct in which the four 488

conserved cysteines were replaced by serine was able to fully rescue AGL15 repression in 489

Arabidopsis hsi2-2 seedlings (Figure 8), indicating that the intact HSI2 CW domain is not 490

required for HSI2-mediated repression of AGL15. While the CW domain of HSL1 was 491

reported to interact with HISTONE DEACETYLASE 19 (HDA19) in vivo to down-492

regulate a subset of seed maturation genes by alteration of histone acetylation at the target 493

gene loci during seed germination, HDA19 binding with the HSI2 CW domain was not 494

detected (Zhou et al. 2013). 495

17

To determine if silencing of AGL15 is the primary HSI2-dependent pathway for the 496

repression of these seed maturation regulatory genes, the expression of LAFL genes in 497

plants with altered expression of AGL15 was analyzed (Figure 9). Expression of LAFL 498

genes was increased in Arabidopsis plants that over-express AGL15, though not as 499

strongly as in hsi2-2. Expression of LAFL genes was strongly decreased in hsi2-2 agl15-4 500

double mutants relative to hsi2-2, but remained somewhat higher than in WT or agl15-4 501

plants. These results provide strong evidence that HSI2-dependent silencing of AGL15 is 502

a major control point in the down-regulation of LAFL gene expression in seedlings, 503

though additional mechanisms appear to be required for full transcriptional repression. 504

These results do not support the hypothesis that HSI2 directly silences LAFL gene 505

expression since, if this were the case, expression of these genes would be expected to 506

remain high in hsi2-2 agl15-4 double mutants. Therefore, AGL15 is required for high-507

level expression of LAFL gene in hsi2 seedlings but it seems likely that another, as yet 508

unidentified HSI2 target gene (or genes) is necessary for full derepression of the seed 509

maturation developmental program in this mutant. 510

Since the derepression of AGL15 in hsi2-2 plants is associated with a loss of H3K27me3 511

marks and the deposition of H3K4me3 at the silenced gene loci (Veerappan et al., 2014), 512

we predicted that PRC2 is involved in the HSI2-dependent silencing of this gene. Using 513

directed yeast 2-hybrid and co-immunoprecipitation assays, we found that the essential 514

PRC2 component MSI1 (Kohler et al., 2003; Schonrock et al., 2006) physically interacts 515

with the HSI2 B3 domain (Figure 10). Analysis of AGL15 expression in msi1-cs mutant 516

seedlings, in which MSI1 expression is reduced by 95% (Hennig et al., 2003) indicated 517

that MSI1 is required for full repression of AGL15 expression in planta. These results 518

suggest two, potentially overlapping, regulatory scenarios. First, HSI2 may be involved 519

in establishing AGL15 silencing by interacting with H3K4me3 marks associated with the 520

actively transcribed AGL15 gene to initiate the formation of a repressive chromatin state. 521

Second, HSI2 could be involved in maintenance of AGL15 silencing by interacting with 522

existing H3K27me3 marks and recruiting PRC2 to add additional silencing-associated 523

modifications, thereby reinforcing transcriptional repression. In animal systems, PRC2 524

activity is inhibited by active chromatin marks such as H3K4me3 and H3K36me3, which 525

block the interaction of the H3 amino-terminal tail with the Nurf55-Su(z)12 complex 526

18

(Schmitges et al., 2011). In Arabidopsis, this interaction can be modulated by 527

incorporation of the Su(z)12 ortholog VRN2 rather than EMF2, which allows PRC2 to 528

autonomously add H3K27me3 without overwriting active chromatin domains (Schmitges 529

et al., 2011). 530

Comparative transcriptomic analysis of hsi2, hsl1, and hsi2 hsl1 double mutants indicated 531

that the transcriptional silencing activities of HSI2 and HSL1 are strongly synergistic 532

(Suzuki et al., 2007). According to those data, expression of AGL15 in hsi2 hsl1 is 4-fold 533

higher than in hsi2, indicating that HSL1 is also involved in the transcriptional silencing 534

of AGL15. A mechanistic explanation for the functional cooperation between HSI2 and 535

HSL1 in the transcriptional silencing of seed maturation genes during germination is yet 536

to be fully formulated. These proteins share significant sequence similarity and, while 537

their functions overlap, they are not fully redundant (Suzuki et al. 2007, Tsukagoshi et al. 538

2007). Chhun et al. (2016) reported that HSI2 and HSL1 can interact with each other and 539

are predicted to form both homodimers and heterodimers. Both HSI2 and HSL1 are also 540

reported to interact with the TRAP250 domain of MED13, a subunit of the MEDIATOR 541

CDK8 repressive module. While the CW domain of HSL1 binds specifically to HDA19 542

(Zhou et al., 2013), HSI2 is reported to specifically bind to the histone deacetylase HDA6 543

(Chhun et al, 2016). Thus, as shown in Figure 11, it seems possible that HSI2 544

homodimers are targeted to the upstream sequences of AGL15 by interaction with RY 545

DNA elements and modified histones, where it recruits PRC2, along with other 546

corepressors, including MED-CDK8 and HDA6, to form a repressive complex. It is also 547

possible that complexes based on HSI2 HSL1 heterodimers, that include HDA19, could 548

also form. However, the importance of the HSL1 B3 and PHD-like domains in targeting 549

this protein to AGL15 or other regulated genes has yet to be directly investigated. 550

551

METHODS 552

Plant materials and growth conditions 553

Arabidopsis (Arabidopsis thaliana) Columbia (Col-0; CS60000) wild-type and loss-of-554

function alleles hsi2-2 (SALK_088606) and agl15-4 (SALK_076234) were obtained 555

19

from Arabidopsis Biological Resources Center. Co-suppression line msi1-cs and 556

35Spro:AGL15 were provided by Dr. Lars Hennig (Department of Plant Biology, 557

Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for 558

Plant Biology, SE-75007 Uppsala, Sweden) and Dr. Donna E. Fernandez (Department of 559

Botany, University of Wisconsin, Madison, Wisconsin 53706), respectively. WTLUC and 560

hsi2-2LUC, which harbors GSTF8pro:LUC HSI2-responsive reporter gene in the WT and 561

HSI2 T-DNA knock-out allele hsi2-2 (SALK_088606) background, respectively, were 562

described previously (Veerappan et al., 2012). For all the experiments, plants were grown 563

under continuous illumination (fluorescent lamps at ~200 μmol m-2 s-1) at 24oC on 0.3% 564

Phytagel plates containing 0.5 X Murashige and Skoog (MS) salt, 0.5 g/L MES (2-(N-565

morpholino) ethanesulfonic acid), 1 X Gamborg vitamin mix, and 1 % sucrose (pH 566

adjusted to 5.7). 567

Plasmid constructs and plant transformation 568

The HSI2 promoter, consisting of a 1500-bp fragment immediately upstream of the HSI2 569

start codon was amplified by PCR and cloned into pGEM T-Easy vector (Promega). 570

After sequencing, the HSI2 promoter was cleaved by NotI restriction enzyme and 571

inserted into pENTR/D TOPO vector containing the HSI2 cDNA. Then, an LR 572

recombination reaction was carried out to introduce HSI2pro:HSI2 into the binary vector 573

pEarleygate301 containing an HA epitope tag at the C-terminus. To generate 574

35Spro:MSI1 constructs, pDONOR207 vector containing full-length MSI1 coding 575

sequence as sub-cloned into destination vector pGWB521. Arabidopsis plants were 576

transformed using the floral dip method (Clough and Bent 1998). The primers used for 577

generating these constructs are listed in Supplemental Data set 1. 578

HSI2-HA complementation 579

The HSI2pro:HSI2-HA construct was introduced into Agrobacterium tumefaciens and 580

used to transform hsi2-2LUC Arabidopsis plant through the floral dip method (Clough and 581

Bent 1998). The resulting T1 generation seeds were plated and screened on MS medium 582

containing 5 µg/ml Glufosinate (BASTA). Genotyping of resistant plants was performed 583

by PCR using specific primers. Expression of the HSI2-HA fusion protein was detected 584

20

by immunoblot analysis. Equal amounts of total protein from WTLUC, hsi2-2LUC and 585

HSI2pro:HSI2-HA Arabidopsis seedlings was resolved by SDS-polyacrylamide gel 586

electrophoresis and electroblotted onto PVDF membranes (BioRad). The processed 587

membranes were probed with anti-HA antibody to detect HSI2-HA fusion protein or with 588

anti-actin to detect endogenous ACTIN for use as a loading standard. 589

Luciferase imaging 590

Imaging of luciferase was performed using Andor iKON-M DU934N-BV CCD camera 591

(Andor Technology). Andor SOLIS (I) imaging software (Andor Technology) was used 592

for image acquisition and processing of luminescence. Prior to luminescence imaging, 593

Arabidopsis seedlings grown on MS plates and infiltrated N. benthamiana leaves were 594

uniformly sprayed with 2 mM D-luciferin (Gold Biotechnology) in 0.01% Triton X-100 595

solution. After spraying with luciferin, seedlings and leaf samples were incubated under 596

dark for 5 min. Exposure time for luminescence imaging was 5 min, unless otherwise 597

specified. 598

Reverse-transcription-quantitative PCR (RT-qPCR) 599

RT-qPCR was performed using a StepOne Plus system (Applied Biosystems) with iTAq 600

SYBR Green Supermix with ROX (BioRad). RNase-free DNase (Qiagen)-treated total 601

RNA was used for cDNA synthesis using iScript cDNA Synthesis Kit (Bio-Rad). For 602

each experiment, cDNA synthesis reactions were carried out on three independent RNA 603

samples prepared from approximately 5 seedlings, each.Three qPCR reactions were 604

performed for each cDNA sample. EF1A (AT5G60390) and HYGROMYCIN 605

PHOSPHOTRANSFERASE (HPT) were used as reference genes for Arabidopsis and N. 606

benthamiana, respectively. The relative expression of genes was calculated according to 607

the ABI PRISM 7700 Sequence Detection System, User Bulletin #2 (Livak and 608

Schmittgen 2001). Primer sequences used for RT-qPCR are listed in Supplemental 609

Dataset 1. 610

611

Chromatin immunoprecipitation analysis 612

21

In transgenic Arabidopsis plants, ChIP assays were performed essentially as described 613

(Yamaguchi et al. 2014; Veerappan et al., 2014) with minor modifications. Chromatin 614

was extracted from 7-day-old seedlings grown in MS medium supplemented with 1% 615

sucrose. The chromatin in these seedlings was cross-linked with 1% formaldehyde. The 616

resulting chromatin was sheared to fragments with 500 bp (200–1,000 bp) average length 617

by sonication and used for immunoprecipitation with commercially available anti-HA 618

(Abcam, ab9110). After reversing the cross-links, immunoprecipitated DNA was 619

analyzed by qPCR using primers for specific regions of the LAFL genes LEC1, ABI3, and 620

FUS3, along with AGL15. Three independent experiments, each using 500 mg of 621

seedlings (25-30 individual plants), were performed. Three technical replications for each 622

qPCR assay were carried out, and ACT2 was used as an internal control for 623

normalization. In Arabidopsis leaf protoplasts, the ChIP assays were performed as 624

described previously (Lee et al., 2007; Du et al., 2009; Xiong et al., 2013; Zhang et al., 625

2014). HSI2pro:HSI2-GFP and HSI2pro:HSI2mB3-GFP DNA were transformed into 626

hsi2-2 Arabidopsis protoplasts from 14-d-old leaves, and 35Spro:MSI1-cmyc DNA was 627

transformed into WT and hsi2-2 Arabidopsis protoplasts using the polyethylene glycol–628

mediated transformation method. Protoplasts were incubated at room temperature for 12 629

h under dark conditions. Protoplast chromatin was cross-linked by 1% formaldehyde in 630

W5 medium for 20 min and quenched with Gly (0.2 M) for 5 min. The protoplasts were 631

then lysed, and the DNA was sheared on ice with sonication. The immunoprecipitation 632

was performed with anti-GFP (ab290) and anti-myc (MA1-980). After reversing the 633

cross-links, the purified DNA was analyzed by qPCR using primers for specific regions 634

of AGL15. Each experiment was repeated at least twice using protoplasts from 635

approximately five leaves. Three technical replications for each qPCR assay were carried 636

out, and ACT2 was used as an internal control for normalization. Primers of target genes 637

used for qPCR in ChIP analysis are listed in Supplemental Data set 1. 638

In vitro histone peptide binding assay 639

Sequences encoding the HSI2 PHD domain (111 amino acids) and mutated PHD domain 640

(C65Y) were amplified from the HSI2 full-length cDNA and cloned into pENTR-D-641

TOPO vector by Gateway cloning system (Invitrogen). The pENTR-PHD and pENTR-642

22

mPHD plasmids was recombined into the destination vector pDEST15 (Invitrogen) to 643

produce fusion proteins with an N-terminal GST tag. The plasmids were transformed 644

into BL21-Rosetta cells. Escherichia coli were grown in LB medium containing 50 μg/ml 645

carbenicillin to a density of OD600=0.6. Expression of recombinant protein was induced 646

for 6 h at 28° C with 0.1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG). Protein was 647

extracted by using CelLytic B cell lysis buffer (Sigma-Aldich). Recombinant fusion 648

proteins were purified by GST-binding kit (Novagen). 649

Peptide binding assays were performed as previously described with minor modification 650

(Lee et al., 2009). Briefly, 1 µg of GST fusion PHD finger domains was incubated with 1 651

µg of various biotinylated histone peptides, H3 (12-403, 12-404), H3K4me3 (12-564), 652

and H3K27me3 (12-565, Millipore) in binding buffer (30 mM HEPES, pH 7.5, 300 mM 653

NaCl, 0.1 % v/v NP-40) overnight at 4º C, followed by addition of streptavidin agarose 654

beads (16-126, Millipore). After 1h incubation, beads were washed with binding buffer 655

five times and eluted with 2x SDS sample loading buffer. The samples were separated by 656

SDS polyacrylamide gel electrophoresis on 10% gels and subjected to immunoblot 657

analysis using anti-GST. 658

DNA binding assay 659

An HSI2 cDNA fragment encoding a peptide (252 amino acids) that contains the B3 660

domain was cloned into pENTR-D-TOPO vector and then recombined into the E. coli 661

expression vector pET59-DEST. Expression of His-Trx-B3 in Rosetta cells was induced 662

with 0.4 mM isopropyl-1-thio-D-galactopyranoside at 37o C for 3 h. The fusion protein 663

was purified using Dynabeads His-tag Isolation & Pulldown kit (#10103D, 664

ThermoFisher) according to the manufacturer's protocol and quantified by the Bio-Rad 665

protein assay reagent. DNA probes derived from the AGL15 promoter sequence, with RY 666

and mutated RY elements, were incubated with purified His-Trx or with the His-Trx-B3 667

fusion protein. The DNA binding assays were performed as previously described (Wang 668

et al., 2017). In brief, 0.3 µg His-B3 protein was bound to His-tag specific magnetic 669

beads (Dynabeads, Thermo Fisher) and incubated with 0.25 µM dsDNA probes for 15 670

minutes at room temperature. Dynabead-DNA complexes were selected with a magnet 671

23

and, after wash and elution steps, the amount of protein-bound probe was determined 672

using qPCR. Primers used are listed in Supplemental Data set 1. 673

Electrophoretic mobility shift assay (EMSA) 674

EMSA was performed using double stranded biotinylated DNA probes and the Lightshift 675

Chemiluminescent EMSA kit (Thermo Scientific). Biotin 3ʹ end-labeled DNA oligomers 676

were prepared using a biotin end-labeling kit (Thermo Scientific), and double-stranded 677

DNA probes were generated by annealing sense and antisense oligomers. Each 20 µl 678

binding reaction contained 50 fmol of biotin-labeled dsDNAs, 5 µg of recombinant His-679

HSI2B3 protein, and 50 ng/µl Poly (dIdC) in binding buffer (10 mM Tris, 30 mM KCl, 680

0.1 mM EDTA, 1 mM DTT, 0.05 % NP-40, 6.5 % glycerol, pH 7.9). Binding reactions 681

were incubated for 20 min at room temperature resolved by electrophoresis of reaction 682

samples on 5% polyacrylamide gels with TBE buffer. Detection of biotin-labeled DNA 683

was performed by chemiluminescence according to the supplier instructions. 684

Yeast two-hybrid assay 685

Yeast two-hybrid assays were performed using the Matchmaker GAL4-based two-hybrid 686

system 3 (Clontech) according to the manufacturer’s instructions. Sequences that encode 687

full-length HSI2 were subcloned into the pGADT7 vector, whereas the full-length CLF, 688

EMF2, FIE and MSI1 coding sequences were subcloned into the pGBKT7 vector. All 689

constructs were transformed into yeast strain AH109 by the lithium acetate method, and 690

yeast cells were grown on a minimal medium/-Leu/-Trp according to the manufacturer’s 691

instructions (Clontech). Transformed colonies were plated onto a minimal medium/-Leu/-692

Trp/-His/-Ade to test for possible interactions. 693

In vivo Co-Immunoprecipitation (CoIP) 694

The MSI1 coding sequence was cloned into the binary vector pGWB521-myc. The 695

construct was introduced into A. tumefaciens strain GV3101 and transformed into 696

Arabidopsis hsi2-2 plants that had been complemented with HSI2pro:HSI2-HA. Total 697

protein was extracted from 9-day-old seedlings using lysis buffer (50mM Tris-HCl 698

pH7.5, 150 mM NaCl, 1mM EDTA, 10% glycerol, 2 mM NaVO4, 25 mM β-699

24

glycerophosphate, 10 mM NaF, 0.1% Tween20, 1 mM PMSF and protease inhibitor 700

cocktail) and incubated 30 minutes at 4o C with gentle agitation. After centrifugation at 701

12,000 x g for 15 minutes at 4o C, supernatant was incubated with protein A 702

agarose/salmon sperm DNA for 1 hour for pre-clearing. The pre-cleared protein solution 703

was incubated with 50 µl HA-antibody conjugated agarose (Thermo Fisher #26181) 704

overnight at 4o C. The supernatant was removed after centrifugation at 2,000 x g for 2 705

minutes at 4o C, and the agarose beads were washed 3 times with lysis buffer. The 706

proteins were eluted with 2x SDS sample loading buffer and analyzed by immunoblotting 707

using c-myc tag antibody (Thermo Fisher MA1-980). 708

Accession Numbers 709

Sequence data from this article can be found in the Arabidopsis Genome Initiative or 710

GenBank/EMBL databases under the accession numbers listed in Supplemental Table 1. 711

712

SUPPLEMENTAL DATA 713

Supplemental Figure 1. Relative expression of AGL15 in multiple independent 714

transgenic lines. (Supports Figures 4, 6, 7, and 8.) 715

Supplemental Table 1. Accession numbers or gene identifiers of sequences used for 716

multiple sequence alignment. 717

Supplemental Data set 1. Sequences of primers used and genes analyzed in this study. 718

Supplemental File 1. Statistical analyses. 719

720

ACKNOWLEDGMENTS 721

We thank Million Tadege for providing Gateway-compatible Y2H vectors, Lars Hennig 722

for providing msi1-cs seeds, Donna E. Fernandez for providing the 35Spro:AGL15 seeds, 723

the Arabidopsis Biological Resource Center at Ohio State University for providing the T-724

DNA insertion mutants. We also thank Fei Zhang, Mohamed Fokar, Angelika Reichert, 725

25

Hui Wang, Tezera Watira, and Tyson Kerr for stimulating discussions, and Million 726

Tadege for critical reading of the manuscript and stimulating discussions. This work was 727

supported by the Oklahoma Agricultural Experiment Station and Oklahoma Center for 728

the Advancement of Science & Technology (Grant # PS16-009 to R.D.A). 729

730

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Figure 1. hsi2-2 plants are rescued by HSI2 complementation. (A) Luminescence images of WTLUC, hsi2-2LUC, and HSI2pro:HSI2-HA Arabidopsis seedlings taken with a cooled CCD camera. (B) RT-qPCR analysis of the relative expression of GSTF8pro:LUC reporter gene in WTLUC, hsi2-2LUC and seedlings from three independently transformed homozygous HSI2pro:HSI2-HA Arabidopsis lines (12, 22, and 26). (C) Relative expression of the seed-specific transcriptional regulator genes ABI3, FUS3, LEC1, LEC2, and AGL15 in WTLUC, hsi2-2LUC and HSI2pro:HSI2-HA Arabidopsis seedlings, assayed by RT-qPCR. Error bars in Figure 1B and C indicate standard deviation (SD) for three independent assays. ** indicates means significantly different from WTLUC at P<0.01 (Student’s t-test; Supplemental File 1). (D) Expression of the HA-HSI2 fusion protein detected by immunoblot analysis with anti-HA antibody in WTLUC, hsi2-2LUC and HSI2pro:HSI2-HA Arabidopsis seedlings, anti-actin was used to detect expression of endogenous ACTIN as a loading standard.

Figure 2. HSI2 accumulation at the AGL15 locus. ChIP-qPCR analysis of HSI2/VAL1 enrichment at LEC1, FUS3, ABI3, LEC2 and AGL15 genes in WT and HSI2‑rescued Arabidopsis plants. Schematic representations of genes and assayed genomic regions are indicated, with P indicating promoter regions and C indicating coding regions. The positions of RY elements within upstream sequences of these genes are indicated. These regions were interrogated by ChIP-qPCR analysis of 7-day-old transgenic plants harboring HSI2pro:HSI2-HA using HA antibody. The amount of DNA after ChIP was normalized using ACT2 as an internal standard. Data represent means of three qPCR reactions from each of three independent ChIP assays. Error bars indicate SD. ** indicates means significantly different from WT at P<0.01 (Student’s t-test; Supplemental File 1).

Figure 3. RY elements are required for HSI2 repression of AGL15 expression. (A) Schematic representation of reporter and effector constructs used to assay the function of RY elements in the AGL15 upstream sequence. In reporter constructs, a luciferase gene is driven by the AGL15 promoter or an RY-mutated AGL15 promoter. Red bars represent mutation sites. (B)

Luminescence images and relative expression of luciferase mRNA, as determined by RT-qPCR assays in

Nicotiana benthamiana leaves co-infiltrated with the reporter and effector gene

combinations indicated. RT-PCR analysis of HSI2 and NbActin gene expression in each infiltrated area. (C)

Luminescence image of

Arabidopsis seedlings that contain either

AGL15pro:LUC or AGL15mpro:LUC reporter genes. (D) Relative expression of luciferase reporter gene, by RT-qPCR, corresponding to the luminescence image above in Arabidopsis seedlings. (E) Luminescence images and relative expression of luciferase reporter genes, assayed by RT-qPCR from N. benthamiana leaves co-infiltrated with combinations of reporter and effector constructs, as indicated. HSI2 and NbActin gene expression was assayed by RT-PCR in each infiltrated area. Data represent means of three qPCR reactions from three independent assays per genotype. Error bars indicate SD. ** indicate that luciferase activity in infiltrations with the 35Spro:HSI2 effector is significantly different from those without the effector at P<0.01 (Student’s t-test; Supplemental File 1).

Figure 4. B3 domain is required for HSI2 activity. (A) Schematic representation of reporter and effector constructs used to assay the function of the HSI2 B3 domain. In the reporter construct, the luciferase gene is driven by the AGL15 promoter. In effector constructs, expression of intact HSI2 or HSI2-ΔB3 is controlled by the CaMV 35S promoter. (B) Luminescence image produced by a cooled CCD camera and relative expression of the luciferase reporter gene from N. benthamiana leaves co-infiltrated with combinations of reporters and effectors, as indicated, was assayed by RT-qPCR. HSI2 and NbActin gene expression in each infiltrated area was assayed by RT-PCR. (C) Amino acid sequence alignment of the N terminal B3 domains among Arabidopsis HSI2, HSL1, VAL3, ABI3, LEC2, FUS3, RAV1, RAV2, and maize VP1. Red stars indicate substitution mutation sites within the HSI2 B3 domain. (D) Relative expression of endogenous AGL15 in WTLUC, hsi2-2LUC, and HSI2pro:HSI2mB3-GFP Arabidopsis lines was assayed by RT-qPCR and normalized by EF1a. HSI2mB3-GFP and EF1a gene expression in WTLUC, hsi2-2LUC and HSI2mB3-GFP Arabidopsis lines was assayed by RT-PCR. Data represent means of three qPCR reactions for each locus from three independent assays. Error bars indicate SD. ** indicate significantly different means at P<0.01 (Student’s t-test; Supplemental File 1).

Figure 5. HSI2 B3 domain interacts with RY elements in the AGL15 promoter. (A) DNA binding assay corresponding to the putative RY binding sites of the HSI2-B3 domain. DNA fragments from AGL15 proximal promoter area containing RY and mutated RY elements bound to His-Trx-B3 fusion protein or His-Trx control were quantified by qPCR after elution. Data represent means of three technical replicates from three independent assays. Boxes and red letters denote RY elements and mutated sites, respectively. Error bars indicate SD. ** indicate means significantly different from control reactions (His-Trx) at P<0.01 (Student’s t-test; Supplemental File 1). (B) Electrophoretic mobility shift assays of interaction between RY1 and RY2, and mutant, mRY1, and mRY2 probes with His-HSI2B3 and mutant His-HSI2mB3 proteins. Free probe and bound probe bands are indicated. (C) Enrichment of HSI2-GFP and HSI2mB3-GFP at the AGL15 locus in Arabidopsis protoplasts assayed by ChIP-qPCR. Interrogated regions are indicated in the gene diagram. Data represent means of three ChIP-qPCR assays from threeindependent assays for each genotype. Error bars indicate SD. ** indicate means significantly different from control (GFP) at P<0.01 (Student’s t-test; Supplemental File 1).

Figure 6. PHD domain is required for HSI2-dependent AGL15 repression. (A) Amino acid sequence alignment of PHD-like finger motifs from HSI2 family proteins and from various Arabidopsis PHD-containing proteins. Multiple amino acid alignments of PHD-like domains were performed using ClustalW Red stars indicate mutation sites. (B) Schematic representation of reporter and effector constructs used to test the function of the PHD domain. In effector constructs, green diagonal shading represents a mutated PHD domain. (C) Luminescence image and relative expression of luciferase reporter genes, measured by RT-qPCR, from N. benthamiana leaves co-infiltrated with combinations of reporter and effector constructs, as indicated. HSI2 and NbActin gene expression in each infiltrated area was assayed by RT-PCR. (D) HSI2mPHD protein was detected by immunoblot assays using anti-HA antibody, anti-actin was used to detect actin as a loading standard. (E) Relative expression of native AGL15 in WTLUC, hsi2-2LUC, and HSI2pro:HSI2mPHD-HA transgenic Arabidopsis plants. All data represent means

of three RT-qPCR reactions from three independent assays for each genotype or treatment. Error bars indicate SD. ** indicate means significantly different from control (WTLUC) at P<0.01 (Student’s t-test; Supplemental File 1). (F) Results of ChIP-qPCR analyses to compare the enrichment of HSI2-HA and HSI2mPHD-HA at the AGL15 locus in Arabidopsis plants. Interrogated regions are indicated in the gene diagram. Data represent means of three ChIP-qPCR assays from three biological replicates. Error bars indicate SD. ** indicate means significantly different from control plants (WT) at P<0.01 (Student’s t-test; Supplemental File 1). (G) In vitro histone peptide binding assay of GST-fused PHD domain of HSI2 with H3K4me3 and H3K27me3 polypeptides. Various biotinylated histone peptides bound to GST-fusion PHD domain of HSI2 were precipitated using streptavidin agarose beads and subjected to immunoblot assay using anti-GST antibody. Input indicates 5% of GST-fusion proteins used for pull-down assay. Control indicates no histone peptide in reaction. H3 (1-21): non-methylated histone H3 amino acid residues 1 to 21, and used as control for H3K4me3 (histone H3, 1–21, tri-methylated at Lys-4). H3 (21-44): non-methylated histone H3 amino acid residues 21 to 44, and used as control for H3K27me3 (histone H3, 21–44 tri-methylated at Lys-27). Amino acid changed by point mutation (C65Y) is indicated based on the position within the full-length protein.

Figure 7. EAR domain is important for full HSI2 activity. (A) Schematic representation of reporter and effector constructs used to test the function of the EAR domain. Effector constructs encode either intact HSI2 or a C-terminal deletion that lacks the EAR domain. (B) Luminescence image and relative expression analysis of the LUC mRNA, by RT-qPCR, from N. benthamiana leaves co-infiltrated with combinations of reporter and effector constructs, as indicated. RT-PCR analysis of HSI2 and NbActin gene expression in infiltrated areas as above. (C) Amino acid sequence alignment of EAR motifs from HSI2 family proteins and from various Arabidopsis EAR-containing proteins aligned using ClustalW. Red star indicates a single substitution mutation used to disrupt the EAR motif of HSI2 protein. (D) HSI2mEAR protein expression in transgenic plants was detected by

immunoblot assays using anti-HA antibody. Anti-actin was used to detect actin as a loading standard. (E) Luminescence images to compare WTLUC, hsi2-2LUC and HSI2pro:HSI2mEAR-HA Arabidopsis seedlings, and relative expression analysis of the LUC reporter gene corresponding to the luminescence image. RT-qPCR assays were normalized by EF1a. (F) Relative expression analysis of endogenous AGL15 in HSI2mEAR transgenic Arabidopsis line, by RT-qPCR, was normalized by EF1a. Data represent means of three technical replicates from three independent assays for each genotype. Error bars indicate SD. Lowercase letters indicate significant difference between genetic backgrounds (Student’s t-test; Supplemental File 1).

Figure 8. CW domain is not required for HSI2 function. (A) Amino acid sequence alignment of CW domain from HSI2 family proteins and from various Arabidopsis CW-containing proteins. Red stars indicate four conserved cysteine residues that were substituted for serine in the CW domain of HSI2 by site directed mutagenesis. (B) Expression of HSI2mCW-HA protein was detected by immunoblot assays using anti-HA antibody in WTLUC, hsi2-2LUC, and HSI2pro:HSI2mCW-HA Arabidopsis seedlings. Actin was analyzed as a loading control. (C) Luminescence images from WTLUC, hsi2-2LUC, and HSI2pro:HSI2mCW-HA Arabidopsis seedlings, obtained with a CCD camera, and relative expression of the LUC reporter gene corresponding to the luminescence image above was assayed by RT-qPCR and normalized by EF1a. (D) Relative expression of endogenous AGL15 in HSI2pro:HSI2mCW-HA transgenic Arabidopsis line, assayed by RT-qPCR and normalized by EF1a. Data represent means of three technical replicates from three independent assays per genotype. Error bars indicate SD. ** indicate significant differences between genetic backgrounds at P<0.01 (Student’s t-test; Supplemental File 1).

Figure 9. Expression of LEC1, ABI3, FUS3, and LEC2 in hsi2 agl15 plants. (A) Relative expression of LEC1, ABI3, FUS3, and LEC2 was measured in WT, agl15-4, AGL15-overexpressor (OE-AGL15), hsi2-2, and hsi2-2 agl15-4 Arabidopsis lines by RT-qPCR. (B) Relative expression of AGL15 in WT, agl15-4, OE-AGL15, hsi2-2, and hsi2-2 agl15-4 Arabidopsis lines. The agl15-4 line is null and expression in the OE-AGL15 is approximately 200X higher than WT. Data were normalized by EF1a and represent means of three technical replicates from three independent RNA samples for each genotype. Error bars indicate SD. Lowercase letters indicate significant differences (P<0.01) between genetic backgrounds (Student’s t-test; Supplemental File 1).

Figure 10. HSI2 interacts with MSI1. (A) Yeast two-hybrid interaction assays with Polycomb Repressive Complex 2 (PRC2) components CLF, EMF2, FIE and MSI1 as baits (fused to BD) and HSI2 as prey (fused to AD). Yeast cells transformed with indicated gene sets were selected on DDO (lacking leu and trp) and QDO (lacking adenine, his, leu, and trp) media. (B) Co-Immunoprecipitation (Co-IP) showing HSI2–MSI1 interaction. Total proteins extracted from Arabidopsis that co-expressed HSI2pro:HSI2-HA and 35Spro:MSI1-myc or HSI2pro:HSI2-HA alone. HSI2-HA was immunoprecipitated with anti-HA antibody and the immunoblot was probed with anti-c-myc antibody to detect interaction between HSI2 and MSI1. (C) Diagram of HSI2 subclone constructs that encode peptides that contain each of the four conserved HSI2 domains used for yeast two hybrid interaction assays with MSI1 as bait. Transformed yeast cells with indicated gene sets were selected on medium of DDO (lacking leu and trp) and QDO (lacking adenine, his, leu, and trp). (D) Relative expression of AGL15 was assayed using RT-qPCR in WT, hsi2-2 and msi1-cs and the assay was normalized by EF1a. Values represent means of three technical replicates from three independent RNA samples. Error bars indicate SD. Lowercase letters indicate significant differences (P<0.01) between genetic backgrounds (Student’s t-test; Supplemental File 1). (E) Analysis of MSI1 enrichment in chromatin at the AGL15 locus in Arabidopsis. ChIP-qPCR analysis was carried out on chromatin from WT and hsi2-2 Arabidopsis protoplasts transformed with 35Spro:MSI1-myc using c-myc antibody. Enrichment of MSI1 at the PYL4 (Mehdi, et al., 2016) was used as a positive control. The amount of DNA after ChIP was normalized using ACT2 as an internal control. Data represent means of three qPCR reactions from three independent ChIP assays. Error bars indicate SD. ** indicate values significantly increased relative to mock treatment (P<0.01, Student’s t-test; Supplemental File 1).

Figure 11. Proposed model for the repression of seed maturation gene expression in Arabidopsis seedlings. According to this model, HSI2 dimers (Chhun et al., 2016) may directly target the AGL15 promoter by binding to RY elements and histone methylation marks through its B3 and PHD domains, respectively. HSI2 recruits PRC2 by interaction with MSI1 to repress AGL15 expression through deposition of H3K27me3 marks. Additional interactions with MED13 and HDA6, reported by Chhun et al. (2016), are also shown. Since AGL15 positively regulates the expression of the LAFL genes that encode the seed maturation transcription factors LEC1, ABI3, FUS3, and LEC2, silencing of AGL15 by HSI2 leads to reduced expression of these regulatory genes. In hsi2null plants, on the other hand, AGL15 expression remains high and LAFL gene expression is activated, leading to ectopic expression of the seed maturation genetic pathway. Solid lines indicate directly up-regulated targets of AGL15 (Zheng et al., 2009). While our results indicate that silencing of AGL15 is a major component of LAFL gene repression in seedlings, another potential target(s) of HSI2 could also play a role in this regulatory mechanism.

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DOI 10.1105/tpc.17.00655; originally published online February 23, 2018;Plant Cell

Naichong Chen, Vijaykumar Veerappan, Haggag Abdelmageed, Miyoung Kang and Randy D AllenVegetative Growth in Arabidopsis

HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition from Seed Maturation to

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