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The second subunit of DNA-polymerase delta is required for genomic stability and 1
epigenetic regulation 2
Jixiang Zhang1, Shaojun Xie2, 3, Jinkui Cheng1, Jinsheng Lai4, Jian-Kang Zhu2, 3, and 3
Zhizhong Gong 1, 5 4
1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological 5
Sciences, China Agricultural University, Beijing 100193, China. 6
2Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, 7
Chinese Academy of Sciences, Shanghai 200032, China 8
3Department of Horticulture and Landscape Architecture, Purdue University, West 9
Lafayette, IN 47906, USA. 10
4 State Key Laboratory of Agrobiotechnology, China National Maize Improvement 11
Center, Department of Plant Genetics and Breeding, China Agricultural University, 12
Beijing 100193, China. 13
5Corresponding author: 14
Zhizhong Gong 15
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological 16
Sciences, China Agricultural University, Beijing 100193, China. 17
Email: [email protected]; Tel: 86-10-62733733 18
Running title: POLD2, Genomic Stability and Epigenetic Regulation 19
One sentence summary 20
Plant Physiology Preview. Published on April 25, 2016, as DOI:10.1104/pp.15.01976
Copyright 2016 by the American Society of Plant Biologists
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Mutation in the subunit of DNA-polymerase delta leads to increased homologous 21
recombination and alters histone modification patterns. 22
*Address correspondence to [email protected] 23
The author responsible for distribution of materials integral to the findings presented in 24
this article in accordance with the policy described in the Instructions for Authors 25
(www.plantphysiol.org) is: Zhizhong Gong ([email protected]). 26
27
This study was supported by the Natural Science Foundation of China (grants 31330041 28
and 31121002). Jixiang Zhang was supported by Chinese Universities Scientific Fund No. 29
2013YJ002. 30
J. Z. and Z. G conceived the original research plans; J. Z performed most of the 31
experiments; S. X, J. C, and J. L provided bioinformatic analysis; J. Z and Z. G designed 32
the project and wrote the article with contributions of all the authors; J-K. Z discussed the 33
data and complemented the writing. 34
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Abstract 36
DNA polymerase δ plays crucial roles in DNA repair and replication, and maintaining 37
genomic stability. However, the function of POLD2, the second small subunit of DNA 38
polymerase δ, has not been characterized yet in Arabidopsis. During a genetic screen for 39
release of transcriptional gene silencing (TGS), we identified a mutation in POLD2. 40
Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the 41
regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-42
mutated and Rad3-related (ATR) and DNA polymerase α (Pol α). The pold2-1 mutant 43
exhibits genomic instability with a high frequency of homologous recombination (HR). It 44
also exhibits hypersensitivity to DNA-damaging reagents, and short telomere length. 45
Whole-genome ChIP-seq and RNA-seq analyses suggest that pold2-1 changes 46
H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene 47
expression levels. Our study suggests that POLD2 is required for maintaining genome 48
integrity, and properly establishing the epigenetic markers during DNA replication to 49
modulate gene expression. 50
Keywords: POLD2, transcriptional gene silencing, DNA replication, homologous 51
recombination, genome stability, histone methylation, DNA methylation 52
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Introduction 54
DNA replication is a fundamental process that duplicates both genetic information (DNA 55
sequence) and epigenetic information (DNA methylation and histone modifications). 56
During DNA replication in each cell cycle, nucleosomes are reassembled onto newly 57
synthesized DNA to maintain the chromatin structures (Probst et al., 2009). Three key 58
DNA polymerases are essential for DNA replication: DNA polymerase α (Pol α), DNA 59
polymerase δ (Pol δ), and DNA polymerase ε (Pol ε). It is a well-accepted concept that, 60
after a short RNA-DNA primer extension by the Pol α-primase complex, Pol ε and Pol δ 61
replace Pol α and perform the bulk of DNA synthesis in the leading and lagging strand, 62
respectively (Burgers, 2009). 63
Pol δ is a highly accurate DNA polymerase that is essential for DNA replication, 64
repair, and recombination, and thus for genome integrity (Prindle and Loeb, 2012). 65
Dysfunction of Pol δ results in genomic instability and cancer (Church et al., 2013; Palles 66
et al., 2013). Pol δ in mammals consists of four subunits: the catalytic subunit p125 67
(POLD1, corresponding to Pol3p in Saccharomyces cerevisiae and CDC6 in S. pombe); 68
the accessory subunit p50 (POLD2, corresponding to Pol31p in S. cerevisiae and CDC1 69
in S. pombe); p68 (POLD3, corresponding to Pol32p in S. cerevisiae and CDC27 in S. 70
pombe); and the smallest subunit p12 (POLD4, corresponding to CDM1 in S. pombe) 71
(Prindle and Loeb, 2012). The Pol31p and Pol32p subunits in yeast also interact with 72
DNA polymerase zeta (Pol ζ) to participate in DNA translesion synthesis (TLS) and 73
mutagenesis (Johnson et al., 2012). Recent studies suggest that Pol δ may replicate both 74
strands (Johnson et al., 2015; Miyabe et al., 2015). 75
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76
Several DNA-replication factors have been reported to help mediate transcriptional 77
gene silencing (TGS) in plants (Liu and Gong, 2011). In Arabidopsis, mutations in 78
replication factor C1 (RFC1), Pol ε, Pol α, and replication protein A2A (RPA2A) can 79
suppress gene silencing in a DNA methylation-independent manner (Elmayan et al., 2005; 80
Kapoor et al., 2005; Xia et al., 2006; Liu et al., 2010; Liu et al., 2010). These mutants are 81
hypersensitive to DNA damage and exhibit reduced telomere length and increased 82
genomic instability. BRUSHY1 (BRU1), TEBICHI, and FASCIATA1 (FAS1, chromatin 83
assembly factor 1) are also related to DNA damage and TGS (Takeda et al., 2004; 84
Ramirez-Parra and Gutierrez, 2007; Inagaki et al., 2009). Because all of these proteins are 85
involved in the DNA replication and repair pathway, we refer to this pathway as the DNA 86
replication and repair-mediated TGS pathway (DRR-TGS pathway). However, the 87
molecular mechanism of this pathway is not well known. 88
Repressor of silencing 1 (ROS1), a 5-meC DNA glycosylase/demethylase, and its 89
homologous proteins DEMETER and DEMETER-like 2-3 (DML2-3), are essential for 90
maintaining the expression of endogenous genes and transgenes through active DNA 91
demethylation (Gong et al., 2002; Zhu, 2009). In a previous study, we performed a 92
genetic screen for additional ROS genes by using a transgenic Arabidopsis line that 93
carries a T-DNA insertion expressing both the ProRD29A:LUC (firefly luciferase 94
reporter driven by the stress-responsive RD29A promoter) gene and the Pro35S:NPTII 95
(neomycin phosphotransferase II driven by the CaMV 35S promoter) gene. Using this 96
screen, we identified several genes in the RNA-directed DNA methylation (RdDM) 97
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pathway whose mutations silence Pro35S:NPTII because of the reduced expression of 98
ROS1, indicating that the RdDM pathway positively modulates ROS1 expression (Huettel 99
et al., 2006; Li et al., 2012). One of these mutants isolated from the screen was defective 100
in meristem silencing 3 (dms3-4) (Li et al., 2012). DMS3 is a silencing factor in RdDM 101
that coordinates the formation of the DDR complex by defective in RNA-directed DNA 102
methylation 1 (DRD1) and RNA-directed DNA methylation 1 (RDM1) and that 103
facilitates Pol V transcript (Law and Jacobsen, 2010). 104
In this study, we used the dms3-4 mutant to identify genes whose mutations release the 105
silencing of Pro35S:NPTII, and we cloned POLD2 from a pold2-1 mutant. Whole-106
genome bisulfite sequencing indicated that pold2-1 does not change DNA methylation. 107
Combining ChIP-seq with RNA-seq data, we found a high correlation between 108
H3K27me3 and H3K4me3 modification and gene expression caused by the pold2-1. We 109
also found that pold2-1 exhibits sensitivity to DNA-damaging reagents, short telomere 110
length, and genomic instability, including a high frequency of homologous recombination 111
(HR). These results suggest that POLD2 is a new component in DRR-TGS pathway, 112
which does not affect the DNA methylation and H3K9me2, but H3K27me3 and 113
H3K4me3 modification with changing the expression of specific genes. 114
Results 115
Isolation of a new component in the DRR-TGS pathway 116
dms3-4 was identified during a genetic screen for mutants that silence Pro35S:NPTII in a 117
transgenic Arabidopsis line carrying expressed ProRD29A:LUC and Pro35S:NPTII (C24 118
accession, which was used as the wild type in this study) (Li et al., 2012). We performed 119
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a forward genetic screen using an ethyl methanesulfonate (EMS)-mutagenized dms3-4 120
population to isolate the mutants that release the silenced Pro35S:NPTII in dms3-4. We 121
isolated several mutants including pold2-1, rfc1-4, ubiquitin-specific protease26 (ubp26-122
5), and histone deacetylase 6 (hda6-11) in this screen (Figure 1A and Supplementary 123
Figure S1). The dms3-4 mutant was kanamycin-sensitive because of the silencing of 124
Pro35S:NPTII caused by the reduced expression of ROS1, while pold2-1, rfc1-4, and 125
ubp26-5 partially rescued the expression of NPTII in the dms3-4 background (Figure 1B) 126
and hda6-11 fully recovered the expression of NPTII. UBP26 is a H2B deubiquitination 127
enzyme that is identified in a genetic screening for releasing silencing of ProRD29A:LUC 128
and Pro35S:NPTII in ros1-1 (Sridhar et al., 2007). HDA6 is a histone deacetylase that is 129
involved in mediating DNA methylation and TGS (Murfett et al., 2001; Aufsatz et al., 130
2002; Probst et al., 2004). However, the expression level of ROS1 was not greatly 131
restored in these mutants (Figure 1C). TSIs are endogenous TGS loci (Steimer et al., 132
2000), the expression of which is altered in some mutants involved in DNA methylation, 133
histone modification, or DNA replication (Steimer et al., 2000; Xia et al., 2006; Yin et al., 134
2009; Liu et al., 2010; Liu et al., 2010). The expression of TSIs was increased in these 135
isolated mutants (Figure 1D). Interestingly, although pold2-1 suppresses the silenced 136
Pro35S:NPTII in the dms3-4 mutant, the expression of NPTII in the pold2-1 single 137
mutant was lower than in the wild type (Figure 1B). To confirm the role of POLD2 in 138
controlling the TGS of Pro35S:NPTII, we crossed pold2-1 with other TGS-related 139
mutants. ROS1 and ROS4 (IDM1) are DNA demethylation factors, and ros1 and ros4 140
exhibited kanamycin sensitivity due to the silencing of Pro35S:NPTII (Li et al., 2012). 141
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The double mutants pold2-1 ros1-1 and pold2-1 ros4-1 were resistant to kanamycin 142
(Figure 1E). pold2-1 also suppressed the kanamycin-sensitive phenotype in nrpd1-8 and 143
nrpe1-14 (Figure 1E). However, pold2-1 had little effect on the ProRD29A:LUC locus in 144
dms3-4. Like mutants of DNA replication-related proteins (Elmayan et al., 2005; Xia et 145
al., 2006; Yin et al., 2009; Liu et al., 2010; Liu et al., 2010), pold2-1 failed to suppress 146
the silenced ProRD29A:LUC in the ros1-1 mutant (Figure 1F). These results indicate that 147
pold2-1 suppresses the TGS of Pro35S:NPTII and that POLD2 is a newly identified 148
component in the DRR-TGS pathway. 149
Map-based cloning of POLD2 150
The F1 progeny of pold2-1 dms3-4 backcrossed with dms3-4 had a kanamycin-sensitive 151
phenotype like that of dms3-4 (Figure 2A), and the F2 progeny showed a kanamycin-152
sensitive to kanamycin-resistant ratio of about 3:1 (177:56) (Figure 2B), indicating that 153
the pold2-1 allele is recessive and caused by a single nuclear gene mutation. pold2-1 154
mutant plants were much smaller and flowered earlier than the wild type (Figure S2B). 155
To clone POLD2, we crossed pold2-1 dms3-4 (C24) with the dms3-1 mutant (Columbia-156
0). The mutants with a kanamycin-resistant phenotype were isolated from the F2 progeny. 157
The mutated position in pold2-1 was mapped at the end of chromosome 2, between BAC 158
clones T28M21 and F18O19. A G-A mutation located at 1170 nucleotides from the 159
putative start codon was identified in AT2G42120 (Figure 2C). Because the mutated 160
nucleotide is located at a splicing site between the fifth intron and the sixth exon, we 161
sequenced the various transcripts using OligodT reverse-transcribed cDNAs. Unlike 162
POLD2 in the wild type, the mis-spliced allele in pold2-1 produced several forms of 163
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transcripts (a total of five transcripts were identified from 23 clones). Among them, four 164
transcripts produced premature stop codons, and one transcript missing 6-bp caused 165
deletion of two amino acids and mutation of one amino acid, which might result in the 166
translation of a protein with reduced function (Figure 2D). To confirm that the 167
kanamycin-resistant and retarded-growth phenotype was caused by the POLD2 mutation, 168
we complemented the pold2-1 dms3-4 double mutant with the full-length POLD2 169
genomic sequence. The transgenic progeny showed kanamycin sensitivity on MS plates 170
containing 50 mg/L kanamycin, and grew normally in the absence of kanamycin (Figure 171
2E). The other two constructs, Pro35S:FLAG-HA-POLD2 and ProPOLD2: POLD2-GFP, 172
could also complement the pold2-1 phenotypes (Figure 2E). The T-DNA insertion mutant 173
pold2-2 (762B02) was embryo lethal (Supplementary Figure S2A), indicating that 174
POLD2 is an essential gene in Arabidopsis. We crossed pold2-1 with pold2-2-/+, and 175
about half of the F1 progeny showed more severe dwarf phenotypes than the pold2-1 176
mutant, indicating that pold2-1 protein dosage influences the plant growth phenotype 177
(Supplementary Figure S2B). To determine whether the transcript missing 6 bp in pold2-178
1 is functional in plants, we transferred this transcript driven by the 35S promoter 179
(Pro35S:pold2-1) into heterozygous pold2-2-/+ plants. Like the pold2-1 mutant, the 180
transgenic plants carrying Pro35S:pold2-1 with homozygous T-DNA insertion were 181
small (Supplementary Figure S2C). These results suggest that this transcript from pold2-1 182
is partially functional in plants. ProPOLD2:GUS expression indicated that POLD2 is 183
highly expressed in the shoot meristem, cotyledons, and older true leaves (Supplementary 184
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Figure S3A). POLD2-GFP localization in transgenic plants indicated that POLD2 is a 185
nuclear protein (Supplementary Figure S3B). 186
Genetic analysis of POLD2 with other DNA-replication proteins 187
To better characterize the genetic interactions between POLD2 and other DNA-188
replication proteins, we crossed pold2-1 with some DNA replication-related mutants and 189
analyzed the phenotypes of the double mutants (Figure 3). pold2-1 mutant plants were 190
much smaller and flowered earlier than the wild type (Figure 3A). pold2-1 polα double 191
mutants were smaller and exhibited more severe growth phenotypes than polα or pold2-1 192
single mutants (Figure 3A), suggesting that the two genes have additive effects on plant 193
growth and development. In contrast, polε polα double mutants exhibited phenotypes 194
similar to polα mutants (Figure 3B), suggesting that Polα and Pol ε do not have additive 195
effect on plant growth and work in the same pathway. The phenotypes of the pold2-1 196
ror1-2 (rpa2a) and polε ror1-2 double mutant were very similar to those of the ror1-2 197
single mutant (Figure 3C, 3D), suggesting that RPA2A is a limiting factor for leading- 198
and lagging-strand DNA replication. The pold2-1 pol ε double mutant was similar to 199
pold2-1 (Figure 3E), indicating that POLD2 acts at or has an epistasis effect on Pol ε for 200
controlling plant development. Furthermore, polα ror1-2 double mutants exhibited more 201
severe growth phenotypes than polα or ror1-2 single mutants (Figure 3F), suggesting that 202
Polα and RPA2A interact genetically in controlling plant growth (Figure 3F). 203
ATR (Ataxia Telangiectasia-mutated and Rad3-related) is a DNA damage-activated 204
protein kinase that is involved in the progression of DNA replication forks, and ATM 205
(Ataxia Telangiectasia-Mutated) is a double-strand break-activated protein kinase. The 206
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atr mutant did not show a clear growth phenotype, and the atm mutant had a partially 207
sterile phenotype relative to the wild type under normal growing conditions (Garcia et al., 208
2003; Culligan et al., 2004). The pold2-1 atr double mutant showed severe defects in leaf 209
development and fertility (Figure 3G), and the pold2 atm double mutant showed growth 210
phenotypes similar to those of pold2-1 (Figure 3H). These results suggest that POLD2 211
and ATR have additive roles in controlling plant development. 212
The pold2-1 mutant exhibits sensitivity to DNA damage, a delayed cell cycle, 213
increased homologous recombination, and a reduced telomere length 214
Previous studies indicated that DNA replication-related proteins have roles in controlling 215
the cell cycle, homologous recombination, and genomic stability (Liu and Gong, 2011). 216
In evaluating the effects of POLD2 on these processes, we found that the pold2-1 mutant 217
was more sensitive to alkylating agent methyl methanesulfonate (MMS) than to the 218
DNA-replication inhibitor hydroxyurea (HU) or to the DNA-interstrand crosslink-reagent 219
cisplatin (Figure 4A, 4B). QRT-PCR assessment of the expression of several genes that 220
are responsible for DNA repair including breast cancer susceptibility 1 (BRCA1), RAD51, 221
poly(ADP-ribose) polymerase 1 (PARP1), and PARP2. These genes were expressed at 222
higher levels in pold2-1 or pold2-1 dms3-4 than in the wild type or dms3-4 (Figure 4C). 223
To investigate cell cycle progression in the pold2-1 mutant, we crossed pold2-1 with the 224
ProCYCB1;1:GUS reporter line (CYCB1;1 is expressed during the G2/M transition and 225
serves as a marker for cell cycle regulation). GUS activity in both the root and the shoot 226
apical meristem was much stronger in pold2-1 than in the wild type (Figure 4Da-d). RT-227
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PCR confirmed that expression of the mitotic cyclin CYCB1;1 was higher in pold2-1 than 228
in the wild type (Figure 4De). This result indicates that pold2-1 delays the cell cycle. 229
We evaluated the frequency of HR in pold2-1 by introducing the reporter line 651 (in 230
C24 accession) into the pold2-1. Reporter line 651 carries an inverted orientation of the 231
inactive GUS gene (Lucht et al., 2002). A blue sector indicates that an intrachromosomal 232
recombination event has reconstituted a functional GUS gene. As shown in Figure 4E, 233
many more GUS staining sectors were observed in the cotyledons in pold2-1 than in the 234
wild type, suggesting that HR is negatively modulated by POLD2. A previous study 235
indicated that mutations in the catalytic subunit of Pol δ (POLD1) result in genome 236
instability and enhance the frequency of somatic intramolecular HR (Schuermann et al., 237
2009). We also found that the telomere was shorter in pold2-1 or pold2-1 dms3-4 than in 238
the wild type or dms3-4 (Figure 4F and 4G), suggesting that POLD2 is involved in 239
modulating telomere length. 240
pold2-1-released NPTII silencing in dms3-4 is independent of DNA methylation 241
We used whole-genome bisulfite sequencing (BS-seq) to determine whether DNA 242
methylation is altered by pold2-1. Consistent with a previous study (Zhao et al., 2014), 243
the 35S promoter region had a high level of DNA methylation (Figure 5A). The level of 244
DNA methylation in 35S was similar among pold2-1, pold2-1 dms3-4, dms3-4, and the 245
wild type (Figure 5B). Researchers have reported that, in ros1 and some other related 246
mutants, the DNA methylation level at the NOS region is increased (Zhao et al., 2014), 247
which leads to the silencing of NPTII. Indeed, we found that DNA methylation and 248
especially CG methylation is increased in the dms3-4 mutant because of the reduced 249
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expression of ROS1 (Li et al., 2012). The methylation level, however, is similar in dms3-250
4 and pold2-1 dms3-4 (Figure 5C). Interestingly, DNA methylation pattern in pold2-1 is 251
much more similar to that in the pold2-1 dms3-4 or dms3-4 mutant than to that in the wild 252
type (Figure 5C). This is probably because the pold2-1 single mutant was obtained from 253
the progeny of pold2-1 dms3-4 crossed with the wild type, and the hyper DNA 254
methylation pattern was maintained after dms3-4 was recovered. These results suggest 255
that DNA methylation is not responsible for the release of the silencing of NPTII in the 256
dms3-4 mutant background. 257
We next checked DNA methylation on a genome-wide scale using the BS-seq. 258
pold2-1 mutant did not show a significantly altered DNA methylation level at the whole-259
genome scale (Figure 5D-E). In agreement with a previous study showing that the DNA 260
single-stranded binding protein RPA2A does not affect DNA methylation (Elmayan et al., 261
2005; Stroud et al., 2013), POLD2 is dispensable for DNA methylation. These results 262
suggest that the DNA replication machinery does not influence DNA methylation. 263
POLD2 mediates H3K27me3 and H3K4me3 histone modification in NPTII 264
Given that POLD2 does not affect DNA methylation and that its mutation changes the 265
expression of NPTII, we suspected that POLD2 may affect histone modification. We 266
performed whole-genome ChIP-seq for H3K27me3, H3K4me3, H3K9me2, and H3 in the 267
wild type and pold2-1. 268
Using ChIP-seq data, we compared the histone modification profiles on the T-DNA 269
locus. The 35S promoter region harbors a slightly higher level of H3K9me2 histone 270
modification in pold2-1 than in the wild type (Figure 6A and 6B). The NPTII region had 271
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decreased H3K27me3 and H3K4me3 but increased H3K9me2 in the pold2-1 mutant 272
comparing to the wild type (Figure 6A and 6B). We next examined histone modification 273
levels among the wild type, dms3-4, pold-1 dms3-4, and pold2-1 using ChIP-PCR. We 274
found that the H3K27me3 level at the NPTII gene body region was much lower in the 275
pold2-1 or pold2-1 dms3-4 mutant than in the wild type or in the dms3-4 mutant (Figure 276
6D), which was consistent with the ChIP-seq data that pold2-1 decreased H3K27me3 277
(Figure 6A and 6B). The H3K9me2 level of both 35S and NPTII was higher in dms3-4 278
than in the wild type (Figure 6C-D), which would lead to a repression of NPTII gene 279
expression in dms3-4. The H3K9me2 level in the pold2-1 dms3-4 double mutant and in 280
the pold2-1 single mutant, however, was similar to that in the dms3-4 mutant (6C, 6D), 281
suggesting that the release of NPTII silencing in the pold2-1 mutant in the dms3-4 282
background was not due to changes in the H3K9me2 level. The H3K4me3 level in the 283
NPTII region was lower in dms3-4 than in the wild type (Figure 6D). pold2-1 mildly 284
increased the H3K4me3 level in the dms3-4 background, but the H3K4me3 level was still 285
lower in the pold2-1 background than in the wild type, which is consistent with the lower 286
expression of NPTII in the pold2-1 mutant (Figure 1B and 6D). Ubiquitin-conjugating 287
enzyme 21 (UBI) was used as a positive control for H3K4me3 and as a negative control 288
for H3K27me3 and H3K9me2, and the histone modification pattern on the UBI region 289
was similar among the four samples (Figure 6E). Together, these results suggest that the 290
release of NPTII silencing in the dms3-4 mutant by pold2-1 results from a reduced level 291
of H3K27me3. 292
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We next sought to determine whether H3K27me3 or H3K4me3 is altered in the other 293
mutants isolated from the same screen. Indeed, we found the H3K27me3 level on the 294
NPTII was decreased in the rfc1-4, ubp26-5 and hda6-11 mutants compared to that in the 295
wild type (Figure S4A). We also found that rfc1-4 and ubp26-5 could partially restore the 296
level of H3K4me3 in the dms3-4 background (Figure S4B). hda6-11 greatly increased 297
H3K4me3, and the expression level of NPTII was higher in the hda6-11 mutant than 298
other mutants (Figure S4B and Figure 1B). These results suggest that RFC1, UBP26 and 299
HDA6 are also involved in mediating H3K27me3 and H3K4me3 modification and NPTII 300
expression. 301
Correlation between changes in levels of H3K27me3 or H3K4me3 and changes in 302
gene expression in the pold2-1 mutant 303
We identified 7786 genes with H3K27me3 (Supplementary Figure S5A, Supplementary 304
table S1), and these genes largely overlap with those identified in two previous studies: 305
they overlap with 85.4% (4,346 of 5,090, Supplementary Figure S5A) of the genes 306
previously identified in a ChIP-seq study (Lu et al., 2011) and with 83.4% (4154 out of 307
4980, Supplementary Figure S5A) of those genes previously identified in another ChIP-308
chip analysis (Zhang et al., 2007). We identified 17,849 genes with H3K4me3, and these 309
genes overlap with 97.8% (13,899 of 14,206, Supplementary table S2) of those reported 310
by Luo et al. (Luo et al., 2012) (Supplementary Figure S5A). H3K9me2 is mainly 311
localized on TEs (Supplementary Figure S5B). We also found 3697 genes that harbor 312
H3K9me2 (Supplementary table S3), and these genes overlap with 73% (836 of 1147) of 313
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those reported by Luo et al. (Supplementary Figure S5A) (Luo et al., 2012). Overall, 314
these results indicate that our ChIP-seq data are reliable. 315
Based on ChIP-seq data, we found 977 genes whose H3K27me3 levels were lower in 316
the pold2-1 mutant than in the wild type (P-value <0.01, false discovery rate (FDR) 317
<0.01, Supplementary table S4). After plotting the epigenetic profile of these 977 genes 318
with decreased levels of H3K27me3, we found that the H3K27me3 level was lower on 319
the gene body in the pold2-1 mutant than in the wild type (Figure 7A). The H3K4me3 320
levels in these 977 genes were relatively low. The H3K4me3 signal is mainly detected 321
after the transcriptional start site (TSS), the level of which was higher in pold2-1 than in 322
the wild type (Figure 7A). The total H3 was enriched on these gene body regions but 323
depleted on the TSS or transcription termination sites (TTS) (Figure 7A), which was 324
consistent with a previous report (Ha et al., 2011). The level of total H3 was slightly 325
lower in the pold2-1 mutant than in the wild type (Figure 7A). To determine whether 326
changes in H3K27me3 were correlated with changes in gene expression, we performed 327
RNA-seq of the wild type and the pold2-1 mutant (Supplementary table S5). Among the 328
977 genes with reduced levels of H3K27me3 in pold2-1, 385 were found to be expressed, 329
of which, 282 genes (73%) were up-regulated. After plotting these 385 expressed genes 330
using a heat map and a box plot, we found that the expression levels of these genes were 331
significantly higher in pold2-1 than in the wild type (P-value <0.0001, paired Student’s t-332
test, Figure 7B-C). These results suggest that pold2-1 leads to a decrease in H3K27me3 333
level, which in turn may increase gene expression. 334
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We found 343 genes that had a significantly higher level of H3K27me3 in the pold2-1 335
mutant than in the wild type (P-value <0.01, FDR <0.01, Supplementary table S6). The 336
increased H3K27me3 modification mainly occurred on the gene body regions (Figure 337
7D). Among these genes, there were no obvious changes in H3K4me3 or in H3 between 338
pold2-1 and the wild type (Figure 7D). Of the 343 genes with increased levels of 339
H3K27me3, 117 were expressed (71 down-regulated and 46 up-regulated), but the 340
expression was not significantly different between pold2-1 and the wild type (Figure 7E-341
F, P-value = 0.2292, paired Student’s t-test). These results suggest that the moderate 342
increase in H3K27me3 level in pold2-1 does not apparently change the gene expression. 343
We found 499 genes that had significantly higher H3K4me3 levels in the pold2-1 344
mutant than in the wild type (P-value <0.01, FDR <0.01, Supplementary table S7), and 345
21.4% (107 of 499) of these genes overlapped with genes with decreased levels of 346
H3K27me3. After plotting the epigenetic profiles of these 499 genes, we found that the 347
increased H3K4me3 signals began to occur just after the TSS (the first nucleosome in the 348
gene body) and then spread to the whole gene body, and that their level was lowest at the 349
TTS (Figure 8A). The H3 level was slightly lower in the pold2-1 mutant than in the wild 350
type (Figure 8A). Most of these 499 genes were expressed (455 of 499), and more than 351
76% (345 out of 455) genes were up-regulated. After statistical analysis, the expression 352
levels were significantly higher in the pold2-1 mutant than in the wild type (Figure 8B, 353
8C, P-value <0.0001, paired Student’s t-test). Only 32 of the genes were found to have a 354
lower H3K4me3 level (P-value <0.01, FDR <0.01, Supplementary table S8, Figure 8D). 355
Among these 32 genes, 30 were found to be expressed, of which 23 showed a lower 356
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expression level in the pold2-1 mutant compared to the wild type. After boxplotting of 357
these 30 genes, they exhibited a lower expression level (Figure 8E-F; P-value = 0.0023, 358
paired Student’s t-test) in pold2-1 than in the wild type. These results suggest that the 359
pold2-1 mutation prominently increases the H3K4me3 level and gene expression. 360
We further selected some loci for validation by ChIP-PCR. SEP3 was recently 361
reported to be a target of the catalytic subunit POLD1, and its H3K4me3 level is 362
increased in the pold1 mutant (Iglesias et al., 2015). Consistent with our ChIP data 363
(Figure 9A), SEP3 together with eight other selected genes had decreased H3K27me3 364
levels and increased H3K4me3 levels. Two genes (AT2G39250 and AT1G06360) with 365
increased H3K27me3 and decreased H3K4me3 in pold2 -1 relative to the wild type were 366
also checked by ChIP-PCR (Figure 9B, 9C). The levels of total histone H3 among these 367
genes were similar between the pold2-1 mutant and the wild type (Figure 9D). UBI, 368
which has only a slight modification in H3K27me3 but a substantial modification in 369
H3K4me3, and H3 were used as controls. Consistently, six of nine genes that had 370
decreased H3K27me3 levels but increased H3K4me3 levels had higher expression levels 371
in pold2-1 than in the wild type (Figure 9E). In contrast, the expression levels of 372
AT2G39250 and AT1G06360 (with an increased level of H3K27me3 and a decreased 373
level of H3K4me3) were lower in pold2-1 than in the wild type (Figure 9E). Together, 374
these results suggest a critical role of POLD2 in mediating H3K27me3 and H3K4me3 375
levels and gene expression. 376
Discussion 377
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Genetic screens for the release of the TGS of Pro35S:NPTII in either the ros1 or dms3-4 378
mutant has identified most of the core DNA-replication proteins including the DNA 379
single-strand-binding protein RPA2A (Xia et al., 2006), Pol α (Liu et al., 2010), Pol ε 380
(Yin et al., 2009), POLD2 (this study), RFC1 (Liu et al., 2010), and TOUSLED protein 381
kinase (Wang et al., 2007). Other studies have identified other DNA replication-related 382
proteins involved in mediating TGS including BRU1 (Takeda et al., 2004), chromatin 383
assembly factor (CAF-1) subunits FAS1 and FAS2 (Ono et al., 2006; Schonrock et al., 384
2006), and TEBICHI (Inagaki et al., 2009). The release of TGS by defects in the DNA-385
replication machinery suggests that these processes must have a common mechanism. In 386
this study, we found that POLD2 does not affect DNA methylation but plays crucial roles 387
in regulating H3K27me3 and H3K4me3 modification according to genome-wide ChIP-388
seq. The released silencing of NPTII in dms3-4 is mainly due to the change in histone 389
modifications by pold2-1. These results suggest that the DRR-TGS pathway is essential 390
for maintaining H3K27me3 and H3K4me3 inheritance and gene expression. 391
DNA Pol δ is conserved in eukaryotes and is important in regulating genomic 392
stability and development in Arabidopsis (Schuermann et al., 2009; Iglesias et al., 2015). 393
To our knowledge, the biological functions of Arabidopsis POLD2 have not been studied 394
yet. In this study, we found that POLD2 is an essential gene that contributes to DNA 395
replication, genomic stability through maintaining HR, DNA damage repair and telomere 396
length. In addition, genetic analysis presents a synergistic role of POLD2 with ATR or 397
Pol α in controlling plant development (Figure 3). Similarly, a previous study reported 398
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20
that TEBICHI protein containing both DNA helicase and polymerase domains genetically 399
interacts with ATR to regulate plant development (Inagaki et al., 2009). 400
DNA replication proteins play crucial roles in maintaining histone inheritance. RNA- 401
and ChIP-seq indicate that POLD2 mediates the expression of genes enriched in 402
H3K27me3 and H3K4me3 in gene bodies. We found a high correlation between reduced 403
H3K27me3 levels or increased H3K4me3 levels and increased gene expression, which is 404
consistent with previous studies in Arabidopsis (Zhang et al., 2009) and Drosophila 405
(Papp and Muller, 2006). However, the expression of genes with increased H3K27me3 406
levels in pold2-1 does not show a clear tendency toward reduced gene expression, 407
probably because their expression is already very low, or because the experimental 408
seedlings have mixed cell types; the obtained results might be over- or under-estimated. 409
In this case, we might not detect the difference if change only occurs in a small 410
proportion of the cells in the whole plant. Perhaps future studies on specific cell types or 411
cell cycles will be able to prove how DNA replication factors participate in epigenetic 412
regulation in Arabidopsis. Interestingly, in embryonic stem cells, a specific chromatin-413
modification pattern (chromatin areas with these patterns are termed bivalent domains 414
and harbor both H3K27me3 and H3K4me3) is important for cell differentiation and 415
memory (Bernstein et al., 2006). Our results indicate that the POLD2 mutation altered the 416
levels of H3K27me3 and H3K4me3 of 107 bivalent genes (Figure S6, Supplementary 417
table S9). These results suggest that POLD2 is involved in regulating the bivalent histone 418
modifications, and that the DNA replication machinery could contribute to cell 419
differentiation and cell-type transition. 420
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So far, the intrinsic mechanism that DNA replication affected H3K27me3 or 421
H3K4me3 was not clear, although several lines of evidence suggest that DNA replication 422
or related factors help regulate PcG target genes. Our RNA-seq data indicate that the 423
POLD2 mutation did not change the expression of genes responsible for the 424
establishment and maintenance of H3K4me3 and H3K27me3 significantly 425
(Supplementary table S10). Western blot analyses indicate that the total levels of 426
H3K4me3 and H3K27me3 were not changed in pold2 mutant (Figure S7). These results 427
suggest that POLD2 does not affect the expression of genes for establishment and 428
maintenance of H3K4me3 and H3K27me3 modification, or the global H3K4me3 and 429
H3K27me3 levels. It was previously proposed that DNA polymerase δ may play a role in 430
preventing replication stress or HR and in depositing active H3K4me3 marks (Iglesias et 431
al., 2015). Meanwhile, some studies indicate that the catalytic subunit of DNA 432
polymerase α interacts with (Barrero et al., 2007) or perturbs the binding of like-433
heterochromatin protein 1 (LHP1) (Hyun et al., 2013), which is the main reader/effector 434
for H3K27me3 in Arabidopsis (Barrero et al., 2007; Zhang et al., 2007). Another link 435
between DNA replication and H3K27me3 is MSI1 (multicopy suppressor of ira1), a 436
chromatin assembly factor in Arabidopsis, which interacts with PcG protein and LHP1 437
(Derkacheva et al., 2013). So, it is possible that a dysfunctional Pol δ may disturb the 438
entire replication machinery, which would interfere with the recruitment of LHP1 or PcG 439
proteins to PcG targets. In mammalian cells, PcG proteins can be phosphorylated by 440
cyclin-dependent kinases (CDK) in a cell cycle-dependent manner (Chen et al., 2010). 441
Because pold2-1 exhibits an abnormal cell cycle (Figure 4D), it is also possible that a 442
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disordered cell cycle machinery in the DNA replication-related mutants may influence 443
the activity of PcG proteins and subsequently epigenetic silencing. Further studies will be 444
needed to dissect the molecular mechanisms for how each component of DNA replication 445
machinery participates in this process. 446
Materials and methods 447
Plant materials and growth conditions 448
pold2-1, hda6-11, ubp26-5, and rfc1-4 were isolated from an EMS-mutagenized 449
population of dms3-4 in this study. ros1 (ros1-1), polε (abo4), polα, rpa2a-2 (ror1-2), 450
dms3-4, nrpd1-8, nrpe1-14, and ros4 are C24 ecotypes carrying homozygous 451
PRORD29A:LUC and 35S-NPTⅡ transgenes. GK-762B02 (pold2-2) (Rosso et al., 2003), 452
atr-2 (SALK_032841C), and atm (SALK_040423C) are T-DNA insertion mutants 453
ordered from ABRC. dms3-1 (Col-0, (Kanno et al., 2008)) is a gift from Dr. Matzke. 454
Seeds were sterilized and grown on MS medium plates supplemented with 20 g/L 455
sucrose and 0.8% agar at 22°C under long-day (23 h) illumination. When 7 days old, 456
seedlings were transferred to soil and were grown in a greenhouse at 22°C under long-457
day (16 h) conditions. 458
Map-based cloning of POLD2 459
Approximately 12,000 dms3-4 (C24 background and kanamycin-sensitive) seeds were 460
mutated with EMS. The putative mutants were isolated from M2 seedlings that survived 461
on MS medium containing 50 mg/L kanamycin. For map-based cloning of the POLD2 462
gene, pold2-1 dms3-4 (in C24) was crossed with dms3-1 (Col-0) (Kanno et al., 2008), and 463
250 kanamycin-resistant plants selected from the F2 progeny were analyzed with SSLP 464
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23
markers based on the polymorphism data (http://1001genomes.org/). POLD2 was 465
narrowed to an area between BAC clone F18O19 and T28M21. Based on previous results 466
with DNA polymerases, the POLD2 gene was selected and sequenced, and a point 467
mutation was determined. 468
For complementation, a 4.9-kbp length of POLD2 genomic DNA containing 1932 bp 469
(promoter + 5’UTR) and 254 bp 3’UTR was cloned into pCAMBIA1391. The plasmid 470
was introduced into pold2-1 dms3-4 mutants by Agrobacterium tumefaciens strain 471
GV3101. 472
Subcellular localization of POLD2–GFP fusion protein 473
Three fragments, i.e., the 1932-bp promoters of POLD2, POLD2 cDNA without the TAA 474
stop codon, and GFP cDNA, were obtained using paired primers and were separately 475
cloned into the pCAMBIA1300 vector with POLD2 fused in frame with GFP. The vector 476
was introduced into pold2-1 dms3-4 plants by A. tumefaciens strain GV3101. This 477
construct was able to complement the pold2-1 mutant, indicating that the POLD2-GFP 478
fusion protein was functional. Three-day-old seedlings of the T2 progeny were examined 479
and photographed with a confocal laser scanning microscope (Leica sp5). 480
GUS staining 481
A 1932-bp genomic fragment of POLD2 upstream of ATG was amplified and cloned into 482
the pCAMBIA1391 vector. This ProPOLD2:GUS plasmid was then transformed into the 483
wild type (C24 ecotype) by A. tumefaciens strain GV3101. At least 25 independent 484
transgenic lines were selected for GUS staining. Transgenic plants at different growth 485
stages were collected and stained with freshly made GUS staining buffer (1× PBS, 0.1% 486
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24
Triton X-100, 0.5 mM K3Fe(CN)6, 1 mg/mL X-Gluc) at 37 °C, and were then washed 487
several times with 70% ethanol. 488
Histochemical assay of ProCYCB1;1:GUS 489
Arabidopsis plants transformed with ProCYCB1;1:GUS (Colon-Carmona et al., 1999) 490
were crossed with the pold2-1 mutant. Three independent lines homozygous for both 491
GUS reporter and pold2-1 were selected from F4 progeny and subjected to histochemical 492
GUS staining. Homozygous GUS reporter wild-type lines selected from F4 progeny were 493
used as the control. 494
Intrachromosomal homologous recombination (HR) assay 495
The intrachromosomal recombination reporter line 651 (Molinier et al., 2006) (in C24) 496
was crossed with the pold2-1dms3-4 double mutant. At least three independent F4 plants 497
homozygous for the mutant pold2-1 (DMS3/DMS3×pold2-1/pold2-1) or wild type 498
(DMS3/DMS3×POLD2/POLD2) and homozygous for the GUS transgene were selected. 499
Seedlings were subjected to histochemical GUS staining as mentioned above. 500
Bisulfite sequencing 501
A whole-genome bisulfite sequencing library was prepared mainly according to (Urich et 502
al., 2015). Genomic DNAs were extracted from seedlings using the DNeasy Plant Mini 503
Kit (QIAGEN) according to the manufacturer’s instructions. A 2-μg quantity of genomic 504
DNAs was sonicated (20 cycles of 30 s on, 30 s off, at low intensity) into 200-bp 505
fragments with a Bioruptor (Diagenode, USA), end repaired, 3’-dA-tailed and ligated to 506
methylated adapters. After purification, the ligated DNA fragments were treated with the 507
EZ Methylation-Gold Kit (Zymo Research). The eluted bisulfite-treated DNAs were 508
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25
amplified using barcoded primers for sequencing (CGCTGT for the wild type and 509
ACAGTG for the pold2 mutant). High-throughput sequencing of the BS-seq library was 510
performed on the Illumina NextSeq 500 System with single-end 75-bp reads. The raw 511
sequence data were demultiplexed and converted to fastq files using bcl2fastq2 512
Conversion Software (version v2.16.0) under default parameters. The DNA methylation 513
was analyzed according to (Zhao et al., 2014)(Wang et al., 2015). 514
For locus-specific bisulfite sequencing, 500-ng of DNA was treated with the EZ 515
Methylation-Gold Kit (Zymo Research). Two eluted fractions (10 µL each) were used as 516
PCR templates for amplifying the target sequence; the primers were the same as 517
previously reported (Zhao et al., 2014). Products were cloned into the pMD18-T vector 518
(TaKaRa), and 10-15 independent clones of each sample were sequenced (Lifetech-519
China). 520
Analysis of RNA-seq data 521
Total RNAs extracted from seedlings using the RNeasy Plant Mini Kit (QIAGEN) were 522
used for sequencing on the Illumina platform. Three biological replicates were performed 523
for pold2-1 and the wild type. Paired-end reads were selected and aligned to the 524
Arabidopsis reference genome (version: TAIR10) (Lamesch et al., 2012) using TopHat 525
and Cuffdiff (Trapnell et al., 2012). Values of each sample used for heatmap and boxplot 526
were log2(FPKM)-transformed. FPKM (fragments per kilobase of transcript per million 527
mapped reads) was generated from the output of the Cuffdiff software. 528
Chromatin immunoprecipitation (ChIP) assay 529
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26
ChIP assay was based on the previous studies (Zhang et al., 2007; Lu et al., 2011) with 530
some modifications. A 2-g quantity of 10-day-old seedlings was fixed in 1% 531
formaldehyde. The seedlings were ground into power with liquid nitrogen and 532
homogenized with nuclei extraction buffer (1 M hexylene glycol, 20 mM Tris-HCl (pH 533
8.0), 0.15 mM spermine, 5 mM 2-mercaptoethanol, 1% Triton X-100, 0.1 mM PMSF, 534
and protease inhibitor cocktail). The homogenates were filtered through Miracloth and 535
centrifuged at 2,000 g for 10 min at 4°C. The pellets were washed two times with nuclei 536
extraction buffer and resuspended in 300 μL of nuclei lysis buffer (50 mM Tris-HCl (pH 537
8.0), 10 mM EDTA, 1% SDS and protease inhibitor cocktail). The chromatin was 538
sonicated (15 cycles of 30 s on with 30 s off, at high intensity) into fragments of about 539
300 bp using a Bioruptor. The antibodies used were anti-H3K27me3 (Ab6002, Abcam), 540
anti-H3 (ab1791, Abcam), anti-H3K4me3 (ab8580, Abcam), and anti-H3K9me2 (ab1220, 541
Abcam). Dynabeads (10001D, Life Technologies) were used for immunoprecipitation. 542
After RNase A and Proteinase K treatment, DNAs were recovered using the QIAquick 543
PCR purification kit (QIAGEN). The DNA concentration was determined using the Qubit 544
2.0 system (Life Technologies). The ChIP and input samples were adjusted with dH2O to 545
a concentration of 50 pg/μL, and used for qRT-PCR. Real-time PCR was performed in 20 546
μL with 1 μL of immunoprecipitated or input DNA in SYBR Green Master Mix 547
(TaKaRa). The reaction conditions were as follows: 95°C for 5 min and 40 cycles of 548
95°C for 30 s and 60°C for 1 min. 549
A 10-ng quantity of ChIP or input DNA was used to prepare a high-throughput 550
sequencing library. End repair, dA-tailing, adapter ligation, and amplification were 551
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27
carried out using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina 552
(E6240, NEB) according to the manufacturer’s protocol. Index primers used for each 553
sample were as follows: ATCACG for WT-H3K27me3, CGATGT for WT-H3K4me3, 554
TTAGGC for WT-H3K9me2, TGACCA for WT-H3, ACAGTG for WT-Input, 555
GCCAAT for pold2-H3K27me3, CAGATC for pold2-H3K4me3, ACTTGA for pold2-556
H3K9me2, GATCAG for pold2-H3, and TAGCTT for pold2-Input. High-throughput 557
sequencing of the ChIP-seq library was carried on the Illumina NextSeq 500 System with 558
single-end 75-bp reads. The raw sequence data were demultiplexed and converted to fastq 559
files using bcl2fastq2 Conversion Software (Version v2.16.0) under default parameters. 560
ChIP-seq analysis 561
Reads were mapped to the Arabidopsis genome (TAIR10) using bowtie1 (version 0.12.9) 562
(Langmead et al., 2009) allowing two nucleotide mismatches using following parameters: 563
bowtie -q -k 1 -n 2 -l 36 --best -S -p 2. Mapped reads were de-duplicated and sorted by 564
samtools (version 0.1.19-44428cd) (Li et al., 2009). Then the reads were normalized to 565
reads depth and used for further analysis. Peaks were found using MACS software 566
(version 1.4.2) (Zhang et al., 2008) using H3 as a background. For H3K27me3 peaks 567
finding in the wild type as an example, the parameters were as follow: macs14 -t 568
WT_K27ME3.sorted.bam –c WT_H3.sorted.bam –f BAM -g 1.30e+8 -n WT_K27ME3 569
--shiftsize 73 --pvalue 1e-5 --bw=300 --mfold=10,30. Gene annotation was used 570
BEDTools (Quinlan and Hall, 2010) according to TAIR10. 571
Histone modification changes between the wild type and pold2-1 mutant were determined 572
with SICER software (version 1.1) (Zang et al., 2009). For H3K4me3, the parameters 573
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28
were as follows: SICER-df.sh pold2-1-H3K4me3.bed pold2-1-input.bed WT-574
H3K4me3.bed WT-input.bed 200 200 0.05 0.05; this means that the Window size and 575
Gap size were set at 200 bp. Then filtered by P-value < 0.01, FDR < 0.01 and fold 576
change > 1.5. For H3K27me3, the parameters were as follows: SICER-df.sh pold2-1-577
H3K27me3.bed pold2-1-input.bed WT-H3K27me3.bed WT-input.bed 200 200 0.05 0.05, 578
then filtered by P-value < 0.01, FDR < 0.01. Different regions were annotated with 579
BEDTools. Figure 7 and 8 were made using R packages (ggplot2, Rmisc, and reshape) 580
and homemade script. 581
Real-time PCR 582
Total RNAs were extracted from 0.1 g of seedlings using the RNeasy Plant Mini Kit 583
(QIAGEN). After RNase-free DNaseⅠ(TaKaRa) digestion at 37°C, 4 µg of RNA was 584
reverse transcribed with M-MLV reverse transcriptase (Promega) in a 30-μL volume. A 585
2-µL volume of five-fold-diluted cDNA was added to a 20-µL real-time PCR reaction 586
system in SYBR Green Master Mix (TaKaRa). The reaction was running by StepOnePlus 587
Real-Time PCR System (Applied Biosystems, USA), and the procedure was as follows: 588
95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Specific primers 589
for each gene are listed in Supplementary table S11. UBIQUITIN-CONJUGATING 590
ENZYME 21 (UBI, AT5G25760) was used as the reference gene as previously described 591
(Huettel et al., 2006). Three technical replicates were performed per biological replicate, 592
and two to three biological replicates were used in all experiments. 593
DNA-damage assay 594
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29
A DNA-damage assay was performed as previously described (Liu et al., 2010). Seeds 595
were sterilized and grown on MS medium or MS medium supplemented with one of three 596
DNA-damaging reagents: 25 µM CIS (cis-diamineplatinum (II) dichloride, SIGMA, 597
479306); 50 ppm (0.005%) MMS (methyl methanesulfonate, SIGMA, 129925); or 200 598
mg/L HU ( , SIGMA, H8627). After they were grown for 15 days at 22°C under long-599
day (23 h) illumination, seedlings were imaged and weighed. The fresh weight relative to 600
untreated controls after treatment with DNA-damaging reagents was calculated. Three 601
biological replicates were carried out. 602
Telomere length 603
Genomic DNAs were extracted from 14-day-old seedlings using the DNaesy Plant Mini 604
Kit (QIAGEN) according to the manufacturer’s instructions. A 3-µg quantity of DNA 605
was digested with HinfI or MseI in a 100-μL system at 37 °C. Southern blot was 606
performed with the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche). 607
The synthesized telomere repeat (TTTAGGG), which was labeled with digoxigenin using 608
the DIG Oligonucleotide 3'-End Labeling Kit (Roche), was used as the probe. 609
Accession numbers 610
Sequence data from this article can be found in the EMBL/GenBank data libraries under 611
accession numbers: POLD2 (AT2G42120), RFC1 (AT5G22010), POLA (AT5G67100), 612
POLE (AT1G08260), ROR1 (AT2G24490), ROS1(AT2G36490), DMS3 (AT3G49250), 613
NRPD1 (AT1G63020), NRPE1 (AT2G40030), ROS4 (AT3G14980), ATM 614
(AT3G48190), ATR (AT5G40820), HDA6 (AT5G63110), UBP26 (AT3G49600), , SEP3 615
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30
(AT1G24260), UBI (AT5G25760), BRCA1 (AT4G21070), RAD51 (AT5G20850), 616
PARP1 (AT2G31320), PARP2 (AT4G02390), and CYCB1;1 (AT4G37490). 617
RNA-seq, BS-seq, and ChIP-seq data have been deposited in the National Center for 618
Biotechnology Information Gene Expression Omnibus (GEO) database under accession 619
number GSE79259. 620
Supplemental Data 621
The following supplemental materials are available. 622
Supplemental Figure S1. Diagrams of hda6-11, ubp26-5, rfc1-4, and pold2-1 mutations. 623
Supplemental Figure S2. Genetic analysis of pold2-1 with pold2-2. 624
Supplemental Figure S3. POLD2 is a nuclear protein and is ubiquitously expressed. 625
Supplemental Figure S4. ChIP-qPCR in rfc1-4, ubp26-5 and hda6-11 samples. 626
Supplemental Figure S5. Validation of ChIP-seq data. 627
Supplemental Figure S6. Epigenetic profile of bivalent genes affected by pold2-1 mutant. 628
Supplemental Figure S7. Western blot to check global H3K27me3 and H3K4me3 in the 629
pold2-1 mutant. 630
Supplementary table S1. List of genes that harbor H3K27me3 in the C24 ecotype. 631
Supplementary table S2. List of genes that harbor H3K4me3 in the C24 ecotype 632
Supplementary table S3. List of genes that harbor H3K9me2 in the C24 ecotype. 633
Supplementary table S4. List of genes with decreased levels of H3K27me3 in the pold2-1 634
mutant. 635
Supplementary table S5. RNA-seq data. 636
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Supplementary table S6. List of genes with increased levels of H3K27me3 in the pold2-1 637
mutant. 638
Supplementary table S7. List of genes with increased levels of H3K4me3 in the pold2-1 639
mutant. 640
Supplementary table S8. List of genes with decreased levels of H3K4me3 in the pold2-1 641
mutant. 642
Supplementary table S9. Bivalent genes changed in the pold2-1 mutant. 643
Supplementary table S10. The expression of genes responsible for H3K27me3 and 644
H3K4me3 in the wild type and pold2-1 mutant from RNA-seq data. 645
Supplementary table S11. Primers used in this study. 646
647
Acknowledgments 648
We would like to thank Dr. Marjori A. Matzke for providing dms3-1 mutant. We also 649
thank Arabidopsis Biological Resource Center for T-DNA mutant. 650
651
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32
Figure legends 652
Figure 1. Isolation of mutants that suppress the kanamycin sensitivity of the dms3-4 653
mutant. 654
A. The phenotype of the wild type (WT), dms3-4, and suppressors of dms3-4 growing on 655
MS medium supplemented with 50 mg/L kanamycin. 656
B. Relative expression of NPTII in the WT, dms3-4, and suppressors of dms3-4. 657
C. Relative expression of ROS1 in the WT, dms3-4, and suppressors of dms3-4. 658
D. Relative expression of TSI in WT, dms3-4, and suppressors of dms3-4. For B, C, and 659
D, RNAs were extracted from 7-day-old seedlings. Values were normalized to the 660
expression level of the references gene (UBI). Two independent experiments (each with 661
three technical replicates) were done with similar results. Values are means ± SE, n=3, 662
from one experiment. 663
E. pold2-1 released the silencing of 35S:NPTII in ros1, ros4, nrpd1, and nrpe1. The 664
growth phenotype of the WT, pold2-1, ros1, ros4, nrpd1-8, nrpe1-14, and double mutants 665
pold2-1 ros1, pold2-1 ros4, pold2-1 nrpd1-8, and pold2-1 nrpe1-14 on MS containing 0 666
or 50 mg/L kanamycin (Kan). 667
F. pold2-1 could not restore the expression of silenced RD29A:LUC caused by the ros1 668
mutant. Luminescence imaging of the RD29A:LUC transgene. The indicated plants were 669
treated with 300 mM NaCl for 3 h, followed by luminescence imaging. 670
Figure 2. Map-based cloning of POLD2 671
A. Phenotypic analysis of F1 seedlings of dms3-4 pold2-1 backcrossed with dms3-4 on 672
kanamycin-containing medium. 673
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33
B. The segregation of F2 seedlings growing on kanamycin-containing medium. 674
C. Map-based cloning of the pold2-1 mutation. The position of the POLD2 mutation was 675
narrowed to the bottom of chromosome 2 between BAC T28M21 and F18O19. The 676
mutation of pold2-1 occurred at splicing sites (G1170A). pold2-2 is a T-DNA insertion 677
allele. The domain structure of the POLD2 protein is indicated. OB, oligonucleotide-678
/oligosaccharide-binding domain; PDE, phosphodiesterase-like domain. 679
D. Spliced forms of cDNA caused by the pold2-1 mutation. Five kinds of transcripts were 680
identified from 23 independent clones amplified from cDNAs. Among them, four 681
transcripts would produce an earlier stop codon, and one would delete 6 bp and lead to 682
the deletion of two amino acids (delete QE) and to the mutation of one amino acid (from 683
original D to H). 684
E. Complementary analyses of pold2-1 with genomic DNA or Pro35S:FLAG-HA-685
POLD2 construct, or native promoter driving cDNA (ProPOLD2: POLD2-GFP). 686
Phenotypes of mutant and complementary lines growing on MS medium containing 0 687
mg/L kanamycin (left) or 50 mg/L kanamycin (right). 688
Figure 3. Phenotypic analyses of pold2-1 with other DNA replication-related 689
mutants. 690
A. Phenotype of the wild type (WT), pold2-1, pol α, and pold2-1 polα double mutant. 691
B. Phenotype of the WT, pol ε, pol α, and polε pol α double mutant. 692
C. Phenotype of the WT, pold2-1, ror1-2 (rpa2a), and pold2-1 ror1-2 double mutant. 693
D. Phenotype of the WT, polε, ror1-2 (rpa2a), and polε ror1-2 double mutant. 694
E. Phenotype of the WT, polε, pold2-1, and pold2-1 polε double mutant. 695
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34
F. Phenotype of the WT, ror1-2 (rpa2a), pol α, and polα ror1-2 double mutant. 696
G. Phenotype of the WT, atr, pold2-1, and atr pold2-1 double mutant. 697
H. Phenotype of the WT, atm, pold2-1, and atm pold2-1 double mutant. Bar = 1 cM. 698
Figure 4. The pold2-1 mutant has increased sensitivity to DNA damage, a delayed 699
cell cycle, increased homologous recombination, and a reduced telomere length. 700
A. pold2-1 is hypersensitive to MMS. Wild-type, dms3-4, pold2-1 dms3-4, and pold2-1 701
seedlings that were grown for 15 days on MS medium alone or MS medium with 200 702
mg/L hydroxyurea (200 HU), 50 ppm methyl methanesulfonate (50 MMS), or 25 µM 703
cisplatin (25 CIS). P values were calculated using Paired Student’s t test. 704
B. Relative fresh weight of seedlings in (D). The fresh weight relative to untreated 705
controls after treatment with DNA-damaging reagents is shown. Values are means ± SE, 706
n=3. 707
C. Relative expression of DNA damage response genes in the wild type, dms3-4, pold2-1 708
dms3-4, and pold2-1 as determined by qRT-PCR. RNAs extracted from 7-day-old 709
seedlings were used for qRT-PCR. Values were normalized to the expression level of the 710
references gene (UBI). Two independent experiments (each with three technical 711
replicates) were done with similar results. Values are means ± SE, n=3, from one 712
experiment. 713
D. pold2-1 increases the expression of ProCYCB1;1:GUS. ProCYCB1;1:GUS was 714
introduced into the pold2-1 mutant by crossing the wild type carrying ProCYCB1;1:GUS 715
with the pold2-1 mutant. a–d show GUS staining in (a) a wild-type seedling; (b) a wild-716
type root tip; (c) a pold2-1 seedling; and (d) a pold2-1 root tip. (e) CYCB1;1 expression 717
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35
as determined by qRT-PCR. RNAs were extracted from 7-day-old seedlings and used for 718
qRT-PCR. Values were normalized to the expression level of the references gene (UBI). 719
Two independent experiments (each with three technical replicates) were done with 720
similar results. Values are means ± SE, n=3, from one experiment. 721
E. pold2-1 increases homologous recombination (HR) in 14-day-old seedlings. The wild-722
type 651 carrying a homologous reporter GUS gene was crossed with the dms3-4 pold2-1 723
mutant. Homozygous pold2-1 plants carrying the homozygous GUS reporter were 724
isolated and used for GUS staining. 725
F-G. pold2-1 exhibits a reduced telomere length. Genomic DNAs of the wild type, dms3-726
4, pold2-1, and pold2-1 dms3-4 digested by HinfI (DNA-methylation sensitive, F) or 727
MseI (DNA-methylation insensitive, G) were subjected to Southern blot with a telomere 728
probe. 729
Figure 5. pold2-1 releases the silencing of NPTII in dms3-4 independent on DNA 730
methylation. 731
A. IGV visualization of the DNA methylation level (CG, CHG, and CHH) at the 732
transgenic T-DNA locus in the wild type (WT) and pold2-1 mutant from whole-genome 733
bisulfite sequencing data. 734
B-C. Methylation level of the 35S (B) and NOS (C) regions of the wild type, dms3-4, 735
pold2-1, and dms3-4 pold2-1 as determined by bisulfite sequencing (10-15 clones were 736
analyzed for each sample). 737
D. Patterns of DNA methylation (CG, CHG, and CHH) across all genes in the wild type 738
(WT) and the pold2-1 mutant. Genes were aligned from the transcriptional start sites 739
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36
(TSS) to the transcriptional termination sites (TTS), and average methylation for all 740
cytosines within each bin is plotted. 741
E. Patterns of DNA methylation (CG, CHG, and CHH) across all transposon elements 742
(TEs) in the wild type (WT) and the pold2-1 mutant. TEs were aligned from the 743
transcriptional start sites (TSS) to the transcriptional termination sites (TTS), and average 744
methylation for all cytosines within each bin is plotted. 745
Figure 6. pold2-1 decreases the H3K27me3 level at NPTII 746
A. Histone modification status of the transgene construct in the wild type (WT) and 747
pold2-1 mutant. Data of Histone H3 lysine 9 dimethylation (K9me2), Histone H3 lysine 748
27 trimethylation (K27me3), Histone H3 lysine 4 trimethylation (K4me3), total histone 749
H3 (H3), and input (Input) level were shown from whole genome ChIP-seq. The data 750
range of each modification was set to the same scale between the wild type and pold2-1 751
mutant. 752
B. Chart depicted the relative quantitative histone modification level of 35S or NPTII 753
region as shown in A. 754
C-E. Histone modification levels at 35S (C), NPTII (D), and UBI (E) in the wild type, 755
dms3-4, pold2-1 dms3-4, and pold2-1 as indicated by ChIP-PCR analysis. The histone 756
modification levels of H3K27me3, H3K4me3, H3K9me2, and total histone H3 on each 757
indicated locus were detected by ChIP-qPCR. UBI was used as a control locus enriched 758
in H3K4me3 but depleted in H3K27me3 or H3K9me2 modification. The error bars 759
represent the standard error of the means. 760
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37
Figure 7. Correlation between H3K27me3 signals across the genes and mRNA 761
expression levels caused by the pold2-1 mutation. 762
A. Altered histone modification profiles of 977 H3K27me3-decreased genes by pold2-1. 763
Genes were aligned from the transcriptional start sites (TSS) to the transcriptional 764
termination sites (TTS) and divided into 60 bins. The 2,000-bp (2-kb) regions either 765
upstream of TSS or downstream TTS were also included and divided into 20 bins 766
respectively. Histone modification levels of each bin are plotted. 767
B. Gene expression profiles of H3K27me3-decreased genes in pold2-1 and the wild type. 768
Values were log2FPKM-transformed. FPKM (fragments per kilobase of transcript per 769
million mapped reads) were generated from RNA-seq using TopHat and Cufflinks 770
software. 771
C. Boxplot showing the expression (log2FPKM-transformed) of genes in B. The mean 772
value of the group was significantly higher in pold2-1 than in the wild type (*** 773
P<0.0001, two-tailed paired Student’s t-test). 774
D. Altered histone modification profiles of 343 H3K27me3-increased genes by pold2-1 775
mutant. Genes were aligned from the transcriptional start sites (TSS) to the transcriptional 776
termination sites (TTS) and divided into 60 bins. The 2,000-bp (2-kb) regions either 777
upstream of TSS or downstream TTS were also included, and each was divided into 20 778
bins. Histone modification levels of each bin are plotted. 779
E. Gene expression profiles of H3K27me3-increased genes in pold2-1. Values shown are 780
log2FPKM-transformed. FPKM (fragments per kilobase of transcript per million mapped 781
reads) were generated from RNA-seq using TopHat and Cufflinks software. 782
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38
F. Boxplot showing the expression (log2FPKM-transformed) of genes in B. The mean 783
value of the group in pold2-1 was not significantly different from that in the wild-type 784
group (n. s., not significant, paired Student’s t-test). 785
Figure 8. Correlation between H3K4me3 signals across the gene and mRNA 786
expression levels caused by pold2 mutant 787
A. Altered histone modification profiles of 499 H3K4me3-increased genes by pold2-1. 788
Genes were aligned from the transcriptional start sites (TSS) to the transcriptional 789
termination sites (TTS) and divided into 60 bins. The 2,000-bp (2-kb) regions either 790
upstream of TSS or downstream TTS were also included, and each was divided into 20 791
bins. Histone modification levels of each bin are plotted. 792
B. Gene expression profiles of H3K4me3-increased genes by pold2-1. Values shown are 793
log2FPKM. FPKM (fragments per kilobase of transcript per million mapped reads) was 794
generated from RNA-seq using TopHat and Cufflinks software. 795
C. Boxplot showing the expression (log2FPKM-transformed) of genes in B. The mean 796
value of the pold2-1 group was significantly higher than that of the wild-type group (*** 797
P<0.0001, two-tailed, paired Student’s t-test). 798
D. Altered histone modification profiles of 32 H3K4me3-decreased genes by pold2-1 799
mutant. Genes were aligned from the transcriptional start sites (TSS) to the transcriptional 800
termination sites (TTS) and divided into 60 bins. The 2,000-bp (2-kb) regions either 801
upstream of TSS or downstream TTS were also included, and each was divided into 20 802
bins. Histone modification levels of each bin are plotted. 803
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39
E. Gene expression profiles of H3K4me3-decreased genes by pold2-1 mutant. Values are 804
log2FPKM-transformed. FPKM (fragments per kilobase of transcript per million mapped 805
reads) was generated from RNA-seq using TopHat and Cufflinks software. 806
F. Boxplot showing the expression (log2FPKM) of genes in B. The mean value of the 807
pold2-1 group was significantly lower than that of the wild-type group (** P<0.001, two-808
tailed paired Student’s t-test). 809
Figure 9. IGV visualization of selected histone modification altered genes, and 810
validation of ChIP-seq results. 811
A. IGV views of reads density (RPKM) of H3K27me3 (K27me3), H3K4me3 (K4me3), 812
H3, and Input from CHIP-seq data in the wild type (WT) and pold2-1 mutant. The data 813
range of each modification was set to the same scale for the WT and pold2-1 mutant. 814
AT2G39250 and AT1G06360 are examples of genes with increased levels of H3K27me3 815
and decreased levels of H3K4me3. UBI is a positive control for H3K4me3 and a negative 816
control for H3K27me3; the level of histone modification for UBI was similar for the wild 817
type (WT) and the pold2-1 mutant. The other genes had decreased levels of H3K27me3 818
and increased levels of H3K4me3. The red bars on the top of each gene indicate the 819
primer location used for validation. 820
B-D. ChIP-qPCR verified the selected histone modification changed genes. The histone 821
modification level of H3K27me3 (B), H3K4me3 (C), and total histone H3 (D) on each 822
indicated gene was measured by ChIP-qPCR between the wild type (WT) and pold2-1 823
mutant. Values presented are relative to the input, the error bars represent the standard 824
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40
error of the mean (n=3). AT5G25760 (UBI) was used as a control locus enriched in 825
H3K4me3 modification but depleted in H3K27me3 modification. 826
E. Relative gene expression level of these selected genes. RNAs extracted from 10-day-827
old seedlings were used for qRT-PCR. Values were normalized to the expression level of 828
the references gene (UBI). Two independent experiments (each with three technical 829
replicates) were done with similar results. Values are means ± SE, n=3, from one 830
experiment. 831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
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41
846
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