B Compartments

Molecular Plant Research Article

3D Chromatin Architecture of Large Plant Genomes Determined by Local A/B Compartments €2, Ning Zhu2, Donald Grierson3, Pengfei Dong1,2,4, Xiaoyu Tu2,4, Po-Yu Chu2,4, Peitao Lu Baijuan Du1, Pinghua Li1,* and Silin Zhong2,* 1

State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai’an, Shandong, China

2

State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China

3

Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Zhejiang University, Hangzhou, Zhejiang, China

4These

authors contributed equally to this article.

*Correspondence: Pinghua Li ([email protected]), Silin Zhong ([email protected]) https://doi.org/10.1016/j.molp.2017.11.005

ABSTRACT The spatial organization of the genome plays an important role in the regulation of gene expression. However, the core structural features of animal genomes, such as topologically associated domains (TADs) and chromatin loops, are not prominent in the extremely compact Arabidopsis genome. In this study, we examine the chromatin architecture, as well as their DNA methylation, histone modifications, accessible chromatin, and gene expression, of maize, tomato, sorghum, foxtail millet, and rice with genome sizes ranging from 0.4 to 2.4 Gb. We found that these plant genomes can be divided into mammalian-like A/B compartments. At higher resolution, the chromosomes of these plants can be further partitioned to local A/B compartments that reflect their euchromatin, heterochromatin, and polycomb status. Chromatins in all these plants are organized into domains that are not conserved across species. They show similarity to the Drosophila compartment domains, and are clustered into active, polycomb, repressive, and intermediate types based on their transcriptional activities and epigenetic signatures, with domain border overlaps with the local A/B compartment junctions. In the large maize and tomato genomes, we observed extensive chromatin loops. However, unlike the mammalian chromatin loops that are enriched at the TAD border, plant chromatin loops are often formed between gene islands outside the repressive domains and are closely associated with active compartments. Our study indicates that plants have complex and unique 3D chromatin architectures, which require further study to elucidate their biological functions. Key words: Hi-C, chromatin loop, compartment domain, local compartment € P., Zhu N., Grierson D., Du B., Li P., and Zhong S. (2017). 3D Chromatin Dong P., Tu X., Chu P.-Y., Lu Architecture of Large Plant Genomes Determined by Local A/B Compartments. Mol. Plant. 10, 1497–1509.

INTRODUCTION Chromatin, the main carrier of eukaryotic genetic information, is non-randomly packaged within the nucleus (Sexton and Cavalli, 2015). Recent developments in chromatin conformation capture technologies enabled us to examine their 3D architectures by proximity ligation (Dekker et al., 2002; Fullwood et al., 2009; Rao et al., 2014). It has been shown that mammalian interphase chromosomes can be partitioned into megabase-sized active and inactive chromosome regions (also known as A/B compartments) based on their genome-wide interaction pattern (Lieberman-Aiden et al., 2009). The A and B compartments are associated with the euchromatic and heterochromatic regions, respectively. They also have distinct genetic and epigenetic

features, such as DNA methylation, open chromatin, transcription, repeats, lamina association and replication timing (Lieberman-Aiden et al., 2009; Ryba et al., 2010). In addition, the A/B compartments are not static but can change during cell differentiation in a lineage-specific manner (Dixon et al., 2015; Fortin and Hansen, 2015). At the sub-megabase scale, mammalian chromatin can be further divided into domains (also known as topologically associated domains [TADs]) based on their local interaction pattern (Dixon et al., 2012; Nora et al., 2012; Sexton et al., 2012; Rao et al., 2014). TADs are defined as

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

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Genome size

Mappable regiona

Rice

373 245 519

305 604 466

rep1 158 211 733 rep2 146 346 947

Foxtail millet

401 300 876

336 502 123

rep1 181 295 773 rep2 193 695 049

130 488 224 1 731 776

9 866 926

Sorghum 683 645 045

410 544 840

rep1 668 541 873

290 179 072 2 237 149

24 166 350 73 234 362 178 342 191

rep2 392 096 855

177 279 238 1 132 880

15 222 137 44 220 561 109 504 442

Tomato

807 224 664

653 250 118

rep1 492 795 112

371 328 273 281 635

4 313 529

53 518 074 311 374 677

rep2 506 235 378

354 497 014 266 594

3 872 830

58 297 891 303 420 399

Maize

2 106 338 117 945 290 415 b

Human

Raw reads

Mapped pairs

Dangling Self-circle end

106 162 746 109 414

PCR duplicate

Valid contact

1 102 015

10 061 490 91 955 592

103 545 233 76 994

282 111

2 917 427

123 358 343 1 376 577

12 933 012 15 086 136 81 608 399

84 982 785

24 588 711 82 902 340

rep1 1 576 115 322 574 949 969 342 234

43 440 977 76 731 244 389 604 472

rep2 1 435 483,497 553 536 682 781 709

77 907 782 86 187 759 386 128 425

3 095 677 412 2 623 721 134 rep1 655 975 359

331 587 795 –

–

–

209 517 391

rep2 1 343 443 711 743 132 905 –

–

–

466 535 676

Table 1. Hi-C Data Summary. a

Calculated according to John et al. (2011). Human embryonic stem cell Hi-C data from Dixon et al. (2015).

b

regions with high intracontact frequency, and it has been suggested that they could spatially confine the interaction between promoter and distal regulatory elements (Jin et al., 2013; Rao et al., 2014). TAD boundaries are enriched for architectural proteins such as cohesin and CCCTC-binding factor (CTCF), and specific epigenetic marks (Dixon et al., 2012; Sexton et al., 2012; Rao et al., 2014; Tang et al., 2015). The metazoan genomes are folded into TADs, and they are considered to be the structural units of the chromosome that are conserved between tissues and even across species (Dixon et al., 2012; Vietri Rudan et al., 2015). Our understanding of the 3D architecture of the plant nucleus is mainly derived from a series of Hi-C analyses in the model plant Arabidopsis. Its chromosome arms can be partitioned into loose or compact structural domains with epigenetic marks that bear some similarities to those of the mammalian A/B compartments (Grob et al., 2014; Liu and Weigel, 2015). Unlike the mammalian genome, however, the strongest interactions in the Arabidopsis genome were found between pericentromeric heterochromatin within and between chromosomes, as well as between small heterochromatin islands (Feng et al., 2014; Grob et al., 2014). Its chromosomes are not segmented into TADs, which are the predominant feature of the mammalian genome. Mutant analysis showed that DNA methylation and histone modification appear to play important roles in shaping its nucleus architecture at both the genome-wide and local levels (Feng et al., 2014). Plant genomes vary considerably in size, chromosome number and length, gene number, and repeat content. The gene-dense and heterochromatin-poor Arabidopsis might not be a suitable model for other plant species. To further our understanding of the 3D organization of plant genomes, we have used in situ HiC to examine the nucleus architectures of five crop species (maize, tomato, sorghum, foxtail millet, and rice) with genome sizes ranging from 2.4 to 0.4 Gb. We observed these plant genomes can be partitioned into mammalian-like A/B compart-

ments with distinct epigenetic features. At higher resolution, the chromosomes of these plants can be further segmented into local compartments, which are consistent with local chromatin state. Different from that of Arabidopsis, these plant genomes contain TAD-like domains that overlap with local compartments, similar to the compartment domains recently found in Drosophila (Rowley et al., 2017). We also found extensive chromatin loops in large-genome plants such as maize and tomato. Unlike the CTCF loops in mammals, these loops are associated with local compartments and are formed between gene islands. Our result suggests that at all levels the local compartments are important structural units in plant genome 3D organization.

RESULTS Distinct Chromosome Contact Patterns in Plant Genomes Instead of extracting the nuclei from the whole plant, which contains multiple cell types at different stages, we used nuclei isolated from the leaf mesophyll protoplasts, which are relatively uniform and consist mainly of differentiated cells. Hence, we were able to obtain tissue-specific Hi-C maps with genome coverage comparable with those of the human embryonic stem cells (Dixon et al., 2015) (Table 1). Our rice protoplast Hi-C data showed high consistency with the recently published whole-seedling Hi-C results (Liu et al., 2017), confirming that the chromatin contact pattern was not affected during the cell isolation process (Supplemental Table 2). In addition, we have also profiled their DNA methylation, histone modifications, and accessible chromatin using the same tissue samples (Supplemental Tables 6–8), which enable us to study the association between epigenetic features and the 3D chromatin architecture. Hi-C data from all five species showed intense signals on the main diagonal, which indicates frequent interactions between adjacent loci (Figure 1). Strong intra- and interchromosome interactions were also observed among euchromatin arms,

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3D Chromatin Architecture of Plant Genomes A

Figure 1. Hi-C Contact Maps.

B

Genome-wide contact matrix of rice (A), foxtail millet (B), sorghum (C), tomato (D), and maize (E). The color intensity represents the frequency of contact between two 1-Mb loci. Intrachromosomal interaction was observed between euchromatin arms in all genomes. Besides the euchromatin interaction, the maize heterochromatin centers also interact with each other. The interaction signals on the two diagonal lines suggest that the maize chromosome arms could interact with each other.

C

ghum (730 Mb), foxtail millet (470 Mb), and rice (375 Mb). In the small Arabidopsis genome (135 Mb), the strongest interaction was observed among the pericentromeric heterochromatin (Feng et al., 2014). On the other hand, the strongest inter- and intrachromosomal contacts in the five genomes we examined are found between their euchromatin arms. This is consistent with previous findings that rice and sorghum adopt typical ‘‘non-Rabl’’ configuration, while that of maize is in between ‘‘non-Rabl’’ and the typical ‘‘Rabl’’ plant such as barley (Dong and Jiang, 1998). In addition, we found widespread scaffolding errors in the maize, tomato, sorghum, and foxtail millet genomes (Supplemental Figures 1–5), suggesting that additional effort is required to refine these crop genomes that were assembled using short-read technology.

D

E

Global A/B Compartments

consistent with the chromosome territory concept that each chromosome occupies limited exclusive nuclear subspace (Tiang et al., 2012). Besides the main diagonal, the Hi-C map of the large maize genome (2.4 Gb) also has intense interaction signal on the anti-diagonal lines, which could be interpreted as enriched cis interactions between the two chromosome arms (Figure 1E). The arms of different maize chromosomes also display high interaction frequency, indicated by the X-shaped trans-interaction signal. Another unique feature of the maize Hi-C map is the intense trans interaction between the centromere regions in different chromosomes. These two features were also observed in the large barley genome (5 Gb), which has recently been reassembled using Hi-C data (Mascher et al., 2017). However, these phenomena are absent in the four mediumsized genomes that we analyzed including tomato (950 Mb), sor-

The mammalian chromosomes could be partitioned into multiple A/B compartments by eigenvector analysis of its genome-wide interaction matrix (Lieberman-Aiden et al., 2009). However, the same approach would generally partition the plant chromosomes to two A compartments at the tips and one B compartment in the center, where interactions within each type are enriched and interactions between each type are depleted (Figure 2A). The A compartments in chromosome arm are associated with higher gene density, active epigenetic marks, and high transcription activity, while the center B compartment has higher transposable element (TE) density and repressive epigenetic marks (Figure 2B). The compartment pattern is consistent with the overall distribution pattern of euchromatin and heterochromatin in the plant chromosomes. For example, 75% of the tomato genome is pericentromeric heterochromatin (B compartment), and two euchromatin arms (A compartment) are located at the distal ends (Sato et al., 2012). However, the genome-wide contact matrix eigenvector analysis is unable to resolve the euchromatin islands in the

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3D Chromatin Architecture of Plant Genomes B

Figure 2. Segregation of Tomato Genome into Global A/B Compartments Using Genome-wide Eigenvector. (A) Pearson correlation matrix illustrates the correlation between the intra- and interchromosomal interaction profiles. The 12 tomato chromosomes (indicated by the yellow boxes) could be segregated into global A/B compartments. The two chromosome arms are grouped as A compartment, while the pericentromeric heterochromatins are grouped as B compartment. (B) Genomic and epigenetic features of the tomato A/B compartments. A compartments are associated with active chromatin marks as well as polycomb mark H3K27me3, while B compartments display heterochromatin features such as low gene density and high DNA methylation level. The cis ratio indicates the precentage of intrachromosome interaction of a given region. A compartments have high interchromosome interaction frequency.

pericentromeric heterochromatin and the TE islands in the euchromatin arms.

Local A/B Compartments Next, we attempted to perform the eigenvector analysis using the individual chromosome’s interaction matrix; a similar approach has been used to partition the Arabidopsis (Grob et al., 2014; Bi et al., 2017) and rice (Liu et al., 2017) chromosomes. Although this chromosome-wide eigenvector is able to identify some euchromatin islands in tomato (Figure 3A), it is not effective in the heterochromatin-rich maize genome (Figure 4A). We then applied constrained clustering to the chromosome interaction matrix in order to segment the chromosomes into blocks with similar contact probability, and calculated the eigenvector separately for each block, which enabled us to infer their local A/B compartments showing typical euchromatin and heterochromatin features (Figures 3 and 4; Supplemental Figures 6 and 7). It should be noted that the chromosome-wide method, which have been successfully applied to the smaller plant genomes such as Arabidopsis and rice to identify global compartments, was scarcely able to identify the local A/B compartments in the species we studied (Supplemental Table 5). To demonstrate that the chromosome block-based eigenvector approach can acutely partition the euchromatin and heterochromatin, we compared the results derived from the genome-wide and the local block-based approaches. Regions classified as local A compartments are euchromatin regions with higher

gene density and active epigenetic marks, while those grouped as local B compartments are heterochromatins with higher TE density and repressive marks (Figures 3 and 4D; Supplemental Figures 8–12). This suggests that the local chromatin contact probability derived from the Hi-C experiment can be used to define chromatin’s euchromatin and heterochromatin status. Intriguingly, we also noticed that the regions classified at the same time as global A compartment and local B compartment have the high average H3K27me3 level, and these regions could be further grouped to either TE-rich or H3K27me3-rich types, suggesting that the polycomb-group protein could be involved in local chromatin organization (Supplemental Figure 13).

Wide Spread of Non-canonical TADs in Plant Genomes The most prominent feature in the mammalian genome are the TADs. These domains are not pervasive in Arabidopsis, where only a few associated with H3K27me3 have been identified (Feng et al., 2014). However, we found widespread TAD-like domains in the five plant species we analyzed (Figure 5). These domains showed enriched cis interactions within domains, and mammalian TAD-like genetic and epigenetic feature changes at the border, which are enriched for active genes, open chromatins (DNase I hypersensitive sites [DHSs]), and active histone marks, and are depleted of TEs and DNA methylation (Figure 5C and Supplemental Figures 14–18). We also found that these plant chromatin domains are associated with different epigenetic signatures. Using unsupervised

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3D Chromatin Architecture of Plant Genomes Tomato chromosome 1

A 0M

100M

Genome Wide Eigene Vector (500K bin) Chromosome Wide Eigen Vector (40K bin) Chromosome Block Eigen Vector (40K bin) Block1

Block2

Block3

B

Block4

Block5

C −0.3

0.4

0.031 −0.031 0.03 CEG −0.03 0.076 BEG −0.076 0.0042 DHS 0 0.029 K4me3 0 0.14 K27me3 0 0.028 K27ac 0 0.33 Gene 0 2.16 mRNA 0 0.94 CG 0 0.93 TE 0 GEG

−0.2

GEG CEG BEG DHS K4me3 K27me3 K27ac Gene mRNA CG TE 6M600k

31M920k

0.6

0.036 −0.036 0.032 −0.032 0.12 −0.12 0.057 0 0.24 0 0.32 0 0.43 0 1 0 23 0 0.85 0 0.39 0 84M200k

98M440k

Figure 3. Segregation of Tomato Chromosome into Local A/B Compartments Using Chromosome Block Eigenvector. (A) Comparison of the genome-wide, chromosome-wide, and block eigenvector in tomato chromosome 1. (B) After partitioning the tomato chromosome 1 into five blocks by constrained clustering of the interaction matrix, block-based eigenvector (BEG) analysis is able to resolve the small euchromatin and heterochromatin islands that the genome-wide (GEG) or chromosome-wide eigenvector (CEG) failed to detect. The top blue-red Pearson correlation matrix is a zoom-in view of block 2, which is rich in heterochromatin. Its euchromatin islands can now be reassigned as A compartment by the block-based method (BEG). (C) Pearson correlation matrix of block 5, which is rich in euchromatin and has heterochromatin islands that are now correctly assigned as B compartment. The genomic and epigenetic feature tracks are shown below the Pearson correlation matrix.

clustering, we have identified four major domain types that are associated with DNA methylation (repressive domain), open chromatin (active domain), and H3K27me3 histone mark (polycomb domain), as well as an intermediate type that lacks features (Figure 5A). These plant domains bear a striking resemblance to those in Drosophila, which can also be clustered into these four categories (Sexton et al., 2012). We also repeated the analysis using only the top 10 percentiles of each domain type, and the genetic and epigenetic feature enrichment results are consistent with our and the previous Drosophila domain definitions (Supplemental Figures 14–18B).

Chromatin Domains in Plants Are Compartment Domains Despite displaying some mammalian TAD-like features, the domains we found are similar to those of Drosophila with asym-

metric features around the domain border (Figure 5D and Supplemental Figures 14–18). The repressive chromatin domains are depleted of active genetic and epigenetic features even at domain borders, the enriched active marks are outside of the domain, while the polycomb ones show sharp H3K27me3 level change at the domain border. These suggest that like the Drosophila domains, active transcription, chromatin, and epigenome change could play an important role in chromatin domain border formation in plants. We also found that plant chromatin domains overlap with local compartments (Figure 5E and Supplemental Table 13). For example, 55.11% (2998/5440) of the tomato domain borders overlap with local compartment borders (p = 9.89E 73, Fisher’s exact test). Recent findings in Drosophila showed that besides CTCF binding, A/B compartments defined by

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A

Maize chromosome 1 0M

300M

Genome Wide Eigene Vector (500K bin) Chromosome Wide Eigen Vector (40K bin)

Chromosome Block Eigen Vector (40K bin) Block1

Block2

Block3

Block4

Block6

Block7

C

B −0.2

GEG

Block5

0.3

−0.3

0.02

GEG

−0.02 0.015 CEG −0.015 0.088 BEG −0.088 0.056 DHS 0 K4me3 0.084 0

0.4

0.02

−0.02 0.02 −0.02 0.076 BEG −0.076 0.018 DHS 0 K4me3 0.058 0 K27me3 0.17 0 CEG

K27me3 0.32 0 K27ac 0.3 0 0.48 Gene 0 mRNA 61 0 1 CG 0 1 TE 0

K27ac Gene mRNA CG TE 8M

0.12 0 0.37 0 19 0 1 0 1 0

20M

155M200k

164M600k

D TE density in local compartment

0.80

0.2

Gene density in local compartment

Genome-wide A Block A compartment Genome-wide A Block B compartment

0.05

Genome-wide B Block A compartment

0.60

Genome-wide B Block B compartment

−40K

border

border

40K

−40K

border

border

40K

Figure 4. Segregation of Maize Chromosome into Local A/B Compartments. (A) Comparison of the genome-wide, chromosome-wide, and block eigenvector in maize chromosome 1. After the 300-Mb maize chromosome 1 is partitioned into seven blocks by constrained clustering of the interaction matrix, block-based eigenvector analysis is able to resolve the small euchromatin and heterochromatin islands. (B) Part of the Pearson correlation matrix of the euchromatin-rich block 1. (C) Part of the Pearson correlation matrix of the heterochromatin-rich block 5. (D) Gene and TE density in four types of genome regions associated with different global and local compartment status. For example, the green line indicates the region classified as B compartment genome-wide and A compartment locally.

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3D Chromatin Architecture of Plant Genomes B

A

Domain DI 0.075 0 K4me3 0.37 0 0.77 K27me3 0 0.58 K27ac 0 34 mRNA 0 0.97 CG 0 0.88 CHG 0 0.19 CHH 0 1 TE 0 DHS

DHS

H3K27me3

CG

C rice

italica

sorghum

5M

tomato

6M

7M

8M

9M

maize active domain polycomb domain intermediate domain repressive domain without domain

E

D TE density

Gene density

CG methylation

Domains overlap with compartments

CHH methylation 100%

0.85

0.6 −100K

100K

DHS

0K

100K

−100K

100K

60% −100K

H3K27me3

0K

100K

H3K27ac

0.15

0.12

0.020

H3K4me3

0K

40%

0.20

0K

80%

0.05

0.2

0.65

0.07

0.5 0.1 −100K

both borders single border no overlap

20%

−100K

0K

100K

−100K

0K

100K

0K

100K

e r ic

lica

ize −100K

ita

100K

tom ato so rgh um

0K

ma

−100K

0

0

0

0

0%

Figure 5. Plant Domains and Their Epigenetic Features. (A) Hierarchical clustering of plant domains using their DHS, CG methylation, and H3K27me3 features. Red, purple, brown, and green colors represent active, polycomb, intermediate, and repressive domains. (B) An example of domains in tomato genome and its epigenetic features as well as correlation with A/B compartments. The interaction frequency of a region in tomato chromosome is shown on top. Domains and their types are indicated by colored lines below, followed by direction of interaction (DI) and (legend continued on next page)

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Molecular Plant chromatin state are responsible for domain structure, and these Drosophila domains are referred to as compartment domains (Rowley et al., 2017). As plants lack a CTCF homolog and the domain we found closely associated with the local A/B compartment, we propose that these plant chromatin domains are also compartment domains.

Lack of Chromatin Domain Synteny between Plant Species When we compared chromatin domains between different plant species, we observed that large plant genomes with high TE content such as maize, sorghum, and tomato have more repressive domains, which are repeat rich, gene poor, and depleted of DHS, and lack active histone marks (Figure 5D). The smaller rice genome contains few retrotransposons, and most of its repeats are class II TE, such as the miniature inverted-repeat TEs located near gene regions, and its genome contains less repressive domains (Figure 5A and Supplemental Figure 18). The foxtail millet genome contains the highest percentage of polycomb domains, especially for chromosome 8, where 73.14% (128/175) of the domains are annotated as the polycomb type. We found that 28.7% (11.1 Mb out of 35.1 Mb) of chromosome 8 and 51.2% (1298/2535) of its genes are associated with H3K27me3. Interestingly, its syntenic sorghum chromosome 5 also exhibits a high H3K27me3 level, while this is not present in the maize genome, suggesting that these polycomb regions could have been lost during evolution (Supplemental Figure 19). One unique feature of the mammalian TADs is that they are highly conserved between different cell types and tissues, and even across species (Dixon et al., 2012). Whether TADs are conserved in other kingdoms remains largely unknown. To test this, we first examined whether genes under the same domain in one plant species could still be located in one domain in another species. To our surprise, no conservation could be observed (Supplemental Tables 10 and 11). For example, none of the sorghum domains has a syntenic counterpart in maize. Only 8.23% (154/1871) of the foxtail millet domains have a syntenic domain in sorghum (12.28% in random, p = 0.9997). No synteny was observed even when the threshold was progressively lowered (Supplemental Tables 10 and 11), and similar results were obtained from the sorghum and maize comparison. Taken together, our findings suggest that the plant domains are not conserved across species. The conservation of mammalian TAD is compatible with conserved CTCF binding (Vietri Rudan et al., 2015); this again implies that without CTCF other factors could mediate domain formation in plant.

Chromatin Loops between Gene Islands in the Large Plant Genomes We detected 5616 and 1650 long-range chromatin interactions (loops) in the large maize and tomato genomes, respectively, while they were almost absent from the smaller genomes

3D Chromatin Architecture of Plant Genomes (Figure 6 and Supplemental Table 3). Genetic and epigenetic feature analysis of the regions involved in loop formation showed that they are enriched for genes and associated with active epigenetic marks such as open chromatin (DHS) and active histone marks, while repressive marks such as CG methylation are depleted (Figure 6F and Supplemental Figure 20). The loops in the mammalian genomes are enriched at the TAD boundary and are referred to as TAD corner loops or peaks, while the maize loops are located outside the domains and correlate well with the local A compartments (Figure 6B and 6G). These results suggest that these plant chromatin loops could result from the interaction between the actively transcribed gene islands (local A compartment) separated by condensed heterochromatin (local B compartment). The mammalian gene’s distal regulatory elements are often hundreds of kilobases upstream, and could be easily visualized as loops in the Hi-C contact map. In our maize dataset, less than 20% of the loops are formed between the gene region and nongene region, and few tomato loops are located within a domain. The distal no-gene region showed high chromatin accessibility (DHS) and H3K27ac signal, suggesting that there might be a distal regulatory element in plant genomes (Figure 6F and Supplemental Figure 21; Supplemental Table 12). It should be noted that one key requirement for loop calling is that the loop position should have higher contact frequency than the surrounding positions, which cannot be a gap in the reference genome or an unmappable region. However, all the large TErich plant genomes have unmappable regions distributed throughout their chromosome, which limits the loop-calling resolution to 10-kb bin size.

DISCUSSION Hi-C analysis of the mammalian genomes revealed their 3D organization at three levels: compartments, domains, and loops. On the megabase scale, the human chromosome is partitioned into active and inactive A/B compartments (Lieberman-Aiden et al., 2009). Each compartment preferentially clusters with regions of the same type, in line with the chromosome territory concept ^ t et al., 2007). We found that plant genome could (Lancto be partitioned into global A/B compartments with A compartments containing mainly euchromatin and located at the euchromatin arm, while B compartments contain mainly heterochromatin and are located at centromeric and pericentromeric heterochromatin regions (Figure 7). These plant chromosomes could be further partitioned into blocks with similar interaction patterns and by using their local Hi-C contact matrix, and chromatin within each block could be partitioned into local A/B compartments. This is most obvious in the largest maize genome, whose chromosomes could be segmented into multiple blocks with local A/B compartments. These block-based local A compartment correlated well with the gene region while the local B compartment comprised mainly TEs (Figures 4D and 7).

different genetic and epigenetic features. The interaction Pearson correlation matrix that indicates local A/B compartment features is shown on the bottom (blue to red indicates correlation from negative to positive). (C) Pie chart showing the genome coverage of each domain type, as well as the genome region without domain. (D) Enrichment of specific epigenetic features of each domain type in the region 100 kb up- and downstream of the domain border. The four domain types are represented by the color lines. (E) Overlap of domain and local A/B compartment borders in the five plant genomes.

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3D Chromatin Architecture of Plant Genomes A

B

Tomato loops

compartment

Maize loops

compartment

C

D

-100K

border

border

100K

E

-100K

border

border

100K

-100K

border

maize domain

tomato domain

border

100K

human domain

F

G H3K27ac

0.4

DHS

0.0

0.00

0.04

Loops overlap with compartments

−100K

0K

100K

−100K

0K

100K

−100K

0K

100K

Gene

−100K

0K

100K

0.4

0.8

TE

−100K

0K

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All maize loops (5616)

−100K

0K

100K

tomato Loop loci separeted by B compartment Within compartment Other

0.2

0.0

maize

−100K

0K

Gene−gene loops (4206, 74.89%)

100K

−100K

0K

100K

Gene−other loops (1118, 19.98%)

Figure 6. Chromatin Loops between Gene Islands. (A) Example of tomato chromatin loops between gene islands and their association with local A/B compartment. The upper triangle shows the interaction frequency and the bottom triangle shows the Pearson correlation matrix. Local A/B compartment is shown in the middle track (red: A compartment; blue: B compartment). One chromatin loop formed between two A compartments is indicated by the green circle. (B) Example of chromatin loops in the maize genome. (C–E) Average interaction matrix of tomato repressive domain (C), maize repressive domain (D), and human domain (E). Mammalian TADs have corner loops inside the domain while plant loops are often observed outside the repressive domain. (F) Epigenetic feature of the genome regions involved in the maize loops. (G) Overlap of the maize and tomato loops with local A/B compartments.

On the sub-megabase scale, mammalian chromosomes are organized as a string of TADs by preferential interactions within them. Cohesin and orientated CTCF are vital for their formation (Zuin et al., 2014; Tang et al., 2015; Nora et al., 2017). Similar

domains have also been found in other eukaryotes and even bacteria (Le et al., 2013). Besides CTCF, transcription and chromatin status played an important role in the domain formation in Drosophila (Sexton et al., 2012; Bonev and Cavalli,

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Figure 7. Schematic Representation of Plant 3D Chromatin Organization. Plant genomes can be partitioned into global A/B compartments, signified by the blue-red checkerboard interaction pattern. The two chromosome arms are grouped as A compartment while the pericentromeric heterochromatin is grouped as B compartment. At a higher resolution, chromosomes can be further segmented into blocks with similar interaction pattern. Within each block, the local Hi-C interaction matrix could be used to define local compartments, which are consistent with their chromatin states. These local compartments overlap with domains, which are similar to the compartment domains recently found in Drosophila. Extensive chromatin loops are formed between active gene loci and are associated with the active A compartments.

2016; Ulianov et al., 2016). The plant domains we found display some TAD-like features, such as enrichment of active genes, DHSs, and epigenetic marks (Figure 5), and are not conserved between plant species. Mammalian TADs are contiguous regions of enriched contact frequency in a Hi-C map insulated from neighboring regions and have sharp contact frequency at the boundaries often bound by CTCF. Plants do not have CTCF, and their genes often do not require distal regulatory elements. Hence, plants are unlikely to have mammalian-type TAD. Our finding and the recent Drosophila Hi-C analysis strongly suggest that plant domains could be established by a noncohesin-CTCF loop extrusion mechanism (Rowley et al., 2017). They are more likely to be defined by the transcription and epigenomic features like the Drosophila compartment domains. We also showed that the plant loops are often formed between active gene islands separated by repressive domains or B compartments. However, further experiments would be required to examine the molecular mechanism underlying their establishment and their biological functions. The loops in the mammalian genomes are enriched at the TAD boundary and hence are referred to as TAD corner loops. However, the maize loops are often enriched outside the domain and are associated with local A compartments (Figure 7). One possible explanation for this is that the large TE-rich plant chromosomes are less prone to decondense in the interphase nuclei, and they remain in their anaphase configuration with centromeres on one side, chromatin arms folded together, and telomeres on the opposite side. Hence, their actively transcribed gene regions need to loop out from the condensed chromatin, which is supported by fluorescence in situ localization analysis of multiple transgenes inserted to distal loci (Shaw, 2002; Abranches et al., 2008). It should be noted that the resolution of Hi-C is not sufficient to detect the interaction between the regulatory elements and plant genes. With a resolution on the upper kilobase scale, the number of

loops detected in our Hi-C dataset is likely to be an underestimate. Site-specific chromosome conformation capture techniques such as ChIA-PET (Tang et al., 2015) would be more suitable for capturing the small plant chromatin loops at the lower-kilobase resolution. In summary, we present a diverse picture of plant 3D nucleus architectures using five model crop species, enabling future studies of their functions during plant growth and development. We also provide valuable epigenomic and chromatin data, and our Hi-C contact matrix could be used to correct their genome assembly errors.

METHODS Illumina Sequencing Library Preparation To isolate mesophyll protoplasts from maize, sorghum, foxtail millet, and rice, we cut their leaves into 1- to 2-mm strips, which were digested for 3–5 h in enzyme solution containing 2% (w/v) cellulose R-10, 0.1% (w/v) macerozyme R-10, 0.1% (w/v) BSA, 1 mM MgCl2, 1 mM CaCl2, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5), and 0.6 M sorbitol. For tomato leaf, 0.1% (w/v) pectolyase Y-23 was also added to the digestion solution. After digestion, the protoplast was filtered and washed twice with W5 buffer containing 154 mM NaCl, 125 mM CaCl2, 2.5 mM KCl, and 2 mM MES (pH 5.5). For Hi-C, formaldehyde was added to 1% (v/v) to fix the protoplasts at room temperate for 10 min, and the fixation was terminated by adding glycine to 0.125 M. Triton X-100 was added to 1% (v/v) to lyse the protoplast membrane and organelles. The Hi-C libraries were constructed according to the published in situ Hi-C protocol (Rao et al., 2014). The isolated nuclei were first lysed with 0.5% SDS at 65 C for 5 min. The SDS was quenched by adding 10 volumes of 1% Triton X-100. The DNA inside the nuclei was digested by MboI for at least 2 h at 37 C. The MobI enzyme was inactivated at 65 C for 20 min. The ends were filled in by Klenow in the presence of biotin-14dATP at 37 C for 45 min to 1.5 h. The biotin fill-in reaction was diluted with five volumes of 13 T4 ligation buffer, and 5 ml of T4 DNA ligase was added. The ligation reaction was performed at room temperature for 4 h. The nuclei pellet was collected by centrifuge and formaldehyde

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3D Chromatin Architecture of Plant Genomes reversed crosslinking was performed at 65 C overnight. The DNA was then ethanol precipitated and sonicated to 300–500 bp using Covaris M220 before binding to the Streptavidin Dynabeads. The Illumina sequencing libraries were constructed on the bead as previously described (Rao et al., 2014). Fleshly prepared nucleus without formaldehyde fixation was directly used for ATAC sequencing (ATAC-seq), and DNase sequencing (DNaseseq) was performed according to the published protocol (Buenrostro et al., 2013; He et al., 2014). The Tn5 transposome was prepared as previously described (Sagasser and Picelli, 2014). After tagmentation, the nuclei were washed twice in 13 PBS containing 1% (v/v) Triton X-100 to remove the plant organelle debris, and the DNA was purified using a Qiagen spin column. After PCR amplification using index primers matching the Illumina Nextra adapter, the ATAC-seq libraries containing DNA insert between 50 and 150 bp were gel purified and sequenced. Bisulfite-sequencing (BS-seq), chromatin immunoprecipitation-sequencing (ChIP-seq), and RNA-sequencing (RNA-seq) libraries were constructed as previously described (Zhong et al., 2013).

Reference Genome and Annotation In this study, we have used the maize ensembl 36, tomato SL3.1, sorghum v3.1, foxtail millet v2.2, and rice v7.0 as reference genomes. We also performed de novo transposable element identification using Repeatmodeler (Smit and Hubley, 2010), and used the Repbase (Jurka et al., 2005) and p-mite database (Chen et al., 2014) to annotate them. The synteny gene pairs for foxtail millet, sorghum, and maize were obtained from CoGe (Lyons et al., 2008).

merged the domain data from all five species and used hierarchal clustering to define the four domain types.

Domain Synteny Analysis We tested whether the syntenic genes within one domain were still located in a domain in other species, and only domains containing more than five genes were kept. The control (random) dataset was generated by shifting the domains 1 Mb along the chromosome, and Fisher’s exact tests were performed with parameter alternative = ‘‘greater.’’

Transcriptome, Methylome, Histone Modification, and DHS Analysis RNA-seq reads were first mapped to ribosomal RNA sequences using bowtie2 (Langmead and Salzberg, 2012). The resulting filtered reads were mapped to the genome by TopHat2 (Langmead et al., 2009), and raw read counts for each gene were further normalized to FPKM (fragments per kilobase per million reads). BS-seq data were processed using BSMAP (Xi and Li, 2009). We mapped the ChIP-seq, DNase-seq, and ATAC-seq data using bowtie2 (Langmead and Salzberg, 2012) with default parameters. Reads were first trimmed to 50 bp and only mapped reads with MAPQ R 10 were used for further analysis. PCR duplicates were removed by samtools (Li et al., 2009). For histone modification data, we used MACS2 (Zhang et al., 2008) for peak calling with parameters ‘‘–nomodel –broad –broad-cutoff 0.01.’’ For DNase-seq and ATAC-seq data, MACS2 was used with parameters ‘‘–nomodel –extsize 70 -q 0.01.’’

SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

Hi-C Data Analysis

FUNDING

We used hiclib (Imakaev et al., 2012) to process the Hi-C data. We independently aligned the paired-end 150-bp reads using bowtie2 with iteratively strategy. Only high-quality alignments (MAPQ > 10) were kept for further analysis. Read pairs within the same restriction enzyme fragments and PCR duplicates were removed.

This work is supported by the Chinese National Key Research and Development Program of China 2016YFD0101003, NSFC 91435108, and Taishan Pandeng Program, as well as Hong Kong UGC GRF 14119814 and 14104515 and Area of Excellence Scheme AoE/M-403/16.

AUTHOR CONTRIBUTIONS For compartment analysis, we first used the whole-genome eigenvector for distance normalized Pearson correlation matrix (bin = 500 kb). To assign A/B compartment we manually reversed the sign of the eigenvector if it negatively correlated with H3K4me3 pattern. Next, we repeated this using a 40-kb bin for each chromosome to obtain higher-resolution chromosome-wide eigenvector. Finally, we used distance normalized interaction matrix (bin = 40 kb) as input employing CONISS (Grimm, 1987), a constrained hierarchical clustering method, to separate each chromosome into blocks. We determined the number of clusters comparing the dispersion, when the dispersion decline is smaller than the maximum from the broken stick model (Bennett, 1996) or 10% of the maximum dispersion decline (for the larger maize matrix, 5% is used). For each chromosome block, we selected the one with maximum correlation with the active mark H3K4me3 (or anti-correlate with DNA CG methylation) from the largest three eigenvectors of the log2 ratios of the distance normalized interaction matrix. We replaced it with the chromosome-wide ones when the correlation was too low (block correlation <0.3, or chromosome correlation minus block correlation >0.1 and block correlation <0.5). We used the Direction Index (DI) method to annotate domains as previously described (Dixon et al., 2012). We performed t-statistics as modified DI (Phillips-Cremins et al., 2013) with a dynamic window size (Wang et al., 2017) at 20-kb bin size. Loop calling was performed using Juicer HICCUPS (Durand et al., 2016) with 10-kb bin size and maximum genomic distance of 2 Mb. To group the domains, we clustered them based on their CG methylation level, chromatin accessibility (ATAC-seq peak coverage), and H3K27me3 (H3K27me3 peak coverage) levels. Due to the uneven distribution of domain types in each plant species, we

S.Z. designed the research; X.T., P.L., N.Z., P.-Y.C., B.D., and P.L. performed the experiments; P.D. and S.Z. analyzed the data; D.G., S.Z., and P.D. wrote the paper.

ACKNOWLEDGMENTS Sequencing data have been deposited in the NCBI Sequence Read Archive under the accession number PRJNA391551. No conflict of interest declared. Received: September 22, 2017 Revised: November 6, 2017 Accepted: November 13, 2017 Published: November 21, 2017

REFERENCES Abranches, R., Santos, A.P., Wegel, E., Williams, S., Castilho, A., Christou, P., Shaw, P., and Stoger, E. (2008). Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase. Plant J. 24:713–723. Bennett, K.D. (1996). Determination of the number of zones in a biostratigraphical sequence. New Phytol. 132:155–170. Bi, X., Cheng, Y., Hu, B., Ma, X., Wu, R., and Wang, J. (2017). Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery. Genome Res. 27:1162–1173. Bonev, B., and Cavalli, G. (2016). Organization and function of the 3D genome. Nat. Rev. Genet. 17:661–678. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Transposition of native chromatin for fast and sensitive

Molecular Plant 10, 1497–1509, December 2017 ª The Author 2017. 1507

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epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10:1213–1218.

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9:357–359.

Chen, J., Hu, Q., Zhang, Y., Lu, C., and Kuang, H. (2014). P-MITE: a database for plant miniature inverted-repeat transposable elements. Nucleic Acids Res. 42:1176–1181.

Langmead, B., Trapnell, C., Pop, M., Salzberg, S.L., Kelley, R., Salzberg, S.L., Sammeth, M., Huang, J., Kirkness, E., Denisov, G., et al. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10:R25.

Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. (2002). Capturing chromosome conformation. Science 295:1306–1311. Dixon, J.R., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M., Liu, J.S., and Ren, B. (2012). Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485:376–380. Dixon, J.R., Jung, I., Selvaraj, S., Shen, Y., Antosiewicz-Bourget, J.E., Lee, A.Y., Ye, Z., Kim, A., Rajagopal, N., Xie, W., et al. (2015). Chromatin architecture reorganization during stem cell differentiation. Nature 518:331–336. Dong, F., and Jiang, J. (1998). Non-Rabl patterns of centromere and telomere distribution in the interphase nuclei of plant cells. Chromosom. Res. 6:551–558. Durand, N.C., Shamim, M.S., Machol, I., Rao, S.S.P., Huntley, M.H., Lander, E.S., and Aiden, E.L. (2016). Juicer provides a one-click system for analyzing loop-resolution Hi-C experiments. Cell Syst. 3:95–98. Feng, S., Cokus, S., Schubert, V., Zhai, J., Pellegrini, M., and Jacobsen, S. (2014). Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis. Mol. Cell 55:694–707. Fortin, J.-P., and Hansen, K.D. (2015). Reconstructing A/B compartments as revealed by Hi-C using long-range correlations in epigenetic data. Genome Biol. 16:180. Fullwood, M.J., Liu, M.H., Pan, Y.F., Liu, J., Xu, H., Bin Mohamed, Y., Orlov, Y.L., Velkov, S., Ho, A., Mei, P.H., et al. (2009). An oestrogen-receptor-a-bound human chromatin interactome. Nature 461:58–64. Grimm, E.C. (1987). CONISS: a FORTRAN 77 program for stratigraphically constrained cluster analysis by the method of incremental sum of squares. Comput. Geosci. 13:13–35.

Le, T.B., Imakaev, M.V., Mirny, L.A., and Laub, M.T. (2013). High-resolution mapping of the spatial organization of a bacterial chromosome. Science 342:731–734. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., and Durbin, R. (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. Lieberman-Aiden, E., van Berkum, N.L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., Lajoie, B.R., Sabo, P.J., Dorschner, M.O., et al. (2009). Comprehensive mapping of longrange interactions reveals folding principles of the human genome. Science 326:289–293. Liu, C., and Weigel, D. (2015). Chromatin in 3D: progress and prospects for plants. Genome Biol. 16:170. Liu, C., Cheng, Y.-J., Wang, J.-W., and Weigel, D. (2017). Prominent topologically associated domains differentiate global chromatin packing in rice from Arabidopsis. Nat. Plants 3:742–748. Lyons, E., Pedersen, B., Kane, J., and Freeling, M. (2008). The value of nonmodel genomes and an example using synmap within CoGe to dissect the hexaploidy that predates the rosids. Trop. Plant Biol. 1:181–190. Mascher, M., Gundlach, H., Himmelbach, A., Beier, S., Twardziok, S.O., Wicker, T., Radchuk, V., Dockter, C., Hedley, P.E., Russell, J., et al. (2017). A chromosome conformation capture ordered sequence of the barley genome. Nature 544:1–43. Nora, E.P., Lajoie, B.R., Schulz, E.G., Giorgetti, L., Okamoto, I., Servant, N., Piolot, T., van Berkum, N.L., Meisig, J., Sedat, J., et al. (2012). Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485:381–385.

Grob, S., Schmid, M., and Grossniklaus, U. (2014). Hi-C analysis in Arabidopsis Identifies the KNOT, a structure with similarities to the flamenco locus of Drosophila. Mol. Cell 55:678–693.

Nora, E.P., Goloborodko, A., Valton, A.-L., Gibcus, J.H., Uebersohn, A., Abdennur, N., Dekker, J., Mirny, L.A., and Bruneau, B.G. (2017). Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169:930–944.e22.

He, H.H., Meyer, C.A., Hu, S.S., Chen, M.W., Zang, C., Liu, Y., Rao, P.K., Fei, T., Xu, H., Long, H., et al. (2014). Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification. Nat. Methods 11:73–78.

Phillips-Cremins, J.E., Sauria, M.E., Sanyal, A., Gerasimova, T.I., Lajoie, B.R., Bell, J.S., Ong, C.T., Hookway, T.A., Guo, C., Sun, Y., et al. (2013). Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153:1281–1295.

Imakaev, M., Fudenberg, G., McCord, R.P., Naumova, N., Goloborodko, A., Lajoie, B.R., Dekker, J., and Mirny, L.A. (2012). Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9:999–1003.

Rao, S.S., Huntley, M.H., Durand, N.C., Stamenova, E.K., Bochkov, I.D., Robinson, J.T., Sanborn, A.L., Machol, I., Omer, A.D., Lander, E.S., et al. (2014). A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680.

Jin, F., Li, Y., Dixon, J.R., Selvaraj, S., Ye, Z., Lee, A.Y., Yen, C.-A., Schmitt, A.D., Espinoza, C.A., and Ren, B. (2013). A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503:290–294. John, S., Sabo, P.J., Thurman, R.E., Sung, M.-H., Biddie, S.C., Johnson, T.A., Hager, G.L., and Stamatoyannopoulos, J.A. (2011). Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat. Genet. 43:264–268.

Rowley, M.J., Nichols, M.H., Lyu, X., Ando-kuri, M., Rivera, I.S.M., Hermetz, K., Wang, P., Ruan, Y., and Corces, V.G. (2017). Evolutionarily conserved principles predict 3D article evolutionarily conserved principles predict 3D chromatin organization. Mol. Cell 67:837–852.

Jurka, J., Kapitonov, V.V., Pavlicek, A., Klonowski, P., Kohany, O., and Walichiewicz, J. (2005). Repbase Update, a database of eukaryotic repetitive elements. Cytogenet. Genome Res. 110:462–467.

Ryba, T., Hiratani, I., Lu, J., Itoh, M., Kulik, M., Zhang, J., Schulz, T.C., Robins, A.J., Dalton, S., and Gilbert, D.M. (2010). Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res. 20:761–770.

^ t, C., Cheutin, T., Cremer, M., Cavalli, G., and Cremer, T. (2007). Lancto Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat. Rev. Genet. 8:104–115.

Sagasser, S., and Picelli, S. (2014). Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24:2033–2040.

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3D Chromatin Architecture of Plant Genomes Sato, S., Tabata, S., Hirakawa, H., Asamizu, E., Shirasawa, K., Isobe, S., Kaneko, T., Nakamura, Y., Shibata, D., Aoki, K., et al. (2012). The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485:635–641. Sexton, T., and Cavalli, G. (2015). The role of chromosome domains in shaping the functional genome. Cell 160:1049–1059. Sexton, T., Yaffe, E., Kenigsberg, E., Bantignies, F., Leblanc, B., Hoichman, M., Parrinello, H., Tanay, A., and Cavalli, G. (2012). Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148:458–472. Shaw, P. (2002). The architecture of interphase chromosomes and nucleolar transcription sites in plants. J. Struct. Biol. 140:31–38. Smit, A.F.A., and Hubley, R. (2010). RepeatModeler Open-1.0. Repeat Masker Website. http://www.repeatmasker.org/RepeatModeler/. Tang, Z., Luo, O.J., Li, X., Zheng, M., Zhu, J.J., Szalaj, P., Trzaskoma, P., Magalska, A., Wlodarczyk, J., Ruszczycki, B., et al. (2015). CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription. Cell 163:1611–1627. Tiang, C., He, Y., and Pawlowski, W.P. (2012). Chromosome organization and dynamics during interphase, mitosis, and meiosis in plants. Plant Physiol. 158:26–34. Ulianov, S.V., Khrameeva, E.E., Gavrilov, A.A., Flyamer, I.M., Kos, P., Mikhaleva, E.A., Penin, A.A., Logacheva, M.D., Imakaev, M.V., Chertovich, A., et al. (2016). Active chromatin and transcription play

a key role in chromosome partitioning into topologically associating domains. Genome Res. 26:70–84. Vietri Rudan, M., Barrington, C., Henderson, S., Ernst, C., Odom, D.T., Tanay, A., and Hadjur, S. (2015). Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep. 10:1297–1309. Wang, X.-T., Cui, W., and Peng, C. (2017). HiTAD: detecting the structural and functional hierarchies of topologically associating domains from chromatin interactions. Nucleic Acids Res. 45:e163. Xi, Y., and Li, W. (2009). BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinformatics 10:232. Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nussbaum, C., Myers, R.M., Brown, M., Li, W., et al. (2008). Model-based analysis of ChIP-seq (MACS). Genome Biol. 9:R137. Zhong, S., Fei, Z., Chen, Y.R., Zheng, Y., Huang, M., Vrebalov, J., McQuinn, R., Gapper, N., Liu, B., Xiang, J., et al. (2013). Singlebase resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat. Biotechnol. 31:154–159. Zuin, J., Dixon, J.R., van der Reijden, M.I., Ye, Z., Kolovos, P., Brouwer, R.W., van de Corput, M.P., van de Werken, H.J., Knoch, T.A., van IJcken, W.F., et al. (2014). Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. Proc. Natl. Acad. Sci. USA 111:996–1001.

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