Principles of 3D Chromosome Folding and Evolutionary Genome Reshuffling in Mammals (PDF)

Article Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals Graphical abstract Authors Lucı́a Álvarez-González,...

Article Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals Graphical abstract Authors Lucı́a Álvarez-González, Cristina Arias-Sardá, Laia Montes-Espuña,..., Paul D. Waters, Marta Farré, Aurora Ruiz-Herrera Correspondence [email protected] In brief A major challenge in genome research is to determine why some species have stable genomes whereas others have undergone extensive rearrangement. Álvarez-González et al. describe fundamental principles of 3D chromosome folding in mammals and show that lineage-specific evolutionary genomic reshuffling can influence EBR EBR patterns of higher-order chromatin organization. Highlights d Vertebrates show different patterns of genome-wide chromosomal interactions d Long marsupial chromosomes arrange their centromeres in clusters d Marsupials and afrotherians show contrasting patterns of genome reshuffling d Evolutionary lineage-specific inversions have distinctive 3D structural features Álvarez-González et al., 2022, Cell Reports 41, 111839 December 20, 2022 ª 2022 The Author(s). https://doi.org/10.1016/j.celrep.2022.111839 ll  ll OPEN ACCESS Article Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals Lucı́a Álvarez-González,1,2 Cristina Arias-Sardá,3 Laia Montes-Espuña,1,2 Laia Marı́n-Gual,1,2 Covadonga Vara,1,2 Nicholas C. Lister,4 Yasmina Cuartero,5 Francisca Garcia,6 Janine Deakin,7 Marilyn B. Renfree,8 Terence J. Robinson,9 Marc A. Martı́-Renom,5,10,11,12 Paul D. Waters,4 Marta Farré,3 and Aurora Ruiz-Herrera1,2,13,* 1Departament de Biologia Cel$lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 2Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 3School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK 4School of Biotechnology and Biomolecular Sciences, Faculty of Science, UNSW Sydney, Sydney, NSW 2052, Australia 5CNAG-CRG, Centre for Genomic Regulation, The Barcelona Institute of Science and Technology, Baldiri Reixac 4, 08028 Barcelona, Spain 6Servei de Cultius Cel.lulars-SCAC, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 7Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia 8School of Biosciences, The University of Melbourne, Victoria, VIC 3010, Australia 9Evolutionary Genomics Group, Department of Botany and Zoology, Faculty of Science, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South Africa 10Centre for Genomic Regulation, The Barcelona Institute for Science and Technology, Carrer del Doctor Aiguader 88, 08003 Barcelona, Spain 11ICREA, Pg. Lluı́s Companys 23, 08010 Barcelona, Spain 12Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain 13Lead contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2022.111839 SUMMARY Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ances- tral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling. INTRODUCTION changes in gene expression caused by genome reshuffling may have a selective advantage through the development of The evolution of chromatin conformation is fundamental for un- new adaptive characters specific to different mammalian line- derstanding the mechanism(s) responsible for the origin and ages.7–10 These data suggest that sequence composition is plasticity of genome architecture. Distant loci within the genome not alone in determining evolutionary plasticity but rather that can interact during the cell cycle to affect function in somatic and the occurrence and subsequent fixation of genome rearrange- germ cells.1–4 Exploring the similarities and differences of these ments are multifaceted, involving (1) repetitive elements genomic interactions across diverse phylogroups is central to (i.e., making DNA more susceptible to chromosomal reorganiza- developing an appreciation of both the dynamics of genome tion)11–13; (2) functional constrains (i.e., genes related to species- function and, ultimately, the effects on speciation. specific phenotypes)14; and (3) genome folding dynamics and its Ancestral genome reconstructions have shown that structural effect on gene regulation/function.3,15,16 This has led to sugges- changes disrupting synteny preferentially cluster in regions that tions that the permissiveness of some genomic regions to are prone to break and reorganize—these are referred to as undergo genomic rearrangements, especially in germ cells, is evolutionary breakpoint regions (EBRs).5,6 It is also known that influenced by chromatin 3D conformation.4,15–18 Cell Reports 41, 111839, December 20, 2022 ª 2022 The Author(s). 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).  ll OPEN ACCESS Article (legend on next page) 2 Cell Reports 41, 111839, December 20, 2022  ll Article OPEN ACCESS Genomes are compartmentalized into different hierarchical tational analysis that included genome-wide chromosome layers including chromosomal territories (CTs), compartments conformation capture (in situ Hi-C) in representative therian (A and B), topologically associated domains (TADs), and looping (eutherian and marsupials) species coupled with comparative interactions.1,19,20 How these different levels of chromatin orga- genomics, transcriptome sequencing (RNA sequencing [RNA- nization are conserved across species underpins their potential seq]), and chromatin immunoprecipitation sequencing (ChIP- evolutionary genome plasticity. For example, chromatin 3D seq) of CTCF and H3K4me3 was performed. We (1) defined organization was recently studied in representative species of patterns of 3D genome folding in interphase nuclei across spe- chordates, plants, and fungi.21 Two types of 3D genome organi- cies; (2) reconstructed ancestral marsupial and Afrotherian zation were found at a chromosomal scale: telomeres and genomes by analyzing whole-genome sequences of 10 species centromeres either (1) clustered across chromosomes adopting that represent the major therian phylogroups; (3) detected line- a Rabl-like configuration or (2) were oriented in a polarized state age-specific chromosome rearrangements; (4) identified the maintaining individual CTs within the cell. However, little is dynamics and structural properties of therian EBRs by applying known about the evolutionary dynamics of 3D genome organiza- integrative computational analyses; and (5) provide evidence of tion, especially at the root of the three major lineages of chromosome folding changes due to inversions and fusions/fis- mammals, Prototheria (monotremes), Metatheria (marsupials), sions. Our results represent a comprehensive catalog of the close Eutheria, and their presumptive ancestor. interplay between chromatin higher-order organization dynamics Monotremes (represented by the duck-billed platypus and the and therian genome evolution. echidnas) are positioned phylogenetically between birds/reptiles and therians and diverged from therian mammals (placentals RESULTS and marsupials) z217 million years ago (mya).22 Monotremes represent a pivotal group with a mix of reptilian and mammalian Patterns of genome-wide chromosomal interactions morphological, physiological, and karyological features.23 Mar- across vertebrates supials, on the other hand, shared a common ancestor with We first explored the characteristic features of 3D genome eutherian mammals z190 mya.22 Karyotyping studies observed evolution across vertebrates performing in situ Hi-C experiments a bimodal distribution of diploid chromosome numbers across in primary fibroblast cell lines from the Afrotheria (African the marsupial phylogeny, with many species having either a elephant with 2n = 56 and aardvark with 2n = 20) and Marsupialia 2n = 14 or a 2n = 22 karyotype.24 (Tasmanian devil with 2n = 14 and tammar wallaby with 2n = 16) In eutherians, Afrotheria represents one of the most ancient (Figure 1A). After filtering the raw Hi-C interactions, an average clades that includes six mammalian orders all with an Afro- of 100 million valid interactions were obtained per species Arabian origin. Afrotherian species exhibit extreme morpholog- (Table S1). The comparison between biological replicates ical diversity and niche preference, which is thought to result resulted in reproducible Hi-C maps (Pearson correlation, from the long period of isolation when Africa was an island conti- R2 > 0.8, p < 0.01; Figure S1). African elephant, aardvark, and nent 105–125 mya.22 Genome organization within Afrotheria is tammar wallaby Hi-C data were mapped against their respective diverse, with diploid numbers ranging from 2n = 56 in the African reference genome available at the DNA Zoo consortium.26,27 (Loxodonta africana) and Asian (Elephas maximus) elephants to These data were combined with publicly available Hi-C data 2n = 20 in the aardvark (Orycteropus afer).25 Given the position for human (2n = 46),1 mouse (2n = 40),3 platypus (2n = 52),28 of Afrotheria and marsupials near the root of therian mammals and chicken (2n = 70)29 (see STAR Methods and Table S1). and their diverse diploid numbers (reflecting extensive genome Comparison of Hi-C matrices revealed different patterns of reshuffling), the analysis of 3D genome architecture provides a chromosomal interactions as reflected by the log2 inter-/intra- unique opportunity to further understand the mechanisms chromosomal interaction ratios in different species (Figure 1B). underpinning mammalian chromosomal evolution. Chicken (a bird representative) presented the lowest log2 inter- To explore the principles of 3D genome folding dynamics and action ratios per chromosome (5% genome size) in all Divergent centromere clustering in marsupials species (Figure 1C). In contrast, marsupials displayed high inter- Close inspection of genome-wide Hi-C contact maps revealed actions between all chromosomes, including the X chromosome striking patterns of chromosomal interactions at centromeres. (Figure 1B), also supporting the view that chromosome compart- Both Hi-C interaction maps (Figure 2A), aggregate peak analysis mentalization is distinct from that observed in other vertebrates. (Figure 2B), and Z score interaction ratio plots between heterol- ogous chromosomes (Figure 2C) detected higher centromeric Diploid numbers determine patterns in chromosome inter-chromosomal interactions (>4 3 10"8 chromosome- folding length-normalized mean interaction value) in marsupials than in Distance-dependent interaction frequencies represented as therian mammals. This contrasted with interactions detected in probability versus distance (P(s)) curves were compared be- the telomeric regions, which were high for all species except tween species to analyze patterns of chromosome folding (Fig- for the aardvark (>3 3 10"8 chromosome length normalized ure 1D). As previously described,19 we detected a general mean interaction value; Figure 2D). The pattern of centromeric decrease in interaction frequencies as genomic distances interaction in marsupials was further demonstrated by the immu- increased. Visual inspection allowed the delineation of distinct nodetection of the centromeric constitutive heterochromatin groups based on the decay of contact probability interactions: (i.e., H3K9me3 signal) in fibroblast cell cultures (Figures 2E– (1) chicken, (2) platypus, (3) human, African elephant, and 2F). Both tammar wallaby and Tasmanian devil fibroblasts mouse, and (4) both marsupial species plus aardvark (Figure 1D). showed shorter relative distances between the centromeres At short genomic distances (90% of which were conserved in on the species, between 1,882 and 2,105 TADs of 1 Mbp average HSBs) irrespective of whether this was for comparisons within length were detected (TAD strength score >6). TAD boundary the same phylogenetic group or between different phylogenetic scores were equivalent between species, and metaborder plots groups (Figures 3E and S3; Table S4). showed clear insulator patterns for all taxa (Figure 3B). All detected TAD boundaries were associated, and enriched, with Evolutionary history of marsupial and afrotherian H3K4me3 and CTCF (permutation test based on 10,000 permu- chromosomes tations, normalized Z score > 0.01, p < 0.05; Figure 3C). To assess whether patterns of chromosome folding correlated with genomic reshuffling during evolution, we reconstructed Conservation of the higher-order chromatin ancestral karyotypes and cataloged the evolutionary history of organization within mammals chromosome rearrangements in afrotherians and marsupials. To test if the higher-order structural organization of mammalian Two ancestral karyotypes were reconstructed for Marsupialia genome architecture was conserved in somatic cells, we estab- using five representative marsupial genomes (Tasmanian devil, lished homologous syntenic blocks (HSBs) between single tammar wallaby, wombat, red kangaroo, and opossum) and representatives of the boreoeutherians (mouse), afrotherians the genomes of five outgroup taxa (sloth, African elephant, (African elephant), and marsupials (Tasmanian devil) using the human, platypus, and chicken) (Figures 4A and S4; Table S2; human genome as reference (see STAR Methods; Table S2). see STAR Methods). The marsupial ancestral karyotype (MAK) At a 300 Kb resolution, a total of 346 HSBs were detected comprised 10 reconstructed ancestral chromosome fragments between human and mouse (ranging from 7.7 to 78.3 Mbp in (RACFs), representing seven ancestral chromosomes that (B) Metaplots for all TAD boundaries detected in human, mouse, African elephant, aardvark, Tasmanian devil, and tammar wallaby. (C) Mean number of CTCF peaks and H3K4me3 relative to TAD boundaries positions in African elephant, aardvark, and Tasmanian devil. The data include two biological replicates per species. (D) Chromosomal synteny between human (HSA) chromosome 12 in mouse (MMU), African elephant (LAF), and Tasmanian devil (SHA). Collinear homologous regions are depicted in light blue and inverted regions in pink. (E) Zoom in of a structural conserved HSB (two-sided, t test, p < 0.01; Tables S3 and S4) human chromosome 12 when compared with mouse (a chromosomal region of chromosome 5), African elephant (a chromosomal region of chromosome 25), and Tasmanian devil (a chromosomal region of chromosome 1). Compartment conservation is represented as a compartment heatmap and first eigenvector distribution. TAD conservation is represented as a contacts heatmap and insulator score distribution. Cell Reports 41, 111839, December 20, 2022 7  ll OPEN ACCESS Article Marsupialia Afrotheria A African elephant Mouse Human Asian elephant Kangaroo Cattle Boar Golden mole Wallaby Manatee Tasmanian devil Tenrec Sloth Wombat Aardvark Hyrax Opossum Platypus Chicken 0 mya 2/3 14/3 78 41 5/22 24 31 50 mya 2/2 MAUK 10 n=7 MAK AFAK 100 mya n=7 n=24 150 mya 200 mya B 1A 1B 2A 2B 2C 3 4 5 6 X MAK 250 mya 300 mya MAUK 1 2A 2B 3 4 5 6 X SHA 1 2 3 4 5 6 X C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 23 X AFAK LAF 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 X Figure 4. Marsupial and afrotherian ancestral karyotypes and genome reshuffling (A) Phylogenetic tree of the species included in the analysis. Marsupial species are shown in red, while afrotherians are in blue. The number of intra-chromosomal rearrangements in each branch is shown in pale blue, while inter-chromosomal (fissions/fusions) rearrangements are in dark green. (B and C) Marsupial ancestral karyotypes (B) and Afrotherian ancestral karyotype (C). Ribbons connecting chromosomes indicate orthologs between ancestors and reference genome chromosomes, with a twist indicating an inversion. MAK, marsupial ancestral karyotype; MAUK, Australian marsupial ancestral karyotype; SHA, Tasmanian devil; LAF, African elephant. 8 Cell Reports 41, 111839, December 20, 2022  ll Article OPEN ACCESS Figure 5. Structural plasticity of lineage-specific chromosomal reorganizations (A) Chromosome 1 and chromosome 2 region-specific 500 Kbp heatmaps, first eigenvector, insulator score, and ancestral specific reorganizations for Tasmanian devil and African elephant. (B) Contact probability P(s) as a function of genomic distance and its derivative for each class of reorganized HSBs in Tasmanian devil and African elephant. (legend continued on next page) Cell Reports 41, 111839, December 20, 2022 9  ll OPEN ACCESS Article covered 95.87% of the Tasmanian devil reference genome (20.83% of the genome is collinear and 6.94% inverted); or (3) (Figures 4B and S4). Our MAK reconstruction was consistent by a fusion of small AFAK chromosomes (24.25% is collinear with previous work,24 recovering 85.16% of the syntenic associ- and 3.54% inverted) (Table S5). These results highlight the ations between MAK and human. Similarly, the ancestor of different genome reshuffling patterns characterizing marsupials Australian marsupials (MAUK) contained eight RACFs, repre- and afrotherian species. senting seven ancestral chromosomes that ranged in size from Differences in lineage-specific chromosomal reorganizations 71.6 to 698 Mb in size. Our results illustrate that the MAK and were also observed in terms of gene content. Approximately MAUK karyotypes were conserved, with three chromosomes 58% of the Tasmanian devil genes were located in collinear maintained as complete syntenic blocks. Ten inversions, two fis- regions and 31.79% in inverted regions. Genes in inverted sions, and two fusions separated MAUK from MAK, whereas 24 regions were related to signaling and response to stimuli, as inversions occurred between MAUK and the Tasmanian devil, well as major histocompatibility complex (MHC) and MHC class while 77 inversions, three fissions, and two fusions separated II protein complexes (Figure S5), whereas genes within collinear MAUK from the tammar wallaby. In summary, these data show regions were enriched in Gene Ontology (GO) terms related to that the marsupial lineage is predominantly characterized by metabolic and developmental processes as well as anatomical intra-chromosomal rearrangements. (structural) development, among others (Figure S5). By contrast, We also reconstructed the ancestral karyotype for Afrotheria only 10.87% of African elephant genes were located in collinear (AFAK) using the African elephant as the reference genome regions; 27.25% were in chromosomes originating from fission and six other afrotherian genomes (Asian elephant, aardvark, followed by fusion of AFAK chromosomes and 21.83% in chro- cape golden mole, rock hyrax, West Indian manatee, and lesser mosomes resulting from fissions only. Genes within collinear hedgehog tenrec), along with those from three outgroup species regions were enriched in GO terms related to nucleic acid trans- (cattle, pig, and human) (Figures 4C and S4; Table S2; see STAR port. Genes within fission-fusion rearrangements were related to Methods). The AFAK consisted of 24 ancestral chromosomes in pheromone responses, whereas genes within fissioned regions 25 RACFs, covering 95.44% of the African elephant genome were enriched in keratin filament and anatomical structure GO (Figures 4C and S4). We recovered 71.43% of the syntenic asso- terms (Figure S5). ciations between human and African elephant that were previ- ously identified by cross-species chromosome painting,35 as Functional and structural characterization of EBRs well as all 23 fissions of human chromosomes, indicating that The reconstruction of ancestral genomes allowed us to identify our reconstruction was accurate. A total of 31 inversions, 22 EBRs in both the Tasmanian devil and the African elephant fissions, and five fusions separate the African elephant from (Figure S6). A total of 34 EBRs were identified within the the AFAK. In the aardvark lineage, 41 inversions, three fissions, Tasmanian devil genome, which correlated with chromosome and 14 fusions occurred after the split from AFAK, showing size (R2 = 0.93, p < 0.001; Figure S6A) and TAD boundaries that afrotherian genomes are predominantly characterized by (permutation test based on 10,000 permutations, normalized inter-chromosomal rearrangements (Figure S4). Z score > 0.01, p < 0.05; Figure S6C). Although EBRs were To analyze the lineage-specific chromosomal reorganiza- embedded in gene-dense regions (Figure S6E), they were nega- tions in the afrotherian and marsupial species, we identified tively associated with gene position (permutation test based on genomic regions that were either collinear or had undergone 10,000 permutations, normalized Z score < "0.01, p < 0.05). reorganization from the most recent ancestor (Figure S4). The same trend was observed in the 45 EBRs identified in the Crucially, for this comparison, all Tasmanian devil chromo- African elephant (Figures S6D–S6F), mirroring previous studies. somes originated from individual MAUK chromosomes, with In both species, no GO term was associated with genes no inter-chromosomal rearrangements, and, consequently, surrounding EBRs. African elephant EBRs, however, were more than 65% of the Tasmanian devil genome is fully enriched in transposable elements (p = 0.04, Z score = 1.82, collinear with that of the MAUK, with 33.58% disrupted due 1,000 permutations). Interestingly, almost all African elephant to inversions. The remaining 1.42% corresponded to un- EBRs contained afroSINEs and L1-LA elements, which are placed sequences (Table S5). specific to afrotherians. In sharp contrast, the African elephant has retained only three chromosomes (LAF10, LAF23, and LAFX) that are fully collinear Lineage-specific chromosomal reorganizations affect with the AFAK; these span 12.60% of the genome. Thus, the genomic evolutionary plasticity majority of African elephant chromosomes originated by either After ancestral genomes were reconstructed and lineage-spe- (1) fissions of larger AFAK chromosomes followed by fusions cific chromosome reorganizations identified, we analyzed the (27.37% of African elephant genome); (2) by fissions only structural and functional features of both conserved (collinear) (C) Distribution of inter-/intra-chromosomal interaction ratio according to HSB length (in Mbp) for each class of reorganization in Tasmanian devil and African elephant: collinear, inverted, fissioned and collinear, fissioned and inverted, fused and collinear, fused and inverted, and fissioned and fused. Boxplots are presented as median values (center line); mean values (dot) ± SD. Asterisks represent statistically significant interaction ratio between HSBs (two-sided t test, ***p < 0.001). (D) Boxplots depicting interactions between the same types of HSBs (cis) and different types of HSBs (trans) in Tasmanian devil and African elephant. Boxplots are presented as median values (center line); mean values (dot) ± SD. Asterisks represent statistically significant interactions between HSBs (two-sided t test, ***p < 0.001). 10 Cell Reports 41, 111839, December 20, 2022  ll Article OPEN ACCESS and rearranged genomic regions (inverted, fused, and fissioned) substantial rearrangement. Here, we describe the fundamental in the Tasmanian devil and the African elephant (Figure 5A). principles of 3D chromosome folding in mammals and show We detected structural differences between collinear and that lineage-specific evolutionary genomic reshuffling can influ- rearranged regions in both species. As a general trend, line- ence patterns of higher-order chromatin organization. age-specific inverted regions displayed higher distance-depen- Our data provide evidence for the existence of different dent contact probabilities than did collinear regions (Figure 5B). chromosome folding patterns within mammals. In the eutherian Differences in contact probability were higher in the Tasmanian mammals (Boreoeutheria and Afrotheria) analyzed herein, the devil, where blocks presented a mean contact probability value chromosomes (irrespective of diploid numbers) were organized of 2.79 and inverted blocks a mean of 6.85 (2.53 fold increase). into CTs during interphase, mirroring observations in other spe- This was translated into different estimated slopes (as a proxy of cies.21 Low inter-/intra-chromosomal interaction ratios observed DNA loop size). Inverted regions had a slope maximum of 6.5 in human, mouse, African elephant, and aardvark were indicative Mbp, whereas the maximum value in conserved blocks of relatively highly compacted chromatin as reflected by high was 7.5 Mbp, indicating that inverted blocks bear slightly distance-dependent interactions and high CTCF density. shorter loops and, therefore, an increase in contact probability In contrast, marsupials (tammar wallaby and Tasmanian devil) (Figure 5B). showed a distinctive Rabl-like chromosomal distribution with In the African elephant, mean contact probability values were centromeres and heterochromatic regions clustering near the 14.2 for collinear regions and 20.38 for the inverted blocks (1.53 center of the nucleus. This chromosome distribution was fold increase). The more complex chromosomal rearrangements accompanied by (1) high inter-/intra-chromosomal interaction between collinear and inverted HSBs (fissions followed by inver- ratios and (2) low distance-dependent interactions. These obser- sions and fusions followed by inversions) showed intermediate vations, together with the detection of low CTCF genomic den- values. Finally, HSBs that were maintained as collinear, but sity in the opossum (an American marsupial representative),30 either fissioned or fused in the African elephant, presented indicate that long marsupial chromosomes (average size 400 equally low contact probability values (mean contacts 3.82, a Mbp) form a ‘‘loose’’ distribution that extends across the 3.53 fold reduction compared with collinear regions). However, nucleus, with presumably lower numbers of longer loops these contacts were never as low as those observed in the Tas- anchored by their centromeres that are orientated toward the manian devil conserved blocks. A similar tendency was detected center of the cell. This probably has implications for the position in the slopes estimated for each type of reorganization, with a of chromosomes within the nucleus. In this context, our genomic maximum at 2 Mbp for inverted blocks, 4.5 Mbp for collinear approach supports initial cytogenetics studies reporting a radial blocks, and 8 Mbp for translocated (fused/fissioned) blocks configuration of marsupial chromosome inside nuclei.36 (Figure 5B). Previous studies have shown the presence of a Rabl-like Differences between types of rearrangements were also noted configuration in yeast (centromeres forming one large focus in when analyzing inter-/intra-chromosomal interaction ratios the vicinity of the spindle pole body), wheat (centromeres clus- (Figures 5C and 5D). In both species, lineage-specific inverted ters at one pole), mosquitos, and sea urchins.21,37 To these spe- blocks presented the lowest inter-/intra-chromosomal interac- cies, we can add marsupials—an ancient mammalian clade with tions, being lower in African elephant (mean 0.05; two-sided t genome plasticity and chromosomal diploid number variation test, p < 0.01) than in Tasmanian devil (mean 0.6; two-sided t that is distinctive from eutherian mammals.38–40 We can only test, p < 0.01). African elephant blocks that were fissioned and speculate on the mechanisms responsible of the Rabl-like subsequently fused displayed the highest inter-/intra-chromo- configuration in marsupials as the function of this chromosome somal interaction (mean 0.35; two-sided, t test, p < 0.01) (Fig- pattern still remains a mystery since its initial first cytological ure 5C). Collectively, these observations point to the presence description.41 Although speculative, it is possible that the Rabl of distinctive genomic architectural features in lineage-specific configuration is a relic from anaphase-segregating chromo- chromosomal reorganizations. This was especially relevant for somes.42 In this case, larger/longer chromosomes would inversions, which presented more intra-chromosomal interac- establish a polarized pattern more readily during interphase tions than surrounding collinear regions (Figure 6). with size and the heterochromatin distribution favoring a Rabl Remarkably, structural reshuffling did not result in gross configuration.43 Further research is needed to fully test these changes in TADs and compartments (Figure 3D). This was in hypotheses. line with the fact that EBRs were positively associated with Importantly, our analysis of the ancestral genomic reconstruc- TAD boundaries (multiple permutation test based on 10,000 per- tions showed contrasting patterns of genome reshuffling in mutations, normalized Z score > 0.01, p < 0.05) in both species marsupials and afrotherians. The Tasmanian devil has retained (Figure S6). Likewise, EBRs were devoid of genes (multiple per- the same chromosome number as has been proposed for both mutation test based on 10,000 permutations, normalized Z the MAK and the MAUK (n = 7). Moreover, it has few species- score > 0.01, p < 0.05; Figure S6), suggesting that gross genome specific inversions. In contrast, the African elephant karyotype reshuffling is less likely to disrupt gene regulation. has been extensively reorganized (inter- and intra-chromoso- mally) compared with the AFAK (n = 24), which, in turn, largely DISCUSSION resembles the eutherian ancestral karyotypic configuration (n = 2338). Based on the homologies shared with the human A major challenge in genome research is to determine why some genome (Figure S3), marsupials have undergone widespread species have stable genomes whereas others have undergone genomic reshuffling after their split from the therian common Cell Reports 41, 111839, December 20, 2022 11  ll OPEN ACCESS Article Figure 6. Model on the influence of evolutionary genome reshuffling in chromatin folding Representation of genomic architectural features detected in lineage-specific chromosomal reorganizations in the Tasmanian devil and African elephant. Left panel: phylogenetic relationship among the afrotherian and marsupial species compared, including the haploid number of chromosomes (n) for each species. Central panel: Tasmanian devil and African elephant chromosome ideograms color coded accordingly to their corresponding MAUK or AFAK chromosomes. Right panel: representation of DNA loops in inverted and fused HSBs in both species, chromosome 6 in the Tasmanian devil, and chromosome 13 in the African elephant. Note that inverted regions have shorter DNA loops than non-inverted regions (either collinear, fissioned, and fusioned). MAK, marsupial ancestral karyotype; MAUK, Australian marsupial ancestral karyotype; AFAK, Afrotheria ancestral karyotype; HSB, homologous synteny blocks; EBRs, evolutionary breakpoint regions. ancestor. This was later stabilized as reflected by the conserved mar wallaby when compared with the MAUK. As centromeres are karyotypes described within the group.24 In sharp contrast, in anchored toward the center of the nucleus in marsupials, the afrotherian species (African elephant and aardvark), and prob- resulting chromosomal distribution could impose structural con- ably those boreoeutherians with highly diverse karyotypes strains that favor intra-chromosomal reorganization rather than (such as n = 3 in the female Indian muntjac and n = 51 in the inter-chromosomal rearrangements. This is supported by data red viscacha rat38), genome evolution involved more complex that suggest centromeres can act as strong topological barriers reorganization (inversions, fusions, and fissions). that prevent contact between the two chromosome arms.37,44 Based on the structural plasticity detected in lineage-specific Should this hold, we can infer that the chromosomal reduction chromosomal reorganizations, we hypothesize that chromo- in marsupials, after their split from the therian ancestor, had some folding patterns have influence on chromosomal evolution already resulted in an ancestral configuration of centromere as- and genome reshuffling in different ways in different phylogroups. sociations that is now reflected in all marsupials. In marsupials, the development of centromeric associations Likewise, the pattern of chromosome reshuffling observed in probably predated the MAUK radiation and most likely influenced modern Afrotheria (that show extensive intra- and inter-chromo- the high occurrence of inversions in the Tasmanian devil and tam- somal reorganizations from the AFAK) can be related to 12 Cell Reports 41, 111839, December 20, 2022  ll Article OPEN ACCESS chromosomes being organized into highly compacted CTs, our results suggest that 3D chromosome folding influences where heterologous chromosomes are more frequently in the patterns of genome reshuffling that are transmitted to contact. This would favor inter-chromosomal, rather than intra- offspring, an observation supported by a recent survey in chromosomal, reorganization. Importantly, chromatin intermin- rodents.4 gles at the peripheral regions of CTs in mammals,45 allowing interactions between non-homologous chromosomes. This can Limitations of the study facilitate the rejoining of broken DNA ends of heterologous chro- As the use of non-model species can be challenging, future mosomes17 and can explain the excess of inter-chromosomal studies with a larger sample sizes and greater species represen- rearrangements detected. tation should be considered a priority. Moreover, understanding Equally unexpected was the observation that inversions the dynamics of chromatin conformation during development in (irrespective of clade) resulted in different DNA loop sizes and other distantly related species will be fundamental to decipher- distance-dependent interaction contact frequencies when ing the structural plasticity of vertebrate genomes. compared with collinear genomic regions (Figure 6). This was observed for inversions in the Tasmanian devil and African STAR+METHODS elephant, where chromatin was packaged differently from neigh- boring, non-reorganized regions on the same chromosome. Detailed methods are provided in the online version of this paper Inverted regions showed (1) differences in slope (an estimate of and include the following: DNA loop size), (2) high interactions at short distances, and (3) high intra-chromosomal interactions. Studies in different taxa d KEY RESOURCES TABLE (i.e., butterflies, mosquitos, pea aphids, fishes, and plants46–50) d RESOURCE AVAILABILITY have revealed clusters of differentiated loci (the so-called B Lead contact ‘‘genomic islands of divergence’’49,51,52) often involved in inver- B Materials availability sions between lineages. In light of our data, we suggest that B Data and code availability lineage-specific inversions may also act as ‘‘structural genomic d EXPERIMENTAL MODEL AND SUBJECT DETAILS islands’’ by imposing structural constrains (i.e., acting as barriers B Cell lines for genomic contacts) with surrounding regions. In contrast, at d METHOD DETAILS higher hierarchical levels (i.e., compartments and TADs), the B Primary fibroblast cell lines 3D genome structure of collinear regions has the same level of B Immunoflourescence and microscopy structural conservation between species as do the inverted B In nuclei Hi-C regions, thereby extending previous observation in the carni- B African elephant Hi-C assisted assembly vores53 to more basal mammals. B Hi-C data processing, binning and normalisation It is tempting to speculate that lineage-specific inversions B Averaged contact probability P(s) and its derivative have resulted in new structural features that are distinct B Inter-chromosome/intra-chromosome interaction ratio from other genomic regions, which have been conserved B Centromere interaction quantification over tens of millions of years. We suggest that these divergent B Centromere aggregate contacts plots structural features can result in topological (and hence ge- B First eigenvector and insulator score calculation netic) barriers that may, at least potentially, have functional B TAD calling and TAD boundaries implications. Inversions could isolate genes from surrounding B ChIP-sequencing regions as they show shorter loops (and hence reduced con- B ChIP-seq peak calling and annotation tacts). This view is consistent with recent evidence provided B African elephant genome annotation by divergent expression profiles in inverted regions within B RNA-seq analysis Cetartiodactyla.14 Such topological barriers could be trans- B Whole-genome pairwise alignments mitted through the germ line. In fact, it was recently shown B Conservation of the higher-order chromatin organiza- that chromosome fusions alter the nuclear architecture in tion between species mouse germ cells.16 This included an increased rate of heter- B Ancestral karyotype reconstructions ologous interactions that, in turn, alter chromosome axis B Detection of evolutionary breakpoint regions (EBRs) length and DNA loop size. Importantly, these disturbances in B Gene ontology enrichment analysis (GOEA) chromosome topology were associated with changes in the d QUANTIFICATION AND STATISTICAL ANALYSIS recombination landscape, resulting in detectable genomic B Multi-association and statistical analysis footprints at a population level. In conclusion, our study provided an evolutionary view of SUPPLEMENTAL INFORMATION the 3D genome folding patterns in distantly related mammals. Through an integrative computational analysis of a compre- Supplemental information can be found online at https://doi.org/10.1016/j. celrep.2022.111839. hensive Hi-C dataset and the use of comparative genomics, it was possible to infer the ancestral karyotypic structure of ACKNOWLEDGMENTS both marsupial and afrotherian genomes. This permitted the reconstruction of lineage-specific chromosome reorganization We thank the specialist and High Performance Computing systems provided that captures the deepest divergences of mammals. Crucially, by Information Services at the University of Kent. Unpublished genome Cell Reports 41, 111839, December 20, 2022 13  ll OPEN ACCESS Article assemblies for aardvark (Orycteropus afer); African elephant (Loxodonta somes have different evolutionary histories. Genome Res. 19, 770–777. africana); Asiatic elephant (Elephas maximus); Cape rock hyrax (Procavia https://doi.org/10.1101/gr.086546.108. capensis); gray short-tailed opossum (Monodelphis domestica); red kangaroo 7. Groenen, M.A.M., Archibald, A.L., Uenishi, H., Tuggle, C.K., Takeuchi, Y., (Macropus rufus/Osphranter rufus); West Indian manatee (Trichechus mana- Rothschild, M.F., Rogel-Gaillard, C., Park, C., Milan, D., Megens, H.J., tus); and wombat (Vombatus ursinus) are used with permission from the et al. (2012). Analyses of pig genomes provide insight into porcine demog- DNA Zoo Consortium (dnazoo.org). This work was supported by the Ministry raphy and evolution. Nature 491, 393–398. https://doi.org/10.1038/ of Economy, Industry and Competitiveness (CGL2017-83802-P to A.R.-H.) nature11622. and the Spanish Ministry of Science and Innovation (PID2020-112557GB-I00 8. Ullastres, A., Farré, M., Capilla, L., and Ruiz-Herrera, A. (2014). Unraveling to A.R.-H. and PID2020-115696RB-I00 to M.A.M.-R.). Research funding to the effect of genomic structural changes in the rhesus macaque - implica- P.D.W. (Australian Research Council grants DP180100931, DP210103512, tions for the adaptive role of inversions. BMC Genom. 15, 530–613. and DP220101429) and T.J.R. (South African National Research Foundation) https://doi.org/10.1186/1471-2164-15-530. are gratefully acknowledged. C.V. and L.A.-G. were supported by FPI predoc- toral fellowships from the Ministry of Economy and Competitiveness (BES- 9. Farré, M., Narayan, J., Slavov, G.T., Damas, J., Auvil, L., Li, C., Jarvis, E.D., 2015-072924 and PRE-2018-083257). L.M.-G. was supported by an FPU Burt, D.W., Griffin, D.K., and Larkin, D.M. (2016). Novel insights into chro- predoctoral fellowship from the Spanish Ministry of Science, Innovation and mosome evolution in birds, archosaurs, and reptiles. Genome Biol. Evol. 8, University (FPU18/03867). C.A.-S. was supported by a GTA fellowship from 2442–2451. https://doi.org/10.1093/gbe/evw166. the University of Kent. 10. Capilla, L., Sánchez-Guillén, R.A., Farré, M., Paytuvı́-Gallart, A., Malin- verni, R., Ventura, J., Larkin, D.M., and Ruiz-Herrera, A. (2016). AUTHOR CONTRIBUTIONS Mammalian comparative genomics reveals genetic and epigenetic features associated with genome reshuffling in rodentia. Genome Biol. A.R.-H. conceived and devised the study. L.A.-G., C.A.-S., P.D.W., M.M.-R., Evol. 8, 3703–3717. https://doi.org/10.1093/gbe/evw276. M.F., and A.R.-H. designed experiments and analysis. L.A.-G., L.M.-G., 11. Longo, M.S., Carone, D.M., NISC Comparative Sequencing Program; C.V., Y.C., F.G., and A.R.-H. performed experiments. L.A.-G., C.A.-S., Green, E.D., O’Neill, M.J., and O’Neill, R.J. (2009). Distinct retroelement L.M.-E., L.M.-G., M.F., and A.R.-H. analyzed the data. J.D., M.B.R., T.J.R., classes define evolutionary breakpoints demarcating sites of evolutionary M.A.M.-R., and A.R.-H. contributed reagents and to data collection. L.A.-G., novelty. BMC Genom. 10, 334–414. https://doi.org/10.1186/1471-2164- C.A.-S., P.D.W., M.F., and A.R.-H. wrote the initial first draft of the manuscript 10-334. with input from all authors. All authors read and approved the final version of the manuscript. 12. Farré, M., Bosch, M., López-Giráldez, F., Ponsà, M., and Ruiz-Herrera, A. (2011). Assessing the role of tandem repeats in shaping the genomic archi- DECLARATION OF INTERESTS tecture of great apes. PLoS One 6, e27239. https://doi.org/10.1371/jour- nal.pone.0027239. M.A.M.-R. serves as a consultant to Acuity Spatial Genomics, Inc., and re- 13. Robinson, T.J., Cernohorska, H., Kubickova, S., Vozdova, M., Musilova, ceives compensation for these services. P., and Ruiz-Herrera, A. (2021). Chromosomal evolution in Raphicerus antelope suggests divergent X chromosomes may drive speciation Received: June 18, 2022 through females, rather than males, contrary to Haldane’s rule. Sci. Rep. Revised: October 1, 2022 11, 3152. https://doi.org/10.1038/s41598-021-82859-0. Accepted: November 24, 2022 14. Farré, M., Kim, J., Proskuryakova, A.A., Zhang, Y., Kulemzina, A.I., Li, Q., Published: December 20, 2022 Zhou, Y., Xiong, Y., Johnson, J.L., Perelman, P.L., et al. (2019). Evolution of gene regulation in ruminants differs between evolutionary breakpoint REFERENCES regions and homologous synteny blocks. Genome Res. 29, 576–589. https://doi.org/10.1101/gr.239863.118. 1. Rao, S.S.P., Huntley, M.H., Durand, N.C., Stamenova, E.K., Bochkov, I.D., 15. Vara, C., and Ruiz-Herrera, A. (2022). Unpacking chromatin remodelling in Robinson, J.T., Sanborn, A.L., Machol, I., Omer, A.D., Lander, E.S., and Ai- germ cells: implications for development and evolution. Trends Genet. 38, den, E.L. 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Protoc. 10, 2004–2015. https://doi.org/10.1038/ 978-1-4939-9173-0_9. nprot.2015.124. Cell Reports 41, 111839, December 20, 2022 17  ll OPEN ACCESS Article STAR+METHODS KEY RESOURCES TABLE REAGENT orRESOURCE SOURCE IDENTIFIER Antibodies Anti-rabbit H3K9me3 Abcam #ab8898; RRID:AB_306848 Anti-human CREST provided by M. Fritzler N/A Anti-rabbit Cy3 Jackson Immunoresearch #115-165-003; RRID:AB_2338680 Anti-mouse FITC Jackson Immunoresearch #115-095-003; RRID:AB_2338589 Anti-rabbit CTCF Millipore #07-729; RRID:AB_441965 Anti-rabbit H3K4me3 Abcam #ab8580; RRID:AB_306649 Chemicals, peptides, and recombinant proteins AmnioMAXTM ThermoFisher Scientific #11269016 Gentamycin ThermoFisher Scientific #15710049 Pencillin-Streptomycin ThermoFisher Scientific #151140122 Amphotericin B ThermoFisher Scientific #15290018 Formaldehyde Sigma-Aldrich #F8775 KaryoMAXTM Colcemid Gibco #15212012 PhotoFlo Kodak #1464510 Trypsin 0.05% Gibco #25300062 NEB2 buffer New England Biolabs #B7002S MboI New England Biolabs #R0147M Proteinase K New England Biolabs #P8107S Phenol/Chloroform/Isoamyl Sigma-Aldrich #P2069-400ML dNTPs Roche #11969064001 Biotin-14-dATP ThermoFisher Scientific #19524-016 DNA Polymerase I, large (Klenow) Fragment New England Biolabs #M0210M T4 DNA Ligase New England Biolabs #M0202M RNAse A ThermoFisher Scientific #EN0531 Dynabeads MyOne Streptavidin T1 ThermoFisher Scientific #65001 T4 Polynucleotides Kinase New England Biolabs #M0201L T4 DNA Polymerase New England Biolabs #M0212M Klenow Fragment 3’ / 50 exo- New England Biolabs #M0203L Nuclease micrococcal from Sigma-Aldrich #N3755 Staphylococcus aureus TRIzol Invitrogen #15596026 Critical commercial assays DynabeadsTM Protein G ThermoFisher Scientific #10007D Immunoprecipitation Kit Truseq ChIP-seq library preparation kit Illumina #IP-202-1012 Deposited data Elephant Hi-C data This paper GEO: GSE206075 Aardvark Hi-C data This paper GEO: GSE206075 Tasmanian devil Hi-C data This paper GEO: GSE206075 Tammar wallaby Hi-C data This paper GEO: GSE206075 Human Hi-C data Rao et al. (2014)1 GEO: GSE63525 Mouse Hi-C data Vara et al. (2019)3 GEO: GSE132054 Platypus Hi-C data Zhou et al. (2021)28 SRA: SRR10530604 Chicken Hi-C data Fishman et al. (2019)29 GEO: GSE96037 (Continued on next page) e1 Cell Reports 41, 111839, December 20, 2022  ll Article OPEN ACCESS Continued REAGENT orRESOURCE SOURCE IDENTIFIER Elephant ChIP-seq data This paper GEO: GSE206075 Aardvark ChIP-seq data This paper GEO: GSE206075 Tasmanian devil ChIP-seq data This paper GEO: GSE206075 Human ChIP-seq data Schmidt et al. (2012)30 ArrayExpress: E-MTAB-437 Mouse ChIP-seq data Schmidt et al. (2012)30 ArrayExpress: E-MTAB-437 Opossum ChIP-seq data Schmidt et al. (2012)30 ArrayExpress: E-MTAB-437 Tasmanian devil RNA-seq data This paper GEO: GSE206075 Elephant RNA-seq data Cortez et al. (2014)54 GEO: GSE50747 Experimental models: Cell lines Elephant XX/XY fibroblast cell lines provided by T. J. Robinson N/A Aardvark XY fibroblast cell line provided by T. J. Robinson N/A Tasmanian Devil XX fibroblast cell line provided by J. Deakin N/A Tammar wallaby XY fibroblast cell line This paper N/A Software and algorithms ImageJ Schneider et al. (2012)55 https://imagej.nih.gov/ij/ Juicer/3D-DNA Dudchenko et al. (2017)26 https://github.com/aidenlab/3d-dna BBDuk (version 09/2019) Bushnell (2014)56 https://github.com/BioInfoTools/BBMap/ blob/master/sh/bbduk.sh TADbit (version 1.0) Serra et al. (2017)34 https://github.com/3DGenomes/TADbit 57 GEM3-Mapper (version 3.0) Marco-Sola et al. (2012) https://github.com/smarco/gem3-mapper HiCExplorer (version 3.6) Wolff et al. (2018)58 https://github.com/deeptools/HiCExplorer/ blob/master/docs/index.rst Cooltools Venev et al. (2021)59 https://github.com/open2c/cooltools Rstats (version 3.6.2) Schwarzer (2007)60 https://stat.ethz.ch/R-manual/R-devel/ library/stats/html/00Index.html Bedtools Quinlan and Hall (2010)61 https://github.com/arq5x/bedtools2 FAN-C (version 0.9.1) Kruse et al. (2020)62 https://github.com/vaquerizaslab/fanc FastQC (version 0.11.9) Andrews et al. (2014)63 https://github.com/s-andrews/FastQC Trimmomatic (version 0.39) Bolger et al. (2014)64 http://www.usadellab.org/cms/?page = trimmomatic BWA-MEM (version 0.7.17) Li et al. (2013)65 https://github.com/lh3/bwa MACS2 (version 2.2.7.1) Zhang et al. (2008)66 https://pypi.org/project/MACS2/ STAR (version 2.7.10a) Dobin et al. (2013)67 https://github.com/alexdobin/STAR featureCounts Liao et al. (2014)68 https://www.rdocumentation.org/ packages/Rsubread/versions/1.22.2/ topics/featureCounts countsToFPKM Alhendi (2019)69 https://github.com/AAlhendi1707/ countToFPKM UCSC Kent Utilities Kent at al. (2010)70 https://github.com/ENCODE-DCC/ kentUtils RepeatMasker (version 4.0.9) Tempel (2012)71 https://github.com/rmhubley/ RepeatMasker LASTZ Harris (2007)72 https://github.com/lastz/lastz maf2Synteny Kolmogorov et al. (2018)73 https://github.com/fenderglass/ maf2synteny syntenyPlotteR Farré et al. (2019)14 https://github.com/marta-fb/ syntenyPlotteR DESCHRAMBLER Kim et al. (2017)74 https://github.com/jkimlab/ DESCHRAMBLER TimeTree Kumar et al. (2017)75 http://www.timetree.org/ (Continued on next page) Cell Reports 41, 111839, December 20, 2022 e2  ll OPEN ACCESS Article Continued REAGENT orRESOURCE SOURCE IDENTIFIER 76 FigTree Rambaut (2009) https://github.com/rambaut/figtree/ releases/tag/v1.4.4 GO::TermFinder Boyle et al. (2004)77 https://metacpan.org/pod/GO::TermFinder RegioneR (version 1.26) Gel el at. (2016)78 https://bioconductor.org/packages/ release/bioc/html/regioneR.html GenoMatriXeR Álvarez-González et al. (2022)4 https://github.com/RMalinverni/ GenoMatriXeR RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to, and will be fulfilled by, the Lead Contact: Aurora Ruiz-Herrera ([email protected]). Materials availability All materials developed in this study will be available from the lead contact upon request. Data and code availability d Raw and processed