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Deep conservation of cis-regulatory elements and chromatin organization in echinoderms uncover ancestral regulatory features of animal genomes

Nature Ecology & Evolution (2026)Cite this article

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Abstract

Despite the growing abundance of sequenced animal genomes, we only have detailed knowledge of regulatory organization for a handful of lineages, particularly flies and vertebrates. These two taxa show contrasting trends in the molecular mechanisms of 3D chromatin organization and long-term evolutionary dynamics of cis-regulatory element (CRE) conservation. Here we study the evolution and organization of the regulatory genome of echinoderms, a lineage whose phylogenetic position and relatively slow molecular evolution have proven particularly useful for evolutionary studies. We generated new reference genome assemblies for two species belonging to two different echinoderm classes: the purple sea urchin Strongylocentrotus purpuratus and the bat sea star Patiria miniata using PacBio and HiC data and characterize their 3D chromatin architecture. We show that these echinoderms have TAD-like domains that, such as in flies, do not seem to be associated with CTCF motif orientation. We systematically profiled CREs during sea star and sea urchin development using ATAC-seq, comparing their regulatory logic and dynamics over multiple developmental stages. Finally, our analysis of sea urchin and sea star CRE evolution across multiple evolutionary distances and timescales showed several thousand elements conserved for hundreds of millions of years, revealing a vertebrate-like pattern of CRE evolution that probably constitutes an ancestral property of the regulatory evolution of animals.

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Fig. 1: Studying the regulatory genomes of the bat sea star and the purple sea urchin.
Fig. 2: Echinoderm TAD boundaries and CTCF sites.
Fig. 3: Regulatory annotation of echinoderm genomes using chromatin accessibility.
Fig. 4: Evolutionary conservation of pCREs in echinoderms.
Fig. 5: Deeply conserved pCREs in deuterostomes.

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Data availability

Genome assemblies used in this work are publicly available at the NCBI genome database GenBank with accession numbers GCA_015706575.1 (P. miniata) and GCA_000002235.4 (S. purpuratus). The HiC (GSE281901) and ATAC-seq (GSE280529) raw and processed sequencing data were deposited at the Gene Expression Omnibus (GEO) database under accession code of the SuperSeries GSE281904, with the exception of ATAC-seq data in S. purpuratus at 48 hpf that were deposited in GSE186363. Files and share links for viewing the multiple genome alignment are available via figshare at https://doi.org/10.6084/m9.figshare.30506378 (ref. 147).

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Acknowledgements

I.M., M.S.M., M.P. and M.F.-M. are supported by grants PID2021-128728NB-I00 and CNS2022-136105 funded by MICIU/AEI/10.13039/501100011033 and by ‘ERDF/EU’ and ‘European Union NextGenerationEU/PRTR’. I.M. was also funded by grants RYC-2016-20089 and PGC2018-099392-A-I00 funded by MICIU/AEI/10.13039/501100011033, ‘ERDF A way of making Europe’ and ‘ESF Investing in your future’. M.S.M. was granted a fellowship of the Program for the Training of Researchers (BES-2014-068494), and M.F.M. holds a FPU fellowship (FPU20/02733), funded by the Spanish Ministry of Science, Innovation and Universities (MICIU). J.L.G.-S. was supported by the European Research Council (grant agreement number 740041) and the Spanish Ministerio de Economía y Competitividad (grant number PID2019-103921GB-I00). P.M.M.-G. was funded by a postdoctoral fellowship from Junta de Andalucía (DOC_00397). V.F.H., S.F., G.A.C. and C.K. were supported by grant P41HD09583106 funded by the National Institutes of Health. Computing was supported via the Bridges2 system by allocation request BIO210137 awarded to S.F. D.V., M.L.R. and C.C. were supported by the Stazione Zoologica Anton Dohrn PhD fellowships. This work was supported by the H2020 Marie Skłodowska-Curie Actions Innovative Training Network EvoCELL (grant number 766053 to M.I.A. and fellowship to P.P.). We thank D. Burguera and I. Almudi for helpful discussions and F. Mantica for advice in orthogroup inference.

Author information

Author notes

  1. These authors contributed equally: Marta S. Magri, Danila Voronov, Saoirse Foley.

  2. These authors jointly supervised this work: Veronica F. Hinman, Maria Ina Arnone, Ignacio Maeso.

Authors and Affiliations

  1. CABD (Centro Andaluz de Biología del Desarrollo), CSIC-Universidad Pablo de Olavide-Junta de Andalucía, Seville, Spain

    Marta S. Magri, Pedro Manuel Martínez-García, Martin Franke, Alejandro Gil-Galvez, Rafael D. Acemel, Panos N. Firbas & José Luis Gómez-Skarmeta

  2. Department of Genetics, Microbiology and Statistics, University of Barcelona, Barcelona, Spain

    Marta S. Magri, Manuel Fernández-Moreno, Marta Portela & Ignacio Maeso

  3. Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Barcelona, Spain

    Marta S. Magri, Manuel Fernández-Moreno, Marta Portela & Ignacio Maeso

  4. Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy

    Danila Voronov, Claudia Cuomo, Periklis Paganos, Jovana Ranđelović, Maria Lorenza Rusciano & Maria Ina Arnone

  5. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

    Saoirse Foley, Gregory A. Cary & Carolyn Ku

  6. Department of Biology, Marian University, Indianapolis, IN, USA

    Saoirse Foley

  7. Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain

    José M. Santos-Pereira

  8. Departamento de Biología Celular, Facultad de Biología, Universidad de Sevilla, Seville, Spain

    José M. Santos-Pereira

  9. Centre for Genomic Regulation, Barcelona, Spain

    Manuel Fernández-Moreno

  10. Champalimaud Centre for the Unknown, Champalimaud Foundation, Lisbon, Portugal

    Panos N. Firbas

  11. Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL, USA

    Veronica F. Hinman

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  1. Marta S. Magri
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  2. Danila Voronov
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  3. Saoirse Foley
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Contributions

DNA library preparation: G.A.C. S. purpuratus genome assembly: G.A.C. and S.F. P. miniata genome assembly: C.K. and S.F. Gene annotation coordination: S.F. ATAC-seq experiments: M.S.M., D.V., J.R. and C.C. ATAC-seq data analyses: M.S.M. and D.V. TF motif analyses: M.S.M., P.N.F. and P.M.M.-G. HiC experiments: M.F. and R.D.A. Preparation of samples for HiC: D.V., C.C. and P.P. HiC data analyses: J.M.S.-P. and P.M.M.-G. Gene family evolution analyses: M.F.-M. Multi-species genome alignments: S.F. and A.G.-G. Sequence conservation analyses: M.S.M., A.G.-G., R.D.A. and M.P. Conservation of pCREs target genes: M.F.-M. and M.P. Preparation of constructs and cis-regulatory reporter assays: D.V., P.P., M.L.R. and M.F.-M. Microinjection experiments: M.I.A. Whole mount in situ hybridization: P.P. Conceptualization and design of the project: I.M., M.I.A., J.L.G.-S. and V.F.H. Project coordination and supervision: I.M., M.I.A. and V.F.H. Funding acquisition: J.L.G.-S., V.F.H., M.I.A. and I.M. Figure preparation: M.S.M., D.V., S.F., P.M.M.-G., J.M.S.-P., M.F.-M., P.P., M.L.R., I.M. and M.I.A. Paper writing–original draft: M.S.M., D.V., S.F. and I.M. Paper writing–review and editing: I.M., D.V., M.S.M., S.F., M.I.A., V.F.H., C.K., J.M.S.-P., P.F., R.D.A., P.P. and M.F.-M. All authors revised and approved the paper. Animal silhouettes for A. planci, P. miniata and A. japonica were drawn by I.M. Sea star and sea urchin embryos were drawn by M.I.A.

Corresponding authors

Correspondence to Veronica F. Hinman, Maria Ina Arnone or Ignacio Maeso.

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Extended data

Extended Data Fig. 1 Comparison of current and previous assembly statistics.

a, BUSCO statistics of current and previous genome assemblies. b, Assembly statistics of the two new genomes along with their older versions for comparison, generated with the assembly-stats perl package. c, d, Snailplots of general metrics (scaffold total length, scaffold count, longest scaffold, N50 and N90 values, and base compositions) of the new assemblies of the bat sea star (GCA_015706575.1) (c, upper panel) and purple sea urchin (GCA_000002235.4) (d, upper panel) versus those of previous assemblies (lower panels). e, f, HiC genome-wide contact maps of the bat sea star (e) and purple sea urchin (f) assemblies.

Extended Data Fig. 2 Chromatin domains and loops in echinoderms.

a, Heatmaps showing normalized HiC signal at 10-kb resolution in A. amurensis. Insulation scores and computationally called TAD boundaries are plotted below the heatmaps. b, Aggregate analysis of the observed versus expected HiC signal around TAD boundaries called at 10-kb resolution in A. amurensis (top) and D. melanogaster (bottom). c, Boxplots showing the distribution of boundary scores (left) and TAD sizes (right) in P. miniata (boundaries n = 2,108, TADs n = 2042), S. purpuratus (boundaries n = 3,009, TADs n = 2880), A. amurensis (boundaries n = 1,596, TADs n = 1566) and D. melanogaster (boundaries n = 476, TADs n = 484). d, Aggregate peak analyses of HiC loops in P. miniata, S. purpuratus, A. amurensis and D. melanogaster. The observed vs. expected HiC signal is shown. e, Boxplots showing the distribution of loop ranges in P. miniata, S. purpuratus, A. amurensis and D. melanogaster. Number of loops in each species as in d. f, Normalized number of ATAC-seq peaks harboring CTCF motifs around HiC loops in P. miniata (left), S. purpuratus (middle) and D. rerio (right). Shuffle control is shown per each graph. Boxplots in c and e show center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; notches, 95% confidence interval of the median.

Extended Data Fig. 3 Examples of ancient conserved microsyntenic blocks located within TAD boundaries.

Sea star (left panels) and sea urchin (right panels) genomic regions around four developmental genes included within ancient microsyntenic blocks (Tbx2/3, Foxa and Pax1/9, and Egr). From top to bottom: heatmaps showing normalized HiC signal at 10-kb resolution in late gastrula sea star and sea urchin embryos, ATAC-seq signal at late gastrula stage embryos of both species, IDR ATAC-seq peaks (pCREs), conserved ATAC-seq peaks (merging peaks conserved at the Valvatida+Asteroidea strata and Odontophora+Echinoidea strata), gene models (with those included within the conserved syntenic blocks colored in green), insulation scores and computationally called TAD boundaries. Note that the conserved block containing Foxa and Pax1/9 is split in two in the case of sea urchin.

Extended Data Fig. 4 Cohesin and CTCF phylogeny and conservation.

a, ML phylogenetic tree of cohesin subunit SA1-3 proteins. Sponge SA proteins were used as outgroups. b, ML phylogenetic tree of CTCF, including the tetrapod-specific paralog CTCFL (also known as BORIS), the outgroup (OG) branch includes the insect proteins CROOKED LEGS, a zinc finger containing family of transcription factors. c, Alignment of CTCF proteins from different bilaterians, showing the region containing its putatively ancestral 11 zinc finger domains, where ambulacrarian species have lost one or two domains. Silhouettes from PhyloPic under a Creative Commons license: sponge (Staurocalyptus solidus), whale shark and Onychophora (CC0 1.0) and sea star (CC BY 3.0).

Extended Data Fig. 5 Functional assays of foxa1 pCREs.

a, Screenshot of sea urchin UCSC browser with ATAC-seq tracks around the foxa1 (LOC110977664) gene and pCREs. b, Relative GFP expression levels driven by foxa1 CREs in sea urchin embryos at mesenchyme blastula stage. c, Scoring table for embryos injected with foxa1 pCRE-Tag constructs at mesenchyme blastula stage. This highlights the distribution of embryos with expression in both ectoderm and endoderm, only ectoderm or only endoderm, as well as the ones exhibiting GFP expression in regions ectopic to foxa1 expression. This was also concordant with scoring done for the FIJ concatenate69 since the majority of the expression is in the endoderm with oral ectoderm expression exhibited by fewer embryos.

Extended Data Fig. 6 Evolutionary strata and developmental dynamics of sea urchin and sea star pCREs.

a, Accession numbers of the genome assemblies included in the alignments. b, Counts of pCREs according to their evolutionary strata. c, Genome browser screenshots around the P. miniata pbx1a (LOC119720117, top panel) and the S. purpuratus six1 (LOC110974175, lower panel) genes showing ATAC-seq tracks from different developmental stages (in orange and purple, respectively), IDR peaks (orange and purple bars) and gene models (dark blue). A conserved Asteroidea pCRE (red bar) with late developmental dynamics and Deuterostome and Odontophora pCREs (green bars) with early developmental dynamics are highlighted with gray boxes.

Extended Data Fig. 7 Sequence and accessibility conservation of transphyletic pCREs and GO term enrichments of their associated genes.

a, Venn diagram with the number of conserved transphyletic pCREs present in each species. pCREs ancestral to deuterostomes and to ambulacrarians are shadowed in blue and orange, respectively. n.a. (not applicable) labels pCRE sharing between species pairs not addressed in this plot: those between amphioxus and hemichordates, which were not directly investigated in the present work, and pCREs shared between sea star and sea urchin, where shared conserved pCREs from the echinoderm stratum were not included to avoid confusions with transphyletic pCREs. b, Venn diagram with the number of transphyletic pCREs showing shared chromatin accessibility in sea star, sea urchin and amphioxus embryos. Deuterostome and ambulacrarian pCREs are indicated in black and gray, respectively. c–e, Top ten significantly enriched GO terms of genes associated with all transphyletic pCREs (c), and with the subsets of pCREs ancestral to deuterostomes (d) and ambulacrarians. Biological Process and Molecular Function ontologies are indicated in the left and right panels, respectively. B. lanceolatum drawing adapted from ref. 7 under a Creative Commons license (CC BY 4.0). Hemichordate (Balanoglossus) silhouette from PhyloPic under a Creative Commons license (CC0 1.0).

Extended Data Fig. 8 Functional assays of Tbx2/3 deeply conserved CRE.

a, Numbers of embryos examined in functional assays with the two different CREs and percentages of expression in S. purpuratus (Sp), P. lividus (Pl), P. miniata (Pm). Microinjection experiments were performed using at least two independent batches of embryos and for each experiment at least 30 embryos were analyzed. b, Expression pattern of tbx2/3 by fluorescent in situ hybridization in S. purpuratus late gastrula (top left, focus on the ectoderm) and functional assays of S. purpuratus and P. miniata CREs in different species (S. purpuratus (Sp), P. lividus (Pl), P. miniata (Pm)) by GFP fluorescence. Scale bar 20 µm; lv, lateral view; vv, ventral view. c, The dot plots highlight the expression of endogenous tbx2/3 as detected from scRNA-seq data analysis of gastrula stages in S. purpuratus51, left column, and from snRNA-seq data analysis of gastrula stages in P. miniata91, right column.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Data 1

MAFFT alignment of SA1-3 proteins.

Supplementary Data 2

MAFFT alignment of CTCF and CROL proteins.

Supplementary Data 3

SA1-3 tree.

Supplementary Data 4

CTCF tree.

Supplementary Data 5

Orthogroups used to assess conservation of pCREs associated genes.

Supplementary Tables

Supplementary Tables 1–14.

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Magri, M.S., Voronov, D., Foley, S. et al. Deep conservation of cis-regulatory elements and chromatin organization in echinoderms uncover ancestral regulatory features of animal genomes. Nat Ecol Evol (2026). https://doi.org/10.1038/s41559-025-02941-y

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