Evolutionary Changes in the Chromatin Landscape Contribute to Reorganization of a Developmental Gene Network During Rapid Life History Evolution in Sea Urchins

Abstract

Chromatin configuration is highly dynamic during embryonic development in animals, exerting an important point of control in transcriptional regulation. Yet there exists remarkably little information about the role of evolutionary changes in chromatin configuration to the evolution of gene expression and organismal traits. Genome-wide assays of chromatin configuration, coupled with whole-genome alignments, can help address this gap in knowledge in several ways. In this study we present a comparative analysis of regulatory element sequences and accessibility throughout embryogenesis in three sea urchin species with divergent life histories: a lecithotroph Heliocidaris erythrogramma, a closely related planktotroph H. tuberculata, and a distantly related planktotroph Lytechinus variegatus. We identified distinct epigenetic and mutational signatures of evolutionary modifications to the function of putative cis-regulatory elements in H. erythrogramma that have accumulated nonuniformly throughout the genome, suggesting selection, rather than drift, underlies many modifications associated with the derived life history. Specifically, regulatory elements composing the sea urchin developmental gene regulatory network are enriched for signatures of positive selection and accessibility changes which may function to alter binding affinity and access of developmental transcription factors to these sites. Furthermore, regulatory element changes often correlate with divergent expression patterns of genes involved in cell type specification, morphogenesis, and development of other derived traits, suggesting these evolutionary modifications have been consequential for phenotypic evolution in H. erythrogramma. Collectively, our results demonstrate that selective pressures imposed by changes in developmental life history rapidly reshape the cis-regulatory landscape of core developmental genes to generate novel traits and embryonic programs.

Keywords: ATAC-seq; Evolution; development; gene regulation; life history; sea urchins.

Fig. 1.

Global variation in chromatin accessibility between sea urchin embryos is largely driven by developmental stage and life history strategy. (A) Three focal sea urchin species of this study, including two planktotrophic species representing the ancestral life history (light green: Lytechinus variegatus; dark green: Heliocidaris tuberculata) and one lecithotrophic species representing the derived life history (orange: Heliocidaris erythrogramma). This color scheme is consistent throughout the study. (B) Developmental stages assayed in this study for each species. The ancestral life history is illustrated above, with corresponding developmental stages of the derived life history below. Developmental times are shown for Heliocidaris tuberculata at 21 °C. Note, however, samples were collected on the basis of developmental stage, not hours postfertilization, for all three species. (C-D) Principal component analysis of variation in normalized chromatin accessibility among (C) all orthologous OCRs and (D) only OCRs near dGRN genes through embryogenesis. OCR: open chromatin region; dGRN: developmental gene regulatory network.

Fig. 2.

Summary statistics of regulatory element gains, losses, accessibility, and positive selection. (A) Union set of all OCRs, including structural differences between species such as lineage-specific gains and losses as well as 35,788 1:1:1 orthologous OCRs shared between all three species. These orthologous OCRs are the primary focus of this study. Breakdown of (B) OCR differentially accessibility between life history strategies and (C) OCR distance to gene models for these orthologous sites. (D) Overlap of OCR differential accessibility and evidence of positive selection on the He, Ht, or both branches. (E) Percentage of each species genome that is ‘open’ at each stage of development. ‘Open’ is defined as the cumulative portion of the genome represented by all OCRs (orthologous and lineage-specific) with significant levels of accessibility for a given stage and species (see supplementary fig. 3 for additional information). Error bars indicate standard deviation of % openness across biological replicates for each species and developmental stage. OCR: open chromatin region; He: Heliocidaris erythrogramma; Ht: Heliocidaris tuberculata.

Fig. 3.

The chromatin landscape of the derived life history is far less accessible than that of species with the ancestral life history mode, particularly near core dGRN genes. (A) Number (flow diagram) and (B) percentage (barplots) of OCRs that are significantly differentially accessible in H. erythrogramma or species with the ancestral life history strategy for all orthologous OCRs (top) and only OCRs near dGRN genes (bottom). A key for flow diagrams is provided to explain how the number of differentially accessible OCRs are gained, retained, and lost between developmental stages. Circle size is proportional to the percentage of each set of OCRs (those near all genes or only dGRN genes) that are differentially accessible at a given stage for either H. erythrogramma or species with the ancestral developmental mode. OCR: open chromatin region; dGRN: developmental gene regulatory network.

Fig. 4.

Example of an evolutionary change in chromatin accessibility during early development. The embryonic chromatin landscape near the gene Patched is shown for the three study species, with gene models on the right and dashed lines indicating orthologous noncoding regions. In the two planktotrophs, the most proximal OCR (likely promoter based on location at the 5’ end of the 5’ UTR) opens substantially after cleavage, and a distal OCR ∼7 kb upstream opens by gastrula stage. In in H. erythrogramma, the proximal OCR is substantially less accessible and the distal OCR is not above background accessibility levels.

Fig. 5.

Sequence changes indicative of positive selection are enriched within cis-regulatory elements near dGRN genes and other transcription factors in the H. erythrogramma genome. (A) Zeta values (ratio of OCR substitution rate to that of neutrally evolving reference sequences) for H. erythrogramma (top) and H. tuberculata (bottom) across the genome. OCRs with significant support for lineage-specific evidence of positive selection are noted for H. erythrogramma and H. tuberculata, and those nearby dGRN genes are outlined in purple. (B) Fold-change in proportion of OCRs with more frequent evidence of positive selection in either H. erythrogramma or H. tuberculata, partitioned by functional categories of nearby genes (see supplementary fig. 4 for all categories). (C) Transcription factor binding motif enrichment results of OCRs near dGRN genes with evidence of positive selection in H. erythrogramma. Detection mode indicates OCR sequences were (i) searched against a ‘known’ set of optimized motif models or (ii) enriched motifs within the OCR were identified ‘de-novo’ and matched to a known set of motifs. (D) Embryonic mRNA expression levels for Nuclear factor erythroid 2-related factor (NRF) for each species. OCR: open chromatin region; dGRN: developmental gene regulatory network.

Fig. 6.

Independent and synergistic changes to regulatory element function have evolved during life history evolution in H. erythrogramma. (A) Proportion of differentially accessible or nondifferentially accessible OCRs with evidence of positive selection on the H. erythrogramma branch (top) or H. tuberculata branch (bottom). Two-sided, Fisher’s exact test: **P ≤ 5 × 10⁻³; ***P ≤ 5 × 10⁻⁴. (B) Proportion of proximal of distal OCRs with evidence of positive selection in either Heliocidaris species. (C) Differential accessibility of proximal and distal OCRs at four stages of development. (D) Diagram describing possible correlative patterns between changes in OCR accessibility and gene expression in either life history strategy (ancestral, planktotroph larva; derived, lecithotroph larva): the layout of this panel is a key for interpreting the density plots in panel (E) below. (E) Fold-change of mRNA expression vs nearby OCR accessibility between life history strategies for proximal (left) and distal (right) OCRs. Relationships are shown for all genes and OCRs (E: top row), significantly differentially expressed genes (E: middle row), and significantly differentially expressed genes and differentially accessible OCRs (E: bottom row). OCR: open chromatin region; DE: differentially expressed (genes); DA: differentially accessible (OCRs).

Fig. 7.

Mechanistic models for the evolutionary origin of an open chromatin region. Three distinct evolutionary time slices are illustrated: t = 1 prior to the appearance of the open chromatin region (OCR), t = 2 the event that precipitates local chromatin opening, and t = 3 many generations after the origin of the OCR. Two general molecular processes could transform a given region of the genome from a relatively closed to a relatively open region of chromatin, based on the location of the genetic basis: cis or local and trans or distant. We illustrate this distinction based on a pioneer transcription factor, a class of proteins capable of sequence-specific binding to DNA in closed chromatin causing a local opening, typically on the scale of 1–2 nucleosomes (Zaret and Carroll 2011). Note, however, that several other specific molecular mechanisms are also possible, involving chromatin remodeling complexes, histone acetylase/deacetylases, histone methylase/demethylases, and other proteins capable of acting to modify local chromatin configuration. (Left) ‘cis mutation first’ model. Here, the precipitating event is a local mutation (yellow; adjacent to the lightning bolt) that allows a pioneer transcription factor (purple protein) to bind to relatively closed chromatin. This pioneer factor was already present in the nucleus (see t = 1), but can only bind locally following the new mutation that creates a binding site for it (t = 2). Because pioneer factors can convert closed to open chromatin, other transcription factors also present in the nucleus (teal protein) may gain access to DNA within the newly opened region without the need for any additional mutations. (Right) ‘trans factor first’ model. Here, the pioneer factor is not originally present (t = 1) in the nucleus at the developmental stage of interest. A mutation elsewhere in the genome (t = 2) results in expression of the pioneer factor transcription factor, which can bind locally due to a preexisting recognition site and thus without the requirement for a local mutation. Once present, the pioneer factor can open the local DNA, potentially allowing other transcription factors (teal protein) to bind without the need for any local mutations. Any impact on the transcription of a local gene could be susceptible to natural selection, since it has a genetic basis and a trait consequence. Over extended evolutionary timescales, additional mutations could accumulate within the OCR (t = 3), potentially altering the suite of interacting transcription factors (for example, green protein now binds, purple protein no longer binds). In exceptional cases, natural selection may favor fixation of several new mutations within the OCR that alter gene expression. This figure illustrates the evolutionary origin of an OCR, but the evolutionary loss of an OCR could similarly occur through distinct molecular mechanisms based on the location of the genetic basis: for example, loss of a binding site for a transcription factor or loss of expression of a pioneer factor.

Fig. 8.

Quantifying developmental expression and accessibility changes in principal component space predicts divergent patterns of gene regulation. (A-D) An example of conserved developmental gene expression and chromatin accessibility for the gene insm. (A) Developmental mRNA expression profile of insm for each sea urchin species and (B) their coordinates in PCA space. (C) Coordinates in PCA space of developmental accessibility of orthologous OCRs near insm and (D) normalized chromatin accessibility coverage of these same OCRs through embryogenesis for each species. (E-H) An example of divergent developmental gene expression and chromatin accessibility associated with life history for the gene brn 1/2/4. (E) Developmental mRNA expression profile of brn 1/2/4 for each sea urchin species and (F) their coordinates in PCA space. (G) Coordinates in PCA space of developmental accessibility of orthologous OCRs near brn 1/2/4 and (H) normalized chromatin accessibility coverage of these same OCRs through embryogenesis for each species. See Supplementary Figure 6 for a summary figure of PCA distances for all genes and OCRs. OCR: open chromatin region; PCA: principal component analysis.

Fig. 9.

Evolutionary changes to cis-regulatory element function associated with life history evolution in sea urchins and expression changes of nearby genes. Proportion of genes that are differentially expressed between developmental life history strategies at four developmental stages, distinguished as having at least one nearby OCR with (A) differential accessibility or (B) evidence of lineage-specific positive selection. OCR: open chromatin region. Values above brackets indicate the percent change in proportion of genes that are differentially expressed between categories.

Fig. 10.

Chromatin accessibility changes, mutational signatures of positive selection, and the coincidence of both evolutionary changes are abundant in the H. erythrogramma genome, especially nearby dGRN genes. Enrichment of regulatory modifications to OCRs near dGRN genes suggests selective pressures imposed by changes in developmental life history can rapidly modify cis-regulatory interactions underlying developmental processes. Tandem accessibility and mutational changes may act as a common gene regulatory method to efficiently modify gene expression for organismal adaptation and innovation. Branch lengths scaled to relative number of changes between Heliocidaris branches for each set of modifications.