tail regeneration (fig. S20, F and G). We con-
clude that AP-1 motifs are required for the
activation of RREs in response to amputation.
Discussion
Activation or inactivation of genes is suspected
to underlie changes in regenerative capacities,
yet how these genetic activities are regulated
remains poorly understood. AP-1 transcription
factors are essential for many biological pro-
cesses, and their diverse functions are part of
complex dynamic networks of signaling path-
ways known to depend on subunit composition
and interactions with other nuclear factors,
which in turn are in part determined by both
cell type and cellular environment ( 33 ). In this
study, AP-1 components were not ubiquitously
expressed in all cell types and were present in
both mesenchymal and epithelial cells, suggest-
ing that specific subunit compositions may be
required to restrict expression of the enhancers
to either the mesenchyme (K-IEN,Z-IEN)or
epithelial cells (H-IEN). Additionally, the pres-
ence of a predicted p53/p63–binding motif
in the human enhancer, which was absent in
K-IENandZ-IEN(fig. S23A), and the abun-
dance ofp63expression in basal epidermal
cells (fig. S23B) suggest that interactions of
AP-1 with other nuclear factors may also play
a role in regulating enhancer activity.Given the ancient evolutionary origin of the
AP-1 complex ( 35 , 36 ), we hypothesize that the
ancestral function of AP-1 motif–enriched en-
hancers was to activate a regenerative response,
and that through the course of evolution and
speciation, regeneration and injury responses
became dissociated from each other in some,
but not all, enhancers (Fig. 6F). Repurposing of
ancestral regulatory sequences to generate new
regulatory functions is not without precedent
( 37 ) and has been well documented in both
vertebrates ( 38 ) and invertebrates ( 39 ). For in-
stance, frequent regulatory element repurpos-
ing was revealed by DNase I–hypersensitive
sites in the mouse and human genomes ( 40 ).Wanget al.,Science 369 , eaaz3090 (2020) 4 September 2020 8of9
Fig. 6. Occupancy of AP-1–binding motifs is essential for RRE activities.
(AandB) Motifs enriched in the conserved (A) and species-specific (B) RREs
identified in killifish (top) and zebrafish (bottom) caudal fin regeneration.
AP-1 motifs are highlighted in red. Each dot in the graph represents a single
binding motif. The sequence of AP-1 motifs is shown in fig. S19. (C) Identification
of AP-1 motifs in the RREsK-IENandZ-IEN.(DandE) Expression of GFP
driven byK-IENM12andZ-IENM12is abolished at 2 dpa in transgenic reporter lines.
Right, quantification of the fluorescence intensity between wild-type and
mutant enhancers.P<0.001(Student’sttest).n= 10. Dashed lines indicate
the amputation site. (F) RRE-based model for the loss of regenerative
capacities during evolution. We propose a regenerative response to injury as
the ancestral function of AP-1 motif–enriched enhancers. In the course of
evolution and speciation, regeneration and injury responses became
dissociated from each other in some, but not all, enhancers. In extant species,
regeneration-competent animals maintain the ancestral enhancer activities
to activate both injury response and regeneration, whereas repurposing
of ancestral enhancers in regeneration-incompetent animals led to loss of
regenerative capacities.RESEARCH | RESEARCH ARTICLE