Co-option of transposable elements (TEs) to become part of existing or new enhancers is an important mechanism for evolution of gene regulation. However, contributions of lineage-specific TE insertions to recent regulatory adaptations remain poorly understood. Gibbons present a suitable model to study these contributions as they have evolved a lineage-specific TE called
Among squamate reptiles, dozens of lineages have independently evolved complete or partial limb reduction. This remarkable convergence of limbless and limb‐reduced phenotypes provides multiple natural replicates of different ages to explore the evolution and development of the vertebrate limb and the gene regulatory network that controls its formation. The most successful and best known of the limb‐reduced squamates are snakes, which evolved a limb‐reduced body form more than 100 million years ago. Recent studies have revealed the unexpected finding that many ancient limb enhancers are conserved in the genomes of snakes. Analyses in limbed animals show that many of these limb enhancers are also active during development of the phallus, suggesting that these enhancers may have been retained in snakes due their importance in regulating transcription in the external genitalia. This hypothesis is substantiated by functional tests of snake enhancers, which demonstrate that snake enhancer elements have lost limb function while retaining genital enhancer function. The large degree of overlap in the gene regulatory networks deployed during limb and phallus development may act to constrain the divergence of shared gene network components and the evolution of appendage morphology. Future studies will reveal whether limb regulatory elements have undergone similar functional changes in other lineages of limb‐reduced squamates.
more » « less- NSF-PAR ID:
- 10045923
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- genesis
- Volume:
- 56
- Issue:
- 1
- ISSN:
- 1526-954X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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LAVA (LINE-Alu Sz-VNTR-Alu LIKE), which is still active in the gibbon genome. The LAVA retrotransposon is thought to have played a role in the emergence of the highly rearranged structure of the gibbon genome by disrupting transcription of cell cycle genes. In this study, we investigated whether LAVA may have also contributed to the evolution of gene regulation by adopting enhancer function. We characterized fixed and polymorphic LAVA insertions across multiple gibbons and found 96 LAVA elements overlapping enhancer chromatin states. Moreover, LAVA was enriched in multiple transcription factor binding motifs, was bound by an important transcription factor (PU.1), and was associated with higher levels of gene expression incis . We found gibbon-specific signatures of purifying/positive selection at 27 LAVA insertions. Two of these insertions were fixed in the gibbon lineage and overlapped with enhancer chromatin states, representing putative co-opted LAVA enhancers. These putative enhancers were located within genes encoding SETD2 and RAD9A, two proteins that facilitate accurate repair of DNA double-strand breaks and prevent chromosomal rearrangement mutations. Co-option of LAVA in these genes may have influenced regulation of processes that preserve genome integrity. Our findings highlight the importance of considering lineage-specific TEs in studying evolution of gene regulatory elements. -
Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis -regulatory sequences (enhancers and promoters), trans -regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis -regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.more » « less
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Abstract Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb‐reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in
Chalcides and limb reduction inScelotes ). The genusBrachymeles , a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake‐like speciesBrachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos ofB .lukbani in early stages of development, on the other hand, show a truncated but well‐developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole‐mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb ofB .lukabni . Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well‐developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well‐developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well‐developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that “limbless” lizards that possess these muscles may have or have had at least transient developing limbs, as we find inB .lukbani . Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation calledcryptomelia . The results of this work add to our growing understanding of clade‐specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes. -
INTRODUCTION Diverse phenotypes, including large brains relative to body size, group living, and vocal learning ability, have evolved multiple times throughout mammalian history. These shared phenotypes may have arisen repeatedly by means of common mechanisms discernible through genome comparisons. RATIONALE Protein-coding sequence differences have failed to fully explain the evolution of multiple mammalian phenotypes. This suggests that these phenotypes have evolved at least in part through changes in gene expression, meaning that their differences across species may be caused by differences in genome sequence at enhancer regions that control gene expression in specific tissues and cell types. Yet the enhancers involved in phenotype evolution are largely unknown. Sequence conservation–based approaches for identifying such enhancers are limited because enhancer activity can be conserved even when the individual nucleotides within the sequence are poorly conserved. This is due to an overwhelming number of cases where nucleotides turn over at a high rate, but a similar combination of transcription factor binding sites and other sequence features can be maintained across millions of years of evolution, allowing the function of the enhancer to be conserved in a particular cell type or tissue. Experimentally measuring the function of orthologous enhancers across dozens of species is currently infeasible, but new machine learning methods make it possible to make reliable sequence-based predictions of enhancer function across species in specific tissues and cell types. RESULTS To overcome the limits of studying individual nucleotides, we developed the Tissue-Aware Conservation Inference Toolkit (TACIT). Rather than measuring the extent to which individual nucleotides are conserved across a region, TACIT uses machine learning to test whether the function of a given part of the genome is likely to be conserved. More specifically, convolutional neural networks learn the tissue- or cell type–specific regulatory code connecting genome sequence to enhancer activity using candidate enhancers identified from only a few species. This approach allows us to accurately associate differences between species in tissue or cell type–specific enhancer activity with genome sequence differences at enhancer orthologs. We then connect these predictions of enhancer function to phenotypes across hundreds of mammals in a way that accounts for species’ phylogenetic relatedness. We applied TACIT to identify candidate enhancers from motor cortex and parvalbumin neuron open chromatin data that are associated with brain size relative to body size, solitary living, and vocal learning across 222 mammals. Our results include the identification of multiple candidate enhancers associated with brain size relative to body size, several of which are located in linear or three-dimensional proximity to genes whose protein-coding mutations have been implicated in microcephaly or macrocephaly in humans. We also identified candidate enhancers associated with the evolution of solitary living near a gene implicated in separation anxiety and other enhancers associated with the evolution of vocal learning ability. We obtained distinct results for bulk motor cortex and parvalbumin neurons, demonstrating the value in applying TACIT to both bulk tissue and specific minority cell type populations. To facilitate future analyses of our results and applications of TACIT, we released predicted enhancer activity of >400,000 candidate enhancers in each of 222 mammals and their associations with the phenotypes we investigated. CONCLUSION TACIT leverages predicted enhancer activity conservation rather than nucleotide-level conservation to connect genetic sequence differences between species to phenotypes across large numbers of mammals. TACIT can be applied to any phenotype with enhancer activity data available from at least a few species in a relevant tissue or cell type and a whole-genome alignment available across dozens of species with substantial phenotypic variation. Although we developed TACIT for transcriptional enhancers, it could also be applied to genomic regions involved in other components of gene regulation, such as promoters and splicing enhancers and silencers. As the number of sequenced genomes grows, machine learning approaches such as TACIT have the potential to help make sense of how conservation of, or changes in, subtle genome patterns can help explain phenotype evolution. Tissue-Aware Conservation Inference Toolkit (TACIT) associates genetic differences between species with phenotypes. TACIT works by generating open chromatin data from a few species in a tissue related to a phenotype, using the sequences underlying open and closed chromatin regions to train a machine learning model for predicting tissue-specific open chromatin and associating open chromatin predictions across dozens of mammals with the phenotype. [Species silhouettes are from PhyloPic]more » « less
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Abstract The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR
HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find thatHACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression ofGbx2 , which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate thatGbx2 is upregulated in the limb chondrogenic mesenchyme ofHACNS1 homozygous embryos, supporting thatHACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.
