Disentangling the determinants of transposable elements dynamics in vertebrate genomes using empirical evidences and simulations.

The interactions between transposable elements (TEs) and their hosts constitute one of the most profound co-evolutionary processes found in nature. The population dynamics of TEs depends on factors specific to each TE families, such as the rate of transposition and insertional preference, the demographic history of the host and the genomic landscape. How these factors interact has yet to be investigated holistically. Here we are addressing this question in the green anole (Anolis carolinensis) whose genome contains an extraordinary diversity of TEs (including non-LTR retrotransposons, SINEs, LTR-retrotransposons and DNA transposons). We observed a positive correlation between recombination rate and frequency of TEs and densities for LINEs, SINEs and DNA transposons. For these elements, there was a clear impact of demography on TE frequency and abundance, with a loss of polymorphic elements and skewed frequency spectra in recently expanded populations. On the other hand, some LTR-retrotransposons displayed patterns consistent with a very recent phase of intense amplification. To determine how demography, genomic features and intrinsic properties of TEs interact we ran simulations using SLiM3. We determined that i) short TE insertions are not strongly counter-selected, but long ones are, ii) neutral demographic processes, linked selection and preferential insertion may explain positive correlations between average TE frequency and recombination, iii) TE insertions are unlikely to have been massively recruited in recent adaptation. We demonstrate that deterministic and stochastic processes have different effects on categories of TEs and that a combination of empirical analyses and simulations can disentangle these mechanisms.

Variation in base composition underlies functional and evolutionary divergence in non-LTR retrotransposons.

BACKGROUND: Non-LTR retrotransposons often exhibit base composition that is markedly different from the nucleotide content of their host's gene. For instance, the mammalian L1 element is AT-rich with a strong A bias on the positive strand, which results in a reduced transcription. It is plausible that the A-richness of mammalian L1 is a self-regulatory mechanism reflecting a trade-off between transposition efficiency and the deleterious effect of L1 on its host. We examined if the A-richness of L1 is a general feature of non-LTR retrotransposons or if different clades of elements have evolved different nucleotide content. We also investigated if elements belonging to the same clade evolved towards different base composition in different genomes or if elements from different clades evolved towards similar base composition in the same genome. RESULTS: We found that non-LTR retrotransposons differ in base composition among clades within the same host but also that elements belonging to the same clade differ in base composition among hosts. We showed that nucleotide content remains constant within the same host over extended period of evolutionary time, despite mutational patterns that should drive nucleotide content away from the observed base composition. CONCLUSIONS: Our results suggest that base composition is evolving under selection and may be reflective of the long-term co-evolution between non-LTR retrotransposons and their host. Finally, the coexistence of elements with drastically different base composition suggests that these elements may be using different strategies to persist and multiply in the genome of their host.

Vertebrate Lineages Exhibit Diverse Patterns of Transposable Element Regulation and Expression across Tissues.

Transposable elements (TEs) comprise a major fraction of vertebrate genomes, yet little is known about their expression and regulation across tissues, and how this varies across major vertebrate lineages. We present the first comparative analysis integrating TE expression and TE regulatory pathway activity in somatic and gametic tissues for a diverse set of 12 vertebrates. We conduct simultaneous gene and TE expression analyses to characterize patterns of TE expression and TE regulation across vertebrates and examine relationships between these features. We find remarkable variation in the expression of genes involved in TE negative regulation across tissues and species, yet consistently high expression in germline tissues, particularly in testes. Most vertebrates show comparably high levels of TE regulatory pathway activity across gonadal tissues except for mammals, where reduced activity of TE regulatory pathways in ovarian tissues may be the result of lower relative germ cell densities. We also find that all vertebrate lineages examined exhibit remarkably high levels of TE-derived transcripts in somatic and gametic tissues, with recently active TE families showing higher expression in gametic tissues. Although most TE-derived transcripts originate from inactive ancient TE families (and are likely incapable of transposition), such high levels of TE-derived RNA in the cytoplasm may have secondary, unappreciated biological relevance.

Finding and extending ancient simple sequence repeat-derived regions in the human genome.

BACKGROUND: Previously, 3% of the human genome has been annotated as simple sequence repeats (SSRs), similar to the proportion annotated as protein coding. The origin of much of the genome is not well annotated, however, and some of the unidentified regions are likely to be ancient SSR-derived regions not identified by current methods. The identification of these regions is complicated because SSRs appear to evolve through complex cycles of expansion and contraction, often interrupted by mutations that alter both the repeated motif and mutation rate. We applied an empirical, kmer-based, approach to identify genome regions that are likely derived from SSRs. RESULTS: The sequences flanking annotated SSRs are enriched for similar sequences and for SSRs with similar motifs, suggesting that the evolutionary remains of SSR activity abound in regions near obvious SSRs. Using our previously described P-clouds approach, we identified 'SSR-clouds', groups of similar kmers (or 'oligos') that are enriched near a training set of unbroken SSR loci, and then used the SSR-clouds to detect likely SSR-derived regions throughout the genome. CONCLUSIONS: Our analysis indicates that the amount of likely SSR-derived sequence in the human genome is 6.77%, over twice as much as previous estimates, including millions of newly identified ancient SSR-derived loci. SSR-clouds identified poly-A sequences adjacent to transposable element termini in over 74% of the oldest class of Alu (roughly, AluJ), validating the sensitivity of the approach. Poly-A's annotated by SSR-clouds also had a length distribution that was more consistent with their poly-A origins, with mean about 35 bp even in older Alus. This work demonstrates that the high sensitivity provided by SSR-Clouds improves the detection of SSR-derived regions and will enable deeper analysis of how decaying repeats contribute to genome structure.

Recent Secondary Contacts, Linked Selection, and Variable Recombination Rates Shape Genomic Diversity in the Model Species Anolis carolinensis.

Gaining a better understanding on how selection and neutral processes affect genomic diversity is essential to gain better insights into the mechanisms driving adaptation and speciation. However, the evolutionary processes affecting variation at a genomic scale have not been investigated in most vertebrate lineages. Here, we present the first population genomics survey using whole genome resequencing in the green anole (Anolis carolinensis). Anoles have been intensively studied to understand mechanisms underlying adaptation and speciation. The green anole in particular is an important model to study genome evolution. We quantified how demography, recombination, and selection have led to the current genetic diversity of the green anole by using whole-genome resequencing of five genetic clusters covering the entire species range. The differentiation of green anole's populations is consistent with a northward expansion from South Florida followed by genetic isolation and subsequent gene flow among adjacent genetic clusters. Dispersal out-of-Florida was accompanied by a drastic population bottleneck followed by a rapid population expansion. This event was accompanied by male-biased dispersal and/or selective sweeps on the X chromosome. We show that the interaction between linked selection and recombination is the main contributor to the genomic landscape of differentiation in the anole genome.

The Mobilome of Reptiles: Evolution, Structure, and Function.

Transposable elements (TE) constitute one of the most variable genomic features among vertebrates, impacting genome size, structure, and composition. Despite their important role in shaping genomic diversity, they have mostly been studied in mammals, which display one of the least diverse genomes in terms of TE diversity. Recent new resources in reptilian genomics have opened a broader perspective about TE evolution in amniotes. We discuss these recent results by showing that TE diversity is high in reptiles, particularly in squamates, with strong heterogeneity in the number of TE classes retained in each lineage, even at short evolutionary scales. More research is needed to uncover the exact mechanisms that regulate TE proliferation in reptiles and to what extent these selfish elements can play a role in local adaptation or in the emergence of barriers to gene flow.

Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals.

Broad paradigms of vertebrate genomic repeat element evolution have been largely shaped by analyses of mammalian and avian genomes. Here, based on analyses of genomes sequenced from over 60 squamate reptiles (lizards and snakes), we show that patterns of genomic repeat landscape evolution in squamates challenge such paradigms. Despite low variance in genome size, squamate genomes exhibit surprisingly high variation among species in abundance (ca. 25-73% of the genome) and composition of identifiable repeat elements. We also demonstrate that snake genomes have experienced microsatellite seeding by transposable elements at a scale unparalleled among eukaryotes, leading to some snake genomes containing the highest microsatellite content of any known eukaryote. Our analyses of transposable element evolution across squamates also suggest that lineage-specific variation in mechanisms of transposable element activity and silencing, rather than variation in species-specific demography, may play a dominant role in driving variation in repeat element landscapes across squamate phylogeny.

Differential Effect of Selection against LINE Retrotransposons among Vertebrates Inferred from Whole-Genome Data and Demographic Modeling.

Variation in LINE composition is one of the major determinants for the substantial size and structural differences among vertebrate genomes. In particular, the larger genomes of mammals are characterized by hundreds of thousands of copies from a single LINE clade, L1, whereas nonmammalian vertebrates possess a much greater diversity of LINEs, yet with orders of magnitude less in copy number. It has been proposed that such variation in copy number among vertebrates is due to differential effect of LINE insertions on host fitness. To investigate LINE selection, we deployed a framework of demographic modeling, coalescent simulations, and probabilistic inference against population-level whole-genome data sets for four model species: one population each of threespine stickleback, green anole, and house mouse, as well as three human populations. Specifically, we inferred a null demographic background utilizing SNP data, which was then exploited to simulate a putative null distribution of summary statistics that was compared with LINE data. Subsequently, we applied the inferred null demographic model with an additional exponential size change parameter, coupled with model selection, to test for neutrality as well as estimate the strength of either negative or positive selection. We found a robust signal for purifying selection in anole and mouse, but a lack of clear evidence for selection in stickleback and human. Overall, we demonstrated LINE insertion dynamics that are not in accordance to a mammalian versus nonmammalian dichotomy, and instead the degree of existing LINE activity together with host-specific demographic history may be the main determinants of LINE abundance.

Growth and stress response mechanisms underlying post-feeding regenerative organ growth in the Burmese python.

BACKGROUND: Previous studies examining post-feeding organ regeneration in the Burmese python (Python molurus bivittatus) have identified thousands of genes that are significantly differentially regulated during this process. However, substantial gaps remain in our understanding of coherent mechanisms and specific growth pathways that underlie these rapid and extensive shifts in organ form and function. Here we addressed these gaps by comparing gene expression in the Burmese python heart, liver, kidney, and small intestine across pre- and post-feeding time points (fasted, one day post-feeding, and four days post-feeding), and by conducting detailed analyses of molecular pathways and predictions of upstream regulatory molecules across these organ systems. RESULTS: Identified enriched canonical pathways and upstream regulators indicate that while downstream transcriptional responses are fairly tissue specific, a suite of core pathways and upstream regulator molecules are shared among responsive tissues. Pathways such as mTOR signaling, PPAR/LXR/RXR signaling, and NRF2-mediated oxidative stress response are significantly differentially regulated in multiple tissues, indicative of cell growth and proliferation along with coordinated cell-protective stress responses. Upstream regulatory molecule analyses identify multiple growth factors, kinase receptors, and transmembrane receptors, both within individual organs and across separate tissues. Downstream transcription factors MYC and SREBF are induced in all tissues. CONCLUSIONS: These results suggest that largely divergent patterns of post-feeding gene regulation across tissues are mediated by a core set of higher-level signaling molecules. Consistent enrichment of the NRF2-mediated oxidative stress response indicates this pathway may be particularly important in mediating cellular stress during such extreme regenerative growth.

LINE Insertion Polymorphisms are Abundant but at Low Frequencies across Populations of Anolis carolinensis.

Vertebrate genomes differ considerably in size and structure. Among the features that show the most variation is the abundance of Long Interspersed Nuclear Elements (LINEs). Mammalian genomes contain 100,000s LINEs that belong to a single clade, L1, and in most species a single family is usually active at a time. In contrast, non-mammalian vertebrates (fish, amphibians and reptiles) contain multiple active families, belonging to several clades, but each of them is represented by a small number of recently inserted copies. It is unclear why vertebrate genomes harbor such drastic differences in LINE composition. To address this issue, we conducted whole genome resequencing to investigate the population genomics of LINEs across 13 genomes of the lizard Anolis carolinensis sampled from two geographically and genetically distinct populations in the Eastern Florida and the Gulf Atlantic regions of the United States. We used the Mobile Element Locator Tool to identify and genotype polymorphic insertions from five major clades of LINEs (CR1, L1, L2, RTE and R4) and the 41 subfamilies that constitute them. Across these groups we found large variation in the frequency of polymorphic insertions and the observed length distributions of these insertions, suggesting these groups vary in their activity and how frequently they successfully generate full-length, potentially active copies. Though we found an abundance of polymorphic insertions (over 45,000) most of these were observed at low frequencies and typically appeared as singletons. Site frequency spectra for most LINEs showed a significant shift toward low frequency alleles compared to the spectra observed for total genomic single nucleotide polymorphisms. Using Tajima's D, FST and the mean number of pairwise differences in LINE insertion polymorphisms, we found evidence that negative selection is acting on LINE families in a length-dependent manner, its effects being stronger in the larger Eastern Florida population. Our results suggest that a large effective population size and negative selection limit the expansion of polymorphic LINE insertions across these populations and that the probability of LINE polymorphisms reaching fixation is extremely low.

Rapid changes in gene expression direct rapid shifts in intestinal form and function in the Burmese python after feeding.

Snakes provide a unique and valuable model system for studying the extremes of physiological remodeling because of the ability of some species to rapidly upregulate organ form and function upon feeding. The predominant model species used to study such extreme responses has been the Burmese python because of the extreme nature of postfeeding response in this species. We analyzed the Burmese python intestine across a time series, before, during, and after feeding to understand the patterns and timing of changes in gene expression and their relationship to changes in intestinal form and function upon feeding. Our results indicate that >2,000 genes show significant changes in expression in the small intestine following feeding, including genes involved in intestinal morphology and function (e.g., hydrolases, microvillus proteins, trafficking and transport proteins), as well as genes involved in cell division and apoptosis. Extensive changes in gene expression occur surprisingly rapidly, within the first 6 h of feeding, coincide with changes in intestinal morphology, and effectively return to prefeeding levels within 10 days. Collectively, our results provide an unprecedented portrait of parallel changes in gene expression and intestinal morphology and physiology on a scale that is extreme both in the magnitude of changes, as well as in the incredibly short time frame of these changes, with up- and downregulation of expression and function occurring in the span of 10 days. Our results also identify conserved vertebrate signaling pathways that modulate these responses, which may suggest pathways for therapeutic modulation of intestinal function in humans.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5-5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

Inference of transposable element ancestry.

Most common methods for inferring transposable element (TE) evolutionary relationships are based on dividing TEs into subfamilies using shared diagnostic nucleotides. Although originally justified based on the "master gene" model of TE evolution, computational and experimental work indicates that many of the subfamilies generated by these methods contain multiple source elements. This implies that subfamily-based methods give an incomplete picture of TE relationships. Studies on selection, functional exaptation, and predictions of horizontal transfer may all be affected. Here, we develop a Bayesian method for inferring TE ancestry that gives the probability that each sequence was replicative, its frequency of replication, and the probability that each extant TE sequence came from each possible ancestral sequence. Applying our method to 986 members of the newly-discovered LAVA family of TEs, we show that there were far more source elements in the history of LAVA expansion than subfamilies identified using the CoSeg subfamily-classification program. We also identify multiple replicative elements in the AluSc subfamily in humans. Our results strongly indicate that a reassessment of subfamily structures is necessary to obtain accurate estimates of mutation processes, phylogenetic relationships and historical times of activity.

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.