Lucía F. Franchini

Exaptation of Transposable Elements into Novel Cis-Regulatory Elements: Is the Evidence Always Strong?

Transposable elements (TEs) are mobile genetic sequences that can jump around the genome from one location to another, behaving as genomic parasites. TEs have been particularly effective in colonizing mammalian genomes, and such heavy TE load is expected to have conditioned genome evolution. Indeed, studies conducted both at the gene and genome levels have uncovered TE insertions that seem to have been co-opted—or exapted—by providing transcription factor binding sites (TFBSs) that serve as promoters and enhancers, leading to the hypothesis that TE exaptation is a major factor in the evolution of gene regulation. Here, we critically review the evidence for exaptation of TE-derived sequences as TFBSs, promoters, enhancers, and silencers/insulators both at the gene and genome levels. We classify the functional impact attributed to TE insertions into four categories of increasing complexity and argue that so far very few studies have conclusively demonstrated exaptation of TEs as transcriptional regulatory regions. We also contend that many genome-wide studies dealing with TE exaptation in recent lineages of mammals are still inconclusive and that the hypothesis of rapid transcriptional regulatory rewiring mediated by TE mobilization must be taken with caution. Finally, we suggest experimental approaches that may help attributing higher-order functions to candidate exapted TEs.

Convergent evolution of two mammalian neuronal enhancers by sequential exaptation of unrelated retroposons

The proopiomelanocortin gene (POMC) is expressed in a group of neurons present in the arcuate nucleus of the hypothalamus. Neuron-specific POMC expression in mammals is conveyed by two distal enhancers, named nPE1 and nPE2. Previous transgenic mouse studies showed that nPE1 and nPE2 independently drive reporter gene expression to POMC neurons. Here, we investigated the evolutionary mechanisms that shaped not one but two neuron-specific POMC enhancers and tested whether nPE1 and nPE2 drive identical or complementary spatiotemporal expression patterns. Sequence comparison among representative genomes of most vertebrate classes and mammalian orders showed that nPE1 is a placental novelty. Using in silico paleogenomics we found that nPE1 originated from the exaptation of a mammalian-apparent LTR retrotransposon sometime between the metatherian/eutherian split (147 Mya) and the placental mammal radiation (≈90 Mya). Thus, the evolutionary origin of nPE1 differs, in kind and time, from that previously demonstrated for nPE2, which was exapted from a CORE-short interspersed nucleotide element (SINE) retroposon before the origin of prototherians, 166 Mya. Transgenic mice expressing the fluorescent markers tomato and EGFP driven by nPE1 or nPE2, respectively, demonstrated coexpression of both reporter genes along the entire arcuate nucleus. The onset of reporter gene expression guided by nPE1 and nPE2 was also identical and coincidental with the onset of Pomc expression in the presumptive mouse diencephalon. Thus, the independent exaptation of two unrelated retroposons into functional analogs regulating neuronal POMC expression constitutes an authentic example of convergent molecular evolution of cell-specific enhancers.

The Developmental Brain Gene NPAS3 Contains the Largest Number of Accelerated Regulatory Sequences in the Human Genome

To identify the evolutionary genetic novelties that contributed to shape human-specific traits such as the use of a complex language, long-term planning and exceptional learning abilities is one of the ultimate frontiers of modern biology. Evolutionary signatures of functional shifts could be detected by comparing noncoding regions that are highly conserved across mammals or primates and rapidly accumulated nucleotide substitutions only in the lineage leading to humans. As gene loci densely populated with human-accelerated elements (HAEs) are more likely to have contributed to human-specific novelties, we sought to identify the transcriptional units and genomic 1 Mb intervals of the entire human genome carrying the highest number of HAEs. To this end, we took advantage of four available data sets of human genomic accelerated regions obtained through different comparisons and algorithms and performed a meta-analysis of the combined data. We found that the brain developmental transcription factor neuronal PAS domain-containing protein 3 (NPAS3) contains the largest cluster of noncoding-accelerated regions in the human genome with up to 14 elements that are highly conserved in mammals, including primates, but carry human-specific nucleotide substitutions. We then tested the ability of the 14 HAEs identified at the NPAS3 locus to act as transcriptional regulatory sequences in a reporter expression assay performed in transgenic zebrafish. We found that 11 out of the 14 HAEs present in NPAS3 act as transcriptional enhancers during development, particularly within the nervous system. As NPAS3 is known to play a crucial role during mammalian brain development, our results indicate that the high density of HAEs present in the human NPAS3 locus could have modified the spatiotemporal expression pattern of NPAS3 in the developing human brain and, therefore, contributed to human brain evolution.

The voltage-gated potassium channel subfamily KQT member 4 (KCNQ4) displays parallel evolution in echolocating bats

Bats are the only mammals that use highly developed laryngeal echolocation, a sensory mechanism based on the ability to emit laryngeal sounds and interpret the returning echoes to identify objects. Although this capability allows bats to orientate and hunt in complete darkness, endowing them with great survival advantages, the genetic bases underlying the evolution of bat echolocation are still largely unknown. Echolocation requires high-frequency hearing that in mammals is largely dependent on somatic electromotility of outer hair cells. Then, understanding the molecular evolution of outer hair cell genes might help to unravel the evolutionary history of echolocation. In this work, we analyzed the molecular evolution of two key outer hair cell genes: the voltage-gated potassium channel gene KCNQ4 and CHRNA10, the gene encoding the α10 nicotinic acetylcholine receptor subunit. We reconstructed the phylogeny of bats based on KCNQ4 and CHRNA10 protein and nucleotide sequences. A phylogenetic tree built using KCNQ4 amino acid sequences showed that two paraphyletic clades of laryngeal echolocating bats grouped together, with eight shared substitutions among particular lineages. In addition, our analyses indicated that two of these parallel substitutions, M388I and P406S, were probably fixed under positive selection and could have had a strong functional impact on KCNQ4. Moreover, our results indicated that KCNQ4 evolved under positive selection in the ancestral lineage leading to mammals, suggesting that this gene might have been important for the evolution of mammalian hearing. On the other hand, we found that CHRNA10, a gene that evolved adaptively in the mammalian lineage, was under strong purifying selection in bats. Thus, the CHRNA10 amino acid tree did not show echolocating bat monophyly and reproduced the bat species tree. These results suggest that only a subset of hearing genes could underlie the evolution of echolocation. The present work continues to delineate the genetic bases of echolocation and ultrasonic hearing in bats.

A fast-evolving human NPAS3 enhancer gained reporter expression in the developing forebrain of transgenic mice

The developmental brain gene NPAS3 stands out as a hot spot in human evolution because it contains the largest number of human-specific, fast-evolving, conserved, non-coding elements. In this paper we studied 2xHAR142, one of these elements that is located in the fifth intron of NPAS3. Using transgenic mice, we show that the mouse and chimp 2xHAR142 orthologues behave as transcriptional enhancers driving expression of the reporter gene lacZ to a similar NPAS3 expression subdomain in the mouse central nervous system. Interestingly, the human 2xHAR142 orthologue drives lacZ expression to an extended expression pattern in the nervous system. Thus, molecular evolution of 2xHAR142 provides the first documented example of human-specific heterotopy in the forebrain promoted by a transcriptional enhancer and suggests that it may have contributed to assemble the unique properties of the human brain.

Enhancer turnover and conserved regulatory function in vertebrate evolution

Mutations in regulatory regions including enhancers are an important source of variation and innovation during evolution. Enhancers can evolve by changes in the sequence, arrangement and repertoire of transcription factor binding sites, but whole enhancers can also be lost or gained in certain lineages in a process of turnover. The proopiomelanocortin gene (Pomc), which encodes a prohormone, is expressed in the pituitary and hypothalamus of all jawed vertebrates. We have previously described that hypothalamic Pomc expression in mammals is controlled by two enhancers—nPE1 and nPE2—that are derived from transposable elements and that presumably replaced the ancestral neuronal Pomc regulatory regions. Here, we show that nPE1 and nPE2, even though they are mammalian novelties with no homologous counterpart in other vertebrates, nevertheless can drive gene expression specifically to POMC neurons in the hypothalamus of larval and adult transgenic zebrafish. This indicates that when neuronal Pomc enhancers originated de novo during early mammalian evolution, the newly created cis- and trans-codes were similar to the ancestral ones. We also identify the neuronal regulatory region of zebrafish pomca and confirm that it is not homologous to the mammalian enhancers. Our work sheds light on the process of gene regulatory evolution by showing how a locus can undergo enhancer turnover and nevertheless maintain the ancestral transcriptional output.

Phylogenetic differences in calcium permeability of the auditory hair cell cholinergic nicotinic receptor

The α9 and α10 cholinergic nicotinic receptor subunits assemble to form the receptor that mediates efferent inhibition of hair cell function within the auditory sensory organ, a mechanism thought to modulate the dynamic range of hearing. In contrast to all nicotinic receptors, which serve excitatory neurotransmission, the activation of α9α10 produces hyperpolarization of hair cells. An evolutionary analysis has shown that the α10 subunit exhibits signatures of positive selection only along the mammalian lineage, strongly suggesting the acquisition of a unique function. To establish whether mammalian α9α10 receptors have acquired distinct functional properties as a consequence of this evolutionary pressure, we compared the properties of rat and chicken recombinant and native α9α10 receptors. Our main finding in the present work is that, in contrast to the high (pCa2+/pMonovalents ∼10) Ca2+ permeability reported for rat α9α10 receptors, recombinant and native chicken α9α10 receptors have a much lower permeability (∼2) to this cation, comparable to that of neuronal α4β2 receptors. Moreover, we show that, in contrast to α10, α7 as well as α4 and β2 nicotinic subunits are under purifying selection in vertebrates, consistent with the conserved Ca2+ permeability reported across species. These results have important consequences for the activation of signaling cascades that lead to hyperpolarization of hair cells after α9α10 gating at the cholinergic–hair cell synapse. In addition, they suggest that high Ca2+ permeability of the α9α10 cholinergic nicotinic receptor might have evolved together with other features that have given the mammalian ear an expanded high-frequency sensitivity.

Genomic approaches to studying human-specific developmental traits.

Changes in developmental regulatory programs drive both disease and phenotypic differences among species. Linking human-specific traits to alterations in development is challenging, because we have lacked the tools to assay and manipulate regulatory networks in human and primate embryonic cells. This field was transformed by the sequencing of hundreds of genomes – human and non-human – that can be compared to discover the regulatory machinery of genes involved in human development. This approach has identified thousands of human-specific genome alterations in developmental genes and their regulatory regions. With recent advances in stem cell techniques, genome engineering, and genomics, we can now test these sequences for effects on developmental gene regulation and downstream phenotypes in human cells and tissues. Summary: As discussed in this Review, genome sequencing and functional genomic approaches enable analysis of the evolutionary and developmental origin of traits unique to our species.

Can a few non-coding mutations make a human brain?

The recent finding that the human version of a neurodevelopmental enhancer of the Wnt receptor Frizzled 8 (FZD8) gene alters neural progenitor cell cycle timing and brain size is a step forward to understanding human brain evolution. The human brain is distinctive in terms of its cognitive abilities as well as its susceptibility to neurological disease. Identifying which of the millions of genomic changes that occurred during human evolution led to these and other uniquely human traits is extremely challenging. Recent studies have demonstrated that many of the fastest evolving regions of the human genome function as gene regulatory enhancers during embryonic development and that the human‐specific mutations in them might alter expression patterns. However, elucidating molecular and cellular effects of sequence or expression pattern changes is a major obstacle to discovering the genetic bases of the evolution of our species. There is much work to do before human‐specific genetic and genomic changes are linked to complex human traits.

Positive selection of co-opted mobile genetic elements in a mammalian gene: If you can’t beat them, join them

The proopiomelanocortin (Pomc) gene encodes a prepropeptide with essential functions in the response to stress and energy balance, which is expressed in the pituitary and hypothalamus of vertebrate animals. Neuronal expression of Pomc is controlled by two distal enhancers named nPE1 and nPE2. Using transgenic mice, we observed that both enhancers drive identical expression patterns in the mammalian hypothalamus, starting at embryonic day 10.5, when endogenous Pomc expression commences. This overlapping enhancer activity is maintained throughout hypothalamic development and into adulthood. We also found that nPE1 and nPE2 were exapted as neuronal enhancers into the POMC locus after the sequential insertion of two unrelated retroposons. Thus, nPE1 and nPE2 are functional analogs and represent an authentic first example of convergent molecular evolution of cell-specific transcriptional enhancers. In this Commentary we discuss the following questions that remain unanswered: (1) how does transcriptional control of POMC operate in hypothalamic neurons of non-mammalian vertebrates? (2) What evolutionary forces are maintaining two discrete neuronal POMC enhancers under purifying selection for the last ~100 million years in all placental mammals? (3) What is the contribution of MaLRs to genome evolution?