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

Abstract

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.

Multiple layers of complexity in cis‐regulatory regions of developmental genes

“…In recent years, numerous cis-regulatory regions of animal genes have been shown to contain multiple enhancers with overlapping or very similar expression patterns (Jeong et al, 2006;Werner et al, 2007;Hong et al, 2008;McGaughey et al, 2008;Corbo et al, 2010;Frankel et al, 2010;Kalay and Wittkopp, 2010;L'Honore et al, 2010;Naranjo et al, 2010;Perry et al, 2010Perry et al, , 2011Franchini et al, 2011;Galindo et al, 2011;Ghiasvand et al, 2011;Lee et al, 2011;McBride et al, 2011;Watts et al, 2011;Pauls et al, 2012). Yet, the first observations of overlapping expression from different enhancers date back from the 1990s (Hoch et al, 1990;Kassis, 1990;Tolias and Kafatos, 1990).…”

Enhancer turnover and conserved regulatory function in vertebrate evolution

“…These neuronal Pomc Enhancers, that we named nPE1 and nPE2, are located at 212 and 210 kb relative to the mouse transcription start site (TSS) [60]. While nPE2 is found in monotremes (egg-laying mammals) and marsupials, as well as placental mammals, nPE1 is only found in placentals [46,47]. No sequences similar to either nPE enhancer can be identified in non-mammalian genomes.…”

“…No sequences similar to either nPE enhancer can be identified in non-mammalian genomes. A detailed phylogenetic analysis of both enhancers revealed that nPE1 and nPE2 are derived from unrelated ancient transposable elements (TEs) that were independently co-opted-or exapted-at different times during early mammalian evolution [46,47,61]. nPE2 was derived from a CORE-SINE retroposon before the mammalian radiation [46], whereas nPE1 is derived from an MaLR retroposon inserted at the time of the placental mammal diversification [47].…”

“…Apart from turnover of TFBS sequence within enhancers, evolution can also lead to the appearance of non-orthologous novel enhancers and, eventually, to enhancer turnover with complete functional loss of the ancestral sequence (figure 1). In practice, however, it is difficult to be certain whether the lack of overt sequence identity of such non-conserved mammal-fish enhancers is the result of TFBS sequence turnover in orthologous enhancers or due to an enhancer appearing de novo, unless a detailed knowledge of the evolutionary history of the enhancer in at least one of the two lineages is found [46,47]. Here, we show that two mouse enhancers of the proopiomelanocortin gene (Pomc) that certainly appeared in the mammalian lineage direct appropriate reporter gene expression to homologous neurons in the ventromedial hypothalamus of transgenic zebrafish.…”

17α‐estradiol acts through hypothalamic pro‐opiomelanocortin expressing neurons to reduce feeding behavior

“…Cooperative interactions between two POMC‐neuron‐specific enhancers, nPE1 and nPE2, promote expression of Pomc transcripts in the mouse ventromedial hypothalamus (Franchini et al., 2011; de Souza et al., 2005). Deletion of nPE2, nPE1, or insertion of a transcription‐blocking neo selection cassette into the vicinity of the two hypothalamic neuronal Pomc enhancers reduces hypothalamic Pomc expression to ~80%, ~30%, or ~2% of wild‐type controls, respectively (Lam et al, 2015).…”