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Link to original content: https://pubmed.ncbi.nlm.nih.gov/17922573
Ancient exaptation of a CORE-SINE retroposon into a highly conserved mammalian neuronal enhancer of the proopiomelanocortin gene - PubMed Skip to main page content
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. 2007 Oct;3(10):1813-26.
doi: 10.1371/journal.pgen.0030166.

Ancient exaptation of a CORE-SINE retroposon into a highly conserved mammalian neuronal enhancer of the proopiomelanocortin gene

Andrea M Santangelo  1 , Flávio S J de Souza, Lucía F Franchini, Viviana F Bumaschny, Malcolm J Low, Marcelo Rubinstein
Affiliations

Ancient exaptation of a CORE-SINE retroposon into a highly conserved mammalian neuronal enhancer of the proopiomelanocortin gene

Andrea M Santangelo et al. PLoS Genet. 2007 Oct.

Abstract

The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5' distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution.

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Conflict of interest statement

Competing interests. FSJdS, MJL, and MR have intellectual property and patent interests in the POMC neuronal-specific enhancers and have received income from the licensing of this intellectual property and related research material to financially interested companies.

Figures

Figure 1
Figure 1. Evolutionary Divergence around Mammalian nPE2 Sequences
(A) Phylogenetic relationships among the orthologous nPE2 enhancers of mammals, including monotremes and marsupials. Branches and nodes are displayed with different colors and arbitrary numbers are indicated on branches for quick reference. Note that this tree was built using the phylogenetic relationships among nPE2 sequences and it is not congruent with the phylogenetic trees of the respective species. (B) Evolutionary divergence sliding-window plot of nPE2. The histogram shows the number of substitutions per site along each branch of the phylogeny displayed in (A), estimated every ten bases in 50-base intervals. Each layer of the plot represents a branch or node of the tree and colors are maintained with respect to the tree. Substitutions were estimated from the best-fit maximum likelihood model, which incorporates unequal equilibrium nucleotide frequencies and unequal rates of transitional and transversional substitutions (K80). The histograms are stacked so that the total height represents the density of substitutions over the entire phylogeny. Between the two black arrows we indicate the most conserved 160 bp that define nPE2. The five regions into which mouse nPE2 was divided for the deletional analysis are indicated with numbered rectangles. The black regions 1 and 3 were demonstrated to be critical for nPE2 enhancer activity (see Figure 4).
Figure 2
Figure 2. ClustalX Alignment of 16 Mammalian Orthologous nPE2 Sequences
Residues highlighted in black and grey indicate different levels of conservation among the orthologs. Mouse nPE2 was subdivided for a deletional analysis into five partially overlapping regions indicated by the brackets and numbered from 1 to 5 within circles. Ms, marsupials, Mt, monotremes.
Figure 3
Figure 3. Spatiotemporal lacZ Expression Pattern Driven by nPE2 during Embryogenesis
A transgene was constructed containing 1.4 kb of mouse Pomc upstream sequences including nPE2 (red) ligated to the chicken minimal β-globin promoter (orange) followed by the E .coli lacZ reporter gene (blue) and the polyadenylation site from SV40 T antigen (yellow). Embryonic stages (dpc) are indicated on the left. (A, C, F, I) whole-mount lacZ staining. (B, D, G, J) lacZ staining of sagital sections. The boxes indicate the areas enlarged in the right panels. (E, H, K) magnified views of the diencephalon-pituitary region after combined enzymatic histochemical lacZ staining (blue) and anti-ACTH immunostaining (brown). Note that lacZ staining is prominent in the basal diencephalon (bd) and later in the basal hypothalamus (bh) but not in Rathke's pouch (Rp) or later in the pituitary (pit). Ectopic expression in somites is present at all developmental stages.
Figure 4
Figure 4. Deletional Analysis of nPE2 Performed in Transgenic Mice
(A) Expression analysis of nPE2 deletion constructs in representative animals. Coronal sections at the level of the arcuate nucleus of mice transgenic for each deletion construct, as indicated (WT, Δ1–Δ5). Sections were subjected to lacZ staining (blue) followed by anti-ACTH immunostaining (brown) to detect endogenous POMC expression. Left panels show low power views of half an arcuate (10×) and right panels are magnified to show individual POMC neurons (40×). Inset on the left panel of deletion 3 shows ectopic lacZ expression in the cerebral cortex of the same brain section, which lacks arcuate expression. For more information see Table S1. (B) Position of the five different deletions (Δ1–Δ5) in relation to WT nPE2 are shown at the left and summaries of lacZ expression data obtained for each transgene at embryonic day 14.5 and adulthood are shown at the right. Arc+, coexpression of lacZ with ACTH immunopositive POMC neurons in the arcuate nucleus; arc−, absence of coexpression in POMC neurons; ect, ectopic expression of lacZ.
Figure 5
Figure 5. POMC Neuronal Enhancer nPE2 Is a CORE-SINE–Derived Sequence
Sequence alignment of the opossum and wallaby nPE2 enhancer sequences and three marsupial CORE-SINE consensus sequences from RepBase: MAR1 MD, MAR1a MD, and a representative nested MAR1 located at Chromosome 3 of the opossum genome (according to UCSC Genome Browser). CORE-SINE functional regions are indicated at the top of the schematic. The shading of the alignment is based on the identity of residues and shows percentage of conservation within each column. One hundred percent identical aligned nucleotides are shaded in black. More than 80% conservation is depicted in dark grey shade. Columns with less than 80% and more than 60% conservation are shaded in light grey, whereas nucleotides with less than 60% are not shaded. Sequences corresponding to boxes A and B are depicted in blue boxes, whereas core sequences are within a red box. Black asterisks are presented at 20-nucleotide intervals. The red asterisk indicates the location of the first core sequence of the nested MAR1 that was removed to simplify the figure.
Figure 6
Figure 6. MAR1s Have Been Active Recently in the Opossum Genome
(A) Core alignment of the ten highest identity instances of MAR1 in the opossum genome. Chromosome locations are given according to the January 2006 opossum (Monodelphis domestica) draft assembly genome. (B) Selected examples of target site duplications, the chromosome location indicated corresponds to the MAR1 elements shown in part A. Conserved nucleotides at both sides of the duplicated site generated by the retroposon insertion are indicated in capital letters. (C) Alignment of the 3′ end of MAR1, a Bov-B LINE, and RTE3 MD consensus sequences from RepBase and RTE3 instances found in the opossum genome.
Figure 7
Figure 7. Examples of Additional Exapted CORE SINEs in Mammalian Genomes
(A) The locations of nine CORE SINE–derived sequences that overlap with ultraconserved elements predicted by the PhasCons program were determined using the Table Browser at the UCSC Genome Bioinformatics website. The position information of the CORE-SINE–derived sequences and the overlapped ultraconserved elements in human were obtained from genomic sequence data in UCSC Genome Bioinformatics Web site (ver. hg17). Note that these exapted elements are annotated as MIRs in the human genome. The type of sequence is indicated in red in the middle column: IG, intergenic; IN, intronic; EX, exon. The example highlighted within a red rectangle has been detected as an exapted element in [16]. (B) An example of a highly conserved element derived from a CORE SINE located between exons 4 and 5 of ZNF384 at Chromosome 12 in humans (chr12:6,656,542–6,656,608 ). Note that the core-derived sequence is conserved in all mammalian ZNF384 orthologs including opossum.
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