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Books By Writers Here. Who's Online? For example, nearly a third of human coding bases 11 Mb out of 34 Mb align to the amphioxus genome. Species are ordered at progressively greater evolutionary distances. Placental mammal CNEs — placental mammal conserved non-coding elements. To screen for enhancers conserved between deuterostome and protostome species, we first defined a set of vertebrate conserved non-coding elements vertCNEs which are human non-coding regions well-conserved in at least a subset of vertebrates see methods.
In total, we constructed a conservative set of 8, vertCNEs. We were especially stringent in filtering out sequences with evidence for potential coding functions exonic or other functional RNA. We used lastz [14] to query these elements against three previously published non-vertebrate deuterostome genomes — ciona [15] , amphioxus [16] , and sea urchin [17] Figure S1. We filtered hits for both quality of alignment and for conserved gene synteny.
We thus found five candidate elements showing conservation between vertebrates and at least one invertebrate Table S2. In order to comprehensively define the extent of conservation of these elements, we searched each against all publicly available sequence data for non-vertebrate metazoans.
Of the five candidates, two elements stood out as having conservation of sequence and gene synteny in protostome species. We call these elements bilaterian conserved regulatory elements Bicores.
Bicore1 is found in two protostome species - Aplysia californica sea hare and Lottia gigantea owl limpet. Bicore2 is found in the protostome Ixodes scapularis tick Figure 2. Together, Bicore1 and Bicore2 are the first examples of human CNEs conserved between deuterostome and protostome species. A Pattern of conservation of Bicores across the metazoan tree.
Green checks denote conservation of sequence and gene synteny. Red crosses denote no detectable sequence conservation. B Characteristics of Bicores. CNS — central nervous system. Id genes encode helix-loop-helix proteins that bind bHLH transcription factors, acting as transcriptional repressors.
Id factors are known to inhibit cells from terminally differentiating, promoting progenitor states [18]. Mammalian genomes contain four Id genes. Bicore1 occurs upstream of Id1. A Multiple alignment of Bicore1. B Conservation profile of Bicore1 showing that conserved blocks in the alignment correspond to transcription factor binding preferences.
C Each instance of Bicore1 green oval is syntenic to a conserved Id gene blue gene structure. D—G Zebrafish transgenic assay showing D human, E zebrafish, F sea urchin, G owl limpet instances of Bicore1 drive expression in the central nervous system at 48 hours. H Whole-mount in situ hybridization of id1 in zebrafish shows expression throughout the central nervous system, courtesy of zfin.
I A human genomic region containing Bicore1 drives expression in the forebrain, midbrain, hindbrain, neural tube, and eye of embryonic day J A sea urchin genomic region drives expression in the aboral ectoderm arrow in the early pluteus stage sea urchin larva.
K Pairwise percent identities of Bicore1 sequences. Green cells indicate a query sequence row that detected Bicore1 in the target column. Red cells indicate query sequences that did not detect Bicore1 in the target. A multiple alignment of Bicore1 shows a striking pattern of sequence conservation Figure 3A—3B. Short stretches of 5—10 base pairs are highly conserved, separated by stretches of non-conserved sequence.
The highly conserved 5—10mers have allowed few substitutions and have completely resisted indels across species. These short conserved sequences match closely to the known binding preference of transcription factors Figure 3B.
Two conserved sites match the primary and secondary binding preference of Smad transcription factors. These studies further showed that a region containing Bicore1 drives expression of a luciferase reporter in response to Bmp.
Mutation of the Smad sites reduces or abolishes this Bmp response [20] , [21]. We also see conserved matches to the E2f binding motif. E2f factors are known to act as Smad cofactors in response to Tgf-beta [22]. Mutations of these conserved E2f sites reduce Bmp responsiveness in a luciferase assay [20] , [21].
ChIP-seq data also supports binding of E2f factors to this region [23]. The most highly conserved region of Bicore1 corresponds to an 8 base pair palindromic sequence that is perfectly conserved across clades. This sequence matches the binding preference of Atf factors. In mammalian cells, Atf forms a complex with Smad and directly binds Bicore1 [24].
This binding event occurs in response to Tgf-beta signaling and leads to the repression of a luciferase reporter. Mutation of the Atf site abolishes the ability of Bicore1 to repress luciferase expression in response to Tgf-beta [24]. Thus, Bicore1 has conserved a series of transcription factor binding sites that have maintained order, orientation, and spacing for over more than million years [25] of evolution. To test if instances of Bicore1 have a conserved capacity to function as an enhancer in vivo , we used a zebrafish transient transgenic enhancer assay.
We tested the human, zebrafish, sea urchin, and owl limpet Bicore1 sequences. At 21 hours post-fertilization hpf , the constructs drove strong expression throughout the embryo. Zebrafish id1 expression at this time point, measured by whole-mount in situ hybridization WISH , is similar Figure S3. At 48 hours, we found that all four constructs drove scattered expression throughout the central nervous system CNS Figure 3D—3G and Table S6 , congruent with id1 expression at this time [26] Figure 3H.
In addition to CNS, we saw expression in the notochord, a structure in which previous WISH experiments have not detected id1 expression.
It is possible that Bicore1 enhances the weak notochord background of our expression vector see methods; Figure S4. Further support for Bicore1 functioning as a CNS enhancer in vertebrates is provided by mouse experiments.
A human construct containing Bicore1 was previously tested in a mouse transgenic enhancer assay [8]. At embryonic day Among the protostome species that have conserved Bicore1 owl limpet and sea hare , transgenic enhancer assays are not yet well developed.
However, such assays have been described in sea urchin [28]. We tested a construct containing the sea urchin Bicore1 in a transgenic sea urchin assay. Whole-mount in situ hybridization experiments in a closely related sea urchin species have shown that id is expressed specifically in the aboral ectoderm during sea urchin development.
Moreover, overexpressing Bmp expands the id expression pattern, and blocking Bmp signaling greatly diminishes id expression [29]. These data provide evidence that Bicore1 functions as a developmental enhancer of id in sea urchins.
Bicore2 is conserved upstream of Znf Figure 4C ; Table S5 , a gene which encodes a zinc-finger transcription factor predicted to act as a transcriptional repressor in deuterostomes and protostomes [30] , [31]. Znf functions as a regulator of vertebrate hindbrain development [32] , [33]. Among vertebrates, these factors are known to form a complex that functions during hindbrain development [34] , [35].
These factors are also known to interact in protostome species [36] , [37]. In both deuterostomes and protostomes, Wnt signaling defines the anterior-posterior axis during development [38]. In vertebrates, Wnt signaling has also been shown to be necessary in defining the midbrain-hindbrain boundary [39].
A Multiple alignment of Bicore2. B Conservation profile of Bicore2 showing that conserved blocks in the alignment correspond to transcription factor binding preferences. C Each instance of Bicore2 green oval is syntenic to a conserved instance of Znf blue gene structure. D—G Zebfrafish transgenic enhancer assay showing D human, E zebrafish, F sea urchin, G tick instances of Bicore2 drive expression in the hindbrain at 48 hours.
H Whole-mount in situ hybridization of Znf shows expression in the hindbrain, courtesy of zfin. I A human region containing Bicore2 drives expression in the hindbrain and the apical ectoderm of the limb in embryonic day J Pairwise percent identities of Bicore2 sequences.
Green cells indicate a query sequence row that detected Bicore2 in the target column. Red cells indicate query sequences that did not detect Bicore2 in the target. We examined human, zebrafish, sea urchin, and tick versions of Bicore2 in a zebrafish transient transgenic enhancer assay.
All four constructs drove consistent expression in the hindbrain Figure 4D—4G , recapitulating the zebrafish znf expression pattern [26] Figure 4H. Interestingly, the zebrafish Bicore2 stood out as having the weakest expression. Zebrafish also stands out in the multiple alignment as having mutated three highly conserved bases, interrupting a predicted Meis-Pbx-Hox binding site Figure 4A—4B.
It is possible that in some fish, Bicore2 function has been modulated. We also tested a human construct containing Bicore2 in a mouse assay. This pattern of hindbrain and limb expression matches previously reported Znf expression in mouse [40]. In this study, we have identified the first examples of cis -regulatory sequence conserved between deuterostomes and protostomes.
These bilaterian conserved regulatory elements Bicores are developmental enhancers that encode complex patterns of transcription factor binding sites. Bicore1 is an enhancer of Id. Binding site analysis predicts that it functions as a Bmp responsive element, and several lines of experimental evidence support this prediction.
In vertebrates, Bicore1 drives expression in the developing central nervous system. In sea urchin, Bicore1 drives expression in the aboral ectoderm, a structure that goes on to form the squamous epithelium of the late larval wall [41]. Although the vertebrate nervous system and the urchin aboral ectoderm are unlikely to be homologous structures, it is reasonable to hypothesize that they might utilize similar genetic circuits. Both are ectodermal structures that define analogous axes dorsal-ventral and aboral-oral through Bmp signaling.
Further work will be needed to determine how Bicore1 functions in those protostome species that have conserved the sequence. We suspect that protostomes like owl limpet and sea hare also use Bicore1 as a Bmp responsive enhancer to drive id expression in ectodermal structures. As enhancer assays are developed in these species, we can begin testing this hypothesis. It is interesting to note that in Drosophila melanogaster , Emc ortholog to Id is not expressed in the ectoderm during development [42].
Further, constitutively active Dpp ortholog to Bmp signaling does not alter Emc expression [43]. Loss of Bicore1 in drosophila is consistent with these observations. Bicore2 is an enhancer of Znf We predict from binding site analysis that it acts as a response element to Wnt signaling. In vertebrate embryos, Bicore2 drives expression in the hindbrain, an ectoderm derived structure. It may seem surprising that CNS enhancers are conserved in species that lack central nervous systems.
In fact, we can infer that Bicores existed in the urbilaterian ancestor, long before the process of neurulation and the vertebrate central nervous system ever emerged. It is well established that as the vertebrate nervous system evolved, it took advantage of preexisting transcription factors and signaling pathways [44] , [45].
We can now appreciate that in addition to these ancient genes, ancient cis -regulatory integrators, in the form of rigid enhancers, were also coopted into vertebrate nervous system development. In fact, it is expected that when key regulatory genes and pathways are activated in a new context, they initially affect downstream targets via pre-existing cis-regulatory regions.
Several past studies have searched for cis -regulatory elements conserved between deuterostomes and protostomes see Text S1. However, these studies focused on searching the genomes of the most commonly used protostome model organisms, drosophila and caenorhabditis. These model organisms correspond to two of the three lineages tunicates, insects, and nematode in which we could detect no Bicore homologies.
Thus, it is possible that Bicores have been lost in the tunicate, insect, and nematode lineages. Corroborating this possibility, genomics studies have shown that these three lineages are perhaps the most diverged among bilaterians [46] — [48].
Even within the protostome genomes that have clear conservation of Bicores, these homologies lie at the cusp of what current computational tools can detect. For example, using human Bicore1 as a query, we can detect Bicore1 deuterostome orthologs in amphioxus, sea urchin, and acorn worm.
However, we miss the critical protostome owl limpet and sea hare elements. Using zebrafish Bicore1 as the query, we detect the owl limpet element but miss the sea hare.
Using amphioxus Bicore1, we detect the sea hare element but miss the owl limpet Figure 3K. We see similar results for Bicore2 Figure 4J. While the full-length Bicores are not identical or ultraconserved even between human and rodents, the binding sites they encode have resisted substitutions, insertions, deletions, and rearrangements for over million years in highly diverged deuterostome and protostome species.
At least two fundamental questions are raised by these observations: First, have Bicores conserved their ancestral sequence while being independently co-opted in different lineages to serve unrelated contexts, or do these conserved sequences also conserve a common ancestral function e.
The second closely related question is what makes Bicores unique? The small number of identified Bicores implies extensive cis-regulatory rewiring. If the Bicores are indeed the only examples of cis-regulatory elements conserved between deuterostomes and protostomes, we are left asking what makes these enhancers different from others. It is, however, currently hard to know what the true number of Bicores is.
More than million years ago, a major group of animals called the bilaterians, animals with bilateral symmetry, underwent an evolutionary event in which they diverged into two groups, the protostomes and the deuterostomes. The deuterostomes include the vertebrates, comprising fish, birds, amphibians, reptiles and mammals, including humans.
The deuterostomes also include some less familiar animals such as sea urchins and starfish. The protostomes include several invertebrate groups such as insects, spiders, lobsters and flatworms. The worm species studied here belong to a special group within the protostomes called the lophotrochozoans. Despite their obscure sounding name, lophotrochozoans represent more than one third of known marine animals.
This group includes earthworms, leeches, snails, oysters, octopuses, and other invertebrate groups and they play many important ecological roles. They found that ribbon worms and horseshoe worms are evolutionarily closely related, despite looking very different.
Surprisingly, the researchers found that these worms, which are protostomes, share many gene families and gene arrangements with the deuterostomes, the group that includes the vertebrates. For example, they share genes that are involved in multicellularity and maintenance of the body's internal environment. They also share a common system for controlling head development; the same mechanism that controls vertebrate head patterning also controls the development of ribbon worm heads and horseshoe worm feeding tentacles.
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