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Dev Biol
2014 Jun 15;3902:261-72. doi: 10.1016/j.ydbio.2014.03.013.
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Dissection of a Ciona regulatory element reveals complexity of cross-species enhancer activity.
Chen WC
,
Pauls S
,
Bacha J
,
Elgar G
,
Loose M
,
Shimeld SM
.
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Vertebrate genomes share numerous conserved non-coding elements, many of which function as enhancer elements and are hypothesised to be under evolutionary constraint due to a need to be bound by combinations of sequence-specific transcription factors. In contrast, few such conserved elements can be detected between vertebrates and their closest invertebrate relatives. Despite this lack of sequence identity, cross-species transgenesis has identified some cases where non-coding DNA from invertebrates drives reporter gene expression in transgenic vertebrates in patterns reminiscent of the expression of vertebrate orthologues. Such instances are presumed to reflect the presence of conserved suites of binding sites in the regulatory regions of invertebrate and vertebrate orthologues, such that both regulatory elements can correctly interpret the trans-activating environment. Shuffling of binding sites has been suggested to lie behind loss of sequence conservation; however this has not been experimentally tested. Here we examine the underlying basis of enhancer activity for the Ciona intestinalis βγ-crystallin gene, which drives expression in the lens of transgenic vertebrates despite the Ciona lineage predating the evolution of the lens. We construct an interactive gene regulatory network (GRN) for vertebrate lens development, allowing network interactions to be robustly catalogued and conserved network components and features to be identified. We show that a small number of binding motifs are necessary for Ciona βγ-crystallin expression, and narrow down the likely factors that bind to these motifs. Several of these overlap with the conserved core of the vertebrate lensGRN, implicating these sites in cross species function. However when we test these motifs in a transgenic vertebrate they prove to be dispensable for reporter expression in the lens. These results show that current models depicting cross species enhancer function as dependent on conserved binding sites can be overly simplistic, with sound evolutionary inference requiring detailed dissection of underlying mechanisms.
Fig. 1. (A) Ciona tadpolelarva, with anterior to the left. The distinction between head (sometimes described as the trunk) and tail is shown. (B) A magnified view of the head, including the sensory vesicle in which the ocellus (oc) and otolith (ot) pigment cells lie, and the palps (p) at the anterior. (C)–(E)Ciona larvae electroporated with Ci-βγcrys pCES. A shows reporter expression in the palps. B shows reporter expression in the otolith. In C, expression includes the otolith but extends more towards the ocellus as described previously (Shimeld et al., 2005). (F, G) Localisation of Ci-βγcrys protein detected by immunohistochemistry. D shows a larval head imaged by DIC microscopy, and E a fluorescent image of the same larva with protein localised in palps and otolith. (H) Initial deletion analysis of the 1225 bp Ci-βγcrys 5′ regulatory region. At the top is the location of this region on scaffold 604 of the version 1C. intestinalis genome assembly (Dehal et al., 2002), between the divergently transcribed cubulin and Ci-βγcrys genes. Schematics of successive 200 bp deletions are underneath. Constructs showed transgene expression in palps and pigment cells in at least 50% of embryos (>50%), or no transgene expression was detected in these tissues in any embryo (0%). Each construct was tested in at least 2 independent electroporations, including concurrent positive and negative controls, with positive control transgenesis levels of at least 50% and with at least thirty surviving embryos per construct. TSS indicates the transcription start site, and numbering is from this point. (I) A transgenic larva with fluorescent reporter detected in palp and otolith. Only one palp is labelled, a common occurrence reflecting mosaicism.
Fig. 2. (A) Alignment of the orthologous Ci-βγcrys regulatory region from C. intestinalis and C. savignyi, with putative conserved binding motifs boxed. The names of these reflect the database entry (JASPAR or TRANSFAC) to which they match and do not necessarily mean that the specific factor binds this site in Ciona. Highest scores for each site are shown with all sites scoring >70%. Core sequences are highlighted in yellow. The translation start site is shown, as is the presumed transcription start site inferred from the 5′ extreme of cDNA sequence. Numbering starts from this point and refers to the C. intestinalis sequence. The start of the minimal −275 bp construct sequence is shown. (B, C) Fine scale deletion and mutation analysis of the Ciona βγ-crystallin minimal region defined as in Fig. 1. Presumptive binding motifs are represented as coloured boxes. (B) Successive approximately 20 bp deletions, each tested by electroporation. Only constructs spanning changes in reporter activity are shown. Controls and use of percentage signs are as in Fig. 1. (C) Result of mutagenesis of selected motifs (indicated by a cross). We targeted four motifs, chosen due to potential overlap with the vertebrate lens gene regulatory network (Sox, Fox and CREB) and/or because the deletion experiments indicated a function for the region in which they lay (Sox and Cdx). Results were more variable than for deletions; hence we show the number of reporter-expressing larvae alongside the total number of larvae that developed. Typical transgenesis rates in positive controls were around 50%.
Fig. 3. (A) The complete vertebrate mouse lens network as illustrated by myGRN. Developmental time flows from top left (7.5 dpc) to bottom right. The outermost layer of the network includes all those interactions that cannot be placed in a specific tissue or time point. Importantly genes appear only once within the network and so interactions that feed forward or back can be clearly seen. Dashed lines indicate interactions for which the existence of intermediates cannot be formally excluded. The circular symbols represent the type of gene: solid circles are transcription factors, solid asterisks are signals, inverted asterisks represent receptors and triangles illustrate terminal markers. This network is available in a fully interactive format at http://public.networks.mygrn.org/. An animated version of this network is provided as Supplementary movie 1. (B, C) Here the complete network is visualised with genes sized according to centrality within the network. Genes with more connections are larger. When including the crystallins, Pax6 and Maf emerge as key regulators (B). However, if the crystallins are excluded from the network, the relative importance of Maf is decreased and Foxe3 appears more important (C).
Fig. 4. (A) The network now shows only those genes with direct connexions to the crystallins. The size of the symbol representing each gene is proportional to the number of connexions it makes in the network. (B) A hypothetical Ciona ‘lens’ network derived from interactions occurring in the mouse. Note that several different candidates exist in Ciona for Integrin Beta-1 like. (C) The network now highlights those interactions required for the expression of Ci-βγcry, as predicted by myGRN based on the mouse lens network.
Fig. 5. GFP in zebrafish lens driven by the Ci-βγcry −275 bp construct (designated WT) and mutant binding site constructs. (A, B) Wild-type (WT) −275 bp constructs in two lines showing the whole fish. The lens is shown, as is the hindbrain (hb). (C, E) Wild-type (WT) −275 bp constructs in three separate lines, showing just the eye with the lens circled in C as a reference. (F)–(N) Lines with constructs with mutant (m) binding motifs as in Fig. 2C; mCDX1 (first Cdx motif mutated, 2 lines). mCDX2 (both Cdx motifs mutated, 2 lines). mCREB (CREB motif mutated, 2 lines). mFOX (Fox motif mutated, 1 line). mSOX (Sox motif mutated, 2 lines).
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