title: Dissection of a Ciona regulatory element reveals complexity of cross-species enhancer activity.
abstract: 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 lens GRN, 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.
authors.affiliation: Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK.
authors.forename: Wei-Chung
authors.initials: WC
authors.lastname: Chen
authors.affiliation: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pauls
authors.affiliation: Centre for Genetics and Genomics, School of Biology, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
authors.forename: Jamil
authors.initials: J
authors.lastname: Bacha
authors.affiliation: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Greg
authors.initials: G
authors.lastname: Elgar
authors.affiliation: Centre for Genetics and Genomics, School of Biology, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK. Electronic address: matt.loose@nottingham.ac.uk.
authors.forename: Matthew
authors.initials: M
authors.lastname: Loose
authors.affiliation: Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. Electronic address: sebastian.shimeld@zoo.ox.ac.uk.
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Shimeld
mesh_terms: D000818:Animals; D005075:Biological Evolution; D002645:Chickens; D002938:Ciona intestinalis / Q000235:genetics*; D003459:Crystallins / Q000235:genetics; D004252:DNA Mutational Analysis; D018274:Electroporation; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D053263:Gene Regulatory Networks / Q000235:genetics*; D018014:Gene Transfer Techniques; D007908:Lens, Crystalline / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D015027:Zebrafish
chemical_list: D003459:Crystallins; D014157:Transcription Factors
keywords: Ciona; Crystallin; Evolution; Gene regulatory network; Lens