Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2013 Jul 01;87:e68548. doi: 10.1371/journal.pone.0068548.
Show Gene links
Show Anatomy links
Interrogating transcriptional regulatory sequences in Tol2-mediated Xenopus transgenics.
Loots GG
,
Bergmann A
,
Hum NR
,
Oldenburg CE
,
Wills AE
,
Hu N
,
Ovcharenko I
,
Harland RM
.
???displayArticle.abstract???
Identifying gene regulatory elements and their target genes in vertebrates remains a significant challenge. It is now recognized that transcriptional regulatory sequences are critical in orchestrating dynamic controls of tissue-specific gene expression during vertebrate development and in adult tissues, and that these elements can be positioned at great distances in relation to the promoters of the genes they control. While significant progress has been made in mapping DNA binding regions by combining chromatin immunoprecipitation and next generation sequencing, functional validation remains a limiting step in improving our ability to correlate in silico predictions with biological function. We recently developed a computational method that synergistically combines genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to predict tissue-specific enhancers in the human genome. We applied this method to 270 genes highly expressed in skeletal muscle and predicted 190 putative cis-regulatory modules. Furthermore, we optimized Tol2 transgenic constructs in Xenopus laevis to interrogate 20 of these elements for their ability to function as skeletal muscle-specific transcriptional enhancers during embryonic development. We found 45% of these elements expressed only in the fast muscle fibers that are oriented in highly organized chevrons in the Xenopus laevis tadpole. Transcription factor binding site analysis identified >2 Mef2/MyoD sites within ~200 bp regions in 6 of the validated enhancers, and systematic mutagenesis of these sites revealed that they are critical for the enhancer function. The data described herein introduces a new reporter system suitable for interrogating tissue-specific cis-regulatory elements which allows monitoring of enhancer activity in real time, throughout early stages of embryonic development, in Xenopus.
???displayArticle.pubmedLink???
23874664
???displayArticle.pmcLink???PMC3713029 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. Characterizing minimal promoters for their ability to drive enhancer expression in a robust and reproducible manner.Three evolutionary conserved and previously characterized enhancers were examined as positive controls (A), in combination with 5 minimal promoters that have been previously employed in transgenic experiments in mice, zebrafish and frog (B). Frog embryos were examined for eye, kidney and muscle expression between stages 30–45 of embryonic development (C) in all enhancer-promoter combinations (D–G).
Figure 3. Validating computational predictions for muscle enhancers in Xenopus trangenics.Predicted SMEs corresponded to evolutionary conserved elements proximal or distal to genes known to function in skeletal muscle (A-F). Six SMEs consistently expressed in skeletal muscle, in addition to eye (A′–F′). Genomic regions are color coded as follows: exons (blue); UTRs (yellow); repeats (green); conserved noncoding sequences (red); predicted SMEs are shaded in purple.
Figure 4. TFBS analysis.Top 20 predicted SMEs were examined for the presence of Mef2 and MyoD clusters. We found clusters of >2 Mef2/MyoD sites over regions >200 bp in all 6 SMEs shown to drive muscle expression in combination with γ-cry promoter (red) and 1/3 of the additional 3 enhancers that were found to drive muscle expression in combination with krt8 promoter only (orange). Two of the negative elements also displayed this cluster (SME6/SME20), but none of the enhancers found to express in tissues other than muscle (green).
Figure 5. Mef2C/MyoD sites are essential for SME8/16 muscle specific expression.Tol2 constructs containing tandem kidney (KE) and muscle (SME) enhancers in front of γ-cry promoter (A) were systematically mutated to remove the predicted Mef2 and MyoD sites (B, H), and compared to the ‘wildtype’ construct in transgenic efficiency and tissue specificity (C, H), as well as expression intensity (G, M). Mutating either Mef2C or MyoD sites reduced the number of embryos expressing in muscle, as well as reduced the expression intensity (D-F; I-K; G, M). [*p-value <0.05; **p-value <0.001].
Figure 1. Comparing fluorescent reporter expression and transgenic efficiency in transgenic embryos generated by nuclear transfer, PiggyBAC or Tol2 transgenesis.Six red fluorescent proteins were examined for bright expression in Xenopus oocytes (A). Using the brightest red fluorescent gene, Katushka, constructs with a ubiquitous promoter (CMV) were examined by three methods: nuclear transfer, PiggyBAC transposition and Tol2 transposition. 250 embryos were injected in four independent experiments using CMV-Katushka constructs, and the number of surviving and transgenic embryos were assessed (Table S1); significantly fewer transgenic embryos (p-value <0.005) were generated by nuclear transfer and PiggyBAC, where large numbers of the surviving Tol2 embryos were transgenic (B). Tol2 transgenics were found to give the brightest and most reliable expression in Xenopus (C).
Allende,
Cracking the genome's second code: enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos.
2006, Pubmed,
Xenbase
Allende,
Cracking the genome's second code: enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos.
2006,
Pubmed
,
Xenbase
Amaya,
Production of transgenic Xenopus laevis by restriction enzyme mediated integration and nuclear transplantation.
2010,
Pubmed
,
Xenbase
Balciunas,
Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.
2006,
Pubmed
Belay,
Transposon-mediated gene transfer into adult and induced pluripotent stem cells.
2011,
Pubmed
Birnbaum,
Coding exons function as tissue-specific enhancers of nearby genes.
2012,
Pubmed
Brancaccio,
Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload.
2003,
Pubmed
Clark,
Transgenic zebrafish using transposable elements.
2011,
Pubmed
Collette,
Targeted deletion of Sost distal enhancer increases bone formation and bone mass.
2012,
Pubmed
Conte,
Promoter competition as a mechanism of transcriptional interference mediated by retrotransposons.
2002,
Pubmed
De Repentigny,
The mouse dystrophin muscle enhancer-1 imparts skeletal muscle, but not cardiac muscle, expression onto the dystrophin Purkinje promoter in transgenic mice.
2004,
Pubmed
Donoghue,
A muscle-specific enhancer is located at the 3' end of the myosin light-chain 1/3 gene locus.
1988,
Pubmed
Donoviel,
Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.
1996,
Pubmed
Gong,
Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.
2002,
Pubmed
Grimaldi,
Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis.
2004,
Pubmed
,
Xenbase
Haeri,
Generation of transgenic Xenopus using restriction enzyme-mediated integration.
2012,
Pubmed
,
Xenbase
Hamlet,
Tol2 transposon-mediated transgenesis in Xenopus tropicalis.
2006,
Pubmed
,
Xenbase
Harada,
The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.
2007,
Pubmed
Hirsch,
Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.
2002,
Pubmed
,
Xenbase
Kawakami,
A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.
2004,
Pubmed
Kawakami,
Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis.
2004,
Pubmed
,
Xenbase
Kroll,
Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.
1996,
Pubmed
,
Xenbase
Kuo,
Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.
2012,
Pubmed
Loots,
Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease.
2005,
Pubmed
Momken,
Impaired voluntary running capacity of creatine kinase-deficient mice.
2005,
Pubmed
Murakami,
Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin.
2005,
Pubmed
Nanda,
Leiomodin 1, a new serum response factor-dependent target gene expressed preferentially in differentiated smooth muscle cells.
2012,
Pubmed
Narlikar,
Genome-wide discovery of human heart enhancers.
2010,
Pubmed
Natarajan,
Predicting cell-type-specific gene expression from regions of open chromatin.
2012,
Pubmed
Neph,
An expansive human regulatory lexicon encoded in transcription factor footprints.
2012,
Pubmed
Ogino,
Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease.
2006,
Pubmed
,
Xenbase
Ohtsuki,
Different core promoters possess distinct regulatory activities in the Drosophila embryo.
1998,
Pubmed
Parinov,
Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.
2004,
Pubmed
Pennacchio,
In vivo enhancer analysis of human conserved non-coding sequences.
2006,
Pubmed
Pennacchio,
Predicting tissue-specific enhancers in the human genome.
2007,
Pubmed
Pletnev,
A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore.
2008,
Pubmed
Qiu,
Assignment and expression patterns of porcine muscle-specific isoform of phosphoglycerate mutase gene.
2008,
Pubmed
Shcherbo,
Bright far-red fluorescent protein for whole-body imaging.
2007,
Pubmed
,
Xenbase
Su,
A gene atlas of the mouse and human protein-encoding transcriptomes.
2004,
Pubmed
Sun,
Molecular cloning and characterization of a novel muscle adenylosuccinate synthetase, AdSSL1, from human bone marrow stromal cells.
2005,
Pubmed
Suster,
Transgenesis in zebrafish with the tol2 transposon system.
2009,
Pubmed
Takada,
Myozenin: an alpha-actinin- and gamma-filamin-binding protein of skeletal muscle Z lines.
2001,
Pubmed
Trinklein,
An abundance of bidirectional promoters in the human genome.
2004,
Pubmed
Wang,
Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.
2012,
Pubmed
Wasserman,
Identification of regulatory regions which confer muscle-specific gene expression.
1998,
Pubmed
Yergeau,
Transgenesis in Xenopus using the Sleeping Beauty transposon system.
2009,
Pubmed
,
Xenbase
de la Calle-Mustienes,
A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts.
2005,
Pubmed
,
Xenbase
