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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.
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???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 30â45 of embryonic development (C) in all enhancer-promoter combinations (DâG).
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 (Aâ²âFâ²). 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 âwildtypeâ 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).
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