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.
Highlights
Many vertebrate genes exhibit highly intricate temporal and spatial expression patterns that span multiple stages of embryonic development, and specific embryonic or adult cell types
Encoded fluorescent proteins have been widely used as transgenic reporters and have been primarily optimized for mammalian in vitro and in vivo applications; among them green fluorescent protein (GFP) has been most widely used
Recent reports have examined a family of far-red fluorescent proteins and have shown that both in tissue culture and in animals, these fluorescent proteins have high fluorescence resonance energy transfer efficiency; they take advantage of the reduced light scattering associated with far-red illumination, and reduced excitation of yolk and other autofluorescent proteins, suggesting that they are well suited for real-time in vivo imaging [13]
Summary
Many vertebrate genes exhibit highly intricate temporal and spatial expression patterns that span multiple stages of embryonic development, and specific embryonic or adult cell types. While transcription is mediated in part by promoters and promoter proximal elements that coordinate the site of initiation and contribute to the levels of transcription, distal cis-regulatory elements such as enhancers, insulators, locus control regions, and silencing elements control the stage and region-specificity of expression. These sequences are frequently positioned at great distances from the promoters of the genes they control. Increasing evidence suggests that a significant proportion of the noncoding sequences, possibly up to 80% (encode papers) contain regulatory elements responsible for the elaborate expression programs in the diverse cell types of the vertebrate body
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