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Interrogating Transcriptional Regulatory Sequences in Tol2-Mediated Xenopus Transgenics

Journal Article · · PLoS ONE
 [1];  [2];  [2];  [3];  [3];  [3];  [4];  [3]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biology and Biotechnology Division; Univ. of California, Merced, CA (United States). School of Natural Sciences; DOE/OSTI
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biology and Biotechnology Division
  3. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology
  4. National Library of Medicine (NLM), Bethesda, MD (United States). National Center for Biotechnology Information. Computational Biology Branch
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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 cisregulatory 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.
Research Organization:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
AC52-07NA27344
OSTI ID:
1627619
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 7 Vol. 8; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English

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