SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics
Identifieur interne : 001779 ( Ncbi/Curation ); précédent : 001778; suivant : 001780SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics
Auteurs : Dana Chen [Israël] ; Yaron Orenstein [Israël] ; Rada Golodnitsky [Israël] ; Michal Pellach [Israël] ; Dorit Avrahami [Israël] ; Chaim Wachtel [Israël] ; Avital Ovadia-Shochat [Israël] ; Hila Shir-Shapira [Israël] ; Adi Kedmi [Israël] ; Tamar Juven-Gershon [Israël] ; Ron Shamir [Israël] ; Doron Gerber [Israël]Source :
- Scientific Reports [ 2045-2322 ] ; 2016.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : Transcription Factors.
- genetics : Nucleotide Motifs.
- methods : Microfluidics, SELEX Aptamer Technique.
- Base Sequence, Binding Sites, Gene Library, Microarray Analysis, Protein Binding, Reproducibility of Results, Sample Size.
Abstract
Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.
Url:
DOI: 10.1038/srep33351
PubMed: 27628341
PubMed Central: 5024299
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PMC:5024299Le document en format XML
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence</term>
<term>Binding Sites</term>
<term>Gene Library</term>
<term>Microarray Analysis</term>
<term>Microfluidics (methods)</term>
<term>Nucleotide Motifs (genetics)</term>
<term>Protein Binding</term>
<term>Reproducibility of Results</term>
<term>SELEX Aptamer Technique (methods)</term>
<term>Sample Size</term>
<term>Transcription Factors (metabolism)</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>Analyse sur microréseau</term>
<term>Banque de gènes</term>
<term>Facteurs de transcription (métabolisme)</term>
<term>Liaison aux protéines</term>
<term>Microfluidique ()</term>
<term>Motifs nucléotidiques (génétique)</term>
<term>Reproductibilité des résultats</term>
<term>Sites de fixation</term>
<term>Séquence nucléotidique</term>
<term>Taille d'échantillon</term>
<term>Technique SELEX ()</term>
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<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Transcription Factors</term>
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<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Nucleotide Motifs</term>
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<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Motifs nucléotidiques</term>
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<term>Microarray Analysis</term>
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<term>Reproductibilité des résultats</term>
<term>Sites de fixation</term>
<term>Séquence nucléotidique</term>
<term>Taille d'échantillon</term>
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<front><div type="abstract" xml:lang="en"><p>Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.</p>
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