De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis.
Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.
Fordyce
Polly M
PM
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA.
Gerber
Doron
D
Tran
Danh
D
Zheng
Jiashun
J
Li
Hao
H
DeRisi
Joseph L
JL
Quake
Stephen R
SR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D002457:Cell Extracts; D005453:Fluorescence; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012156:Reticulocytes / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D002457:Cell Extracts; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
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