title: Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.
abstract: Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.
authors.affiliation: Mina and Evrard Goodman life science faculty, Bar Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Yair
authors.initials: Y
authors.lastname: Glick
authors.affiliation: Blavatnik School of Computer Science, Tel-Aviv University, Tel-Aviv, 69978, Israel.
authors.forename: Yaron
authors.initials: Y
authors.lastname: Orenstein
authors.affiliation: Mina and Evrard Goodman life science faculty, Bar Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Chen
authors.affiliation: Mina and Evrard Goodman life science faculty, Bar Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Dorit
authors.initials: D
authors.lastname: Avrahami
authors.affiliation: Department of Biochemistry & Molecular Biology, Life Sciences Institute, Tel-Aviv University, Tel-Aviv, 69978, Israel.
authors.forename: Tsaffrir
authors.initials: T
authors.lastname: Zor
authors.affiliation: Blavatnik School of Computer Science, Tel-Aviv University, Tel-Aviv, 69978, Israel.
authors.forename: Ron
authors.initials: R
authors.lastname: Shamir
authors.affiliation: Mina and Evrard Goodman life science faculty, Bar Ilan University, Ramat-Gan, 5290002, Israel Doron.Gerber@biu.ac.il.
authors.forename: Doron
authors.identifier: http://orcid.org/0000-0002-0532-2928
authors.initials: D
authors.lastname: Gerber
mesh_terms: D051697:Activating Transcription Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057166:High-Throughput Screening Assays; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D018808:Transcription Factor AP-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C531110:ATF1 protein, human; D051697:Activating Transcription Factor 1; D016760:Proto-Oncogene Proteins c-fos; D018808:Transcription Factor AP-1; D004247:DNA; D048031:JNK Mitogen-Activated Protein Kinases