Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities

Yair Glick, Yaron Orenstein, Dana Chen, Dorit Avrahami, Tsaffrir Zor, Ron Shamir, Doron Gerber

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


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.

Original languageEnglish
Article number1327
JournalNucleic Acids Research
Issue number6
StatePublished - 3 Dec 2015

Bibliographical note

Funding Information:
European council [3309600 to D.G.]; Israel Science Foundation [715/11 to D.G.]; The Israel Science Foundation [317/13 to R.S.]; Raymond and Beverly Sackler chair in Bioinformatics [to R.S.]; Edmond J. Safra Center for Bioinformatics at Tel Aviv University [to Y.O.]. Funding for open access charge: ERC [3309600].

Publisher Copyright:
© 2016 The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


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