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. 2010 Sep;28(9):970-5.
doi: 10.1038/nbt.1675. Epub 2010 Aug 29.

De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis

Polly M Fordyce  1 , Doron Gerber, Danh Tran, Jiashun Zheng, Hao Li, Joseph L DeRisi, Stephen R Quake
Affiliations

De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis

Polly M Fordyce et al. Nat Biotechnol. 2010 Sep.

Abstract

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.

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Figures

Figure 1
Figure 1
Overall experimental design and procedure. (a) Microfluidic device hybridized to glass slide. Unit cells contain two chambers (a ‘DNA chamber’ and a ‘protein’ chamber) controlled by three valves: a ‘neck’ valve (green) to separate the two chambers, a ‘sandwich’ valve (orange) to isolate unit cells, and a ‘button’ valve (blue) to protect molecular interactions. (b) DNA 8mer library design. Each 70 bp oligonucleotide contains 45 overlapping 8mers, a 3 bp GC-clamp at the 5′ end, and an identical 14-bp sequence at the 3′ end for Cy5 labeling and primer extension. (c) PCR generation of linear templates for protein expression. In PCR1, template-specific primers attach a Kozak sequence, 6× His tag, and universal overhangs. In PCR2, universal primers add a T7 promoter, poly-A tail, and T7 terminator. In vitro transcription/translation (ITT) of this template in rabbit reticulocyte lysate (RR) with BODIPY-labeled lysine charged tRNA produces labeled, His-tagged protein. (d) Overview of experimental procedure. Devices are manually aligned to a spotted microarray. Neck valves are closed to protect DNA within chambers, and slide surfaces are derivatized with anti-pentaHis antibodies below the button (white) and passivated elsewhere (grey). Lysate containing fluorescently labeled His-tagged TFs is introduced and neck valves are opened to allow interaction between transcription factors and DNA; sandwich valves are closed to isolate each unit cell. Following an incubation, button valves are pressurized to protect protein:DNA interactions, unbound DNA and proteins are washed out, and the device is scanned. (e) Scanned picture showing final protein (BODIPY, left) and DNA (Cy5, right) intensities in the chamber and under the button. (f) Arrays showing example protein intensities (left) and DNA intensities (right) under the button for each unit cell within a device.
Figure 2
Figure 2
Detailed analysis of measured Cy5 intensities and fluorescence intensity ratios (Cy5/BODIPY-FL) for rabbit reticulocyte lysate alone, Reb1p, Cin5p, and Cup9p. (a) Distribution of measured Cy5 intensities for all oligonucleotides. Light grey box indicates measurements within 4 standard deviations of the mean (as determined by a Gaussian fit). Measured Cy5 intensities for rabbit reticulocyte lysate alone are well-fit by a Gaussian (reduced χ2 = 1.0, p = 0.47). For all TFs, measured Cy5 intensities deviate significantly from a Gaussian distribution, with measured events many standard deviations above the mean. (b)Distribution of measured intensity ratios for all oligonucleotides. Light grey box indicates measurements within 4 standard deviations of the mean (as determined by a Gaussian fit). Measured intensity ratios in the presence of TFs deviate significantly from a normal distribution (Supplementary Table 2). (c) Correlation between ratios measured for the same oligonucleotide at two separate locations within the device.
Figure 3
Figure 3
Comparison between Kd values derived from direct measurements of concentration-dependent binding and Kd values calculated from ratio measurements at a single concentration. (a) Cin5p measurements. Measured ratio signals for all oligonucleotides (grey) and selected oligonucleotides (blue) (left); concentration-dependent binding for selected oligonucleotides fit to a single-site binding model (right). (b) Kd calculated from single-concentration measurements vs. Kd derived from fits concentration-dependent binding for Cin5p (blue), Pho4p (red), Yap1p (grey) and Cbf1p (green). (c) Calculated Kd values for all oligonucleotides for Cin5p. (d) Calculated Kd values for all oligonucleotides for Pho4p.
Figure 4
Figure 4
Comparison between motifs found for all 28 S. cerevisiae TFs and previous literature results (SWISS: SwissRegulon, ChIP-chip: Harbison library, PBM′: protein binding microarray, and PBM: protein binding microarray15). For ChIP-chip data, boxes shaded in grey represent literature-derived motifs. For PBM results, white boxes represent proteins applied to arrays that did not yield motifs; boxes shaded in grey represent proteins that did not express sufficiently to be applied to arrays. fREDUCE Seeds: 7-and 8-bp fREDUCE motifs that correlate most strongly with measured intensities; Optimized PSAM: MatrixREDUCE PSAM represented as an AffinityLogo; r2: Pearson correlation coefficient between all measured ratio values and protein occupancies predicted by the optimized PSAM.
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