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Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities

Nature Biotechnology volume 24, pages 1429–1435 (2006)Cite this article

Abstract

Transcription factors (TFs) interact with specific DNA regulatory sequences to control gene expression throughout myriad cellular processes. However, the DNA binding specificities of only a small fraction of TFs are sufficiently characterized to predict the sequences that they can and cannot bind. We present a maximally compact, synthetic DNA sequence design for protein binding microarray (PBM) experiments1 that represents all possible DNA sequence variants of a given length k (that is, all 'k-mers') on a single, universal microarray. We constructed such all k-mer microarrays covering all 10–base pair (bp) binding sites by converting high-density single-stranded oligonucleotide arrays to double-stranded (ds) DNA arrays. Using these microarrays we comprehensively determined the binding specificities over a full range of affinities for five TFs of different structural classes from yeast, worm, mouse and human. The unbiased coverage of all k-mers permits high-throughput interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution.

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Figure 1: Design of a universal microarray for PBM experiments.
Figure 2: Relating PBM signal intensity to individual k-mers.
Figure 3: Determination of motifs and logos for five TFs.

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Acknowledgements

We thank T.V.S. Murthy, Leo Brizuela and Josh LaBaer for providing the Cbf1 and Rap1 clones, Gwenael Badis-Breard and Tim Hughes for providing the Zif268 DNA-binding domain clone and Shufen Meng for assistance with the Biacore technology. We also thank Stephen Gisselbrecht, Amy Donner and Rachel McCord for critical reading of the manuscript. This work was funded in part by grants R01 HG003985 and R01 HG003420 from National Institutes of Health/National Human Genome Research Institute to M.L.B. M.F.B. was supported in part by a National Science Foundation Graduate Research Fellowship. A.A.P. was supported in part by a National Defense Science and Engineering Graduate Fellowship, a National Science Foundation Graduate Research Fellowship and an Athinoula Martinos Fellowship.

Author information

Author notes

  1. Michael F Berger and Anthony A Philippakis: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, 02115, Massachusetts, USA

    Michael F Berger, Anthony A Philippakis, Aaron M Qureshi, Fangxue S He, Martha L Bulyk & Martha L Bulyk

  2. Harvard University Graduate Biophysics Program, Cambridge, 02138, Massachusetts, USA

    Michael F Berger, Anthony A Philippakis & Martha L Bulyk

  3. Harvard/MIT Division of Health Sciences and Technology (HST), Harvard Medical School, Boston, 02115, Massachusetts, USA

    Anthony A Philippakis, Fangxue S He & Martha L Bulyk

  4. Department of Mathematics, University of Maryland, College Park, Maryland, 20742, USA

    Aaron M Qureshi

  5. Longenity, Inc., Waltham, 02451, Massachusetts, USA

    Preston W Estep III

  6. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, 02115, Massachusetts, USA

    Martha L Bulyk

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Contributions

M.F.B. participated in the array design, experimental design, analysis of results and drafting of the manuscript, and performed the experiments; A.A.P. conceived the idea of using de Bruijn sequences and participated in the binding site survey, array design, experimental design, analysis of results and drafting of the manuscript; A.M.Q. provided linear feedback shift register expertise and assisted in the array design; F.S.H. participated in the binding site survey; P.W.E. III conceived the concept of the compact universal array; M.L.B. conceived the concept of the compact universal array and participated in the array design, experimental design, analysis of the results and drafting of the manuscript.

Corresponding author

Correspondence to Martha L Bulyk.

Ethics declarations

Competing interests

A subset of the authors—M.L.B., A.A.P and P.W.E. 3rd—have filed a patent application, through Brigham and Women's Hospital, covering the sequence design described in this paper.

Supplementary information

Supplementary Fig. 1

Survey of binding sites in the JASPAR database. (PDF 570 kb)

Supplementary Fig. 2

Reproducibility of Cy3 dUTP signal intensities. (PDF 512 kb)

Supplementary Fig. 3

Correlation of PBM signal intensities with affinities. (PDF 679 kb)

Supplementary Fig. 4

Effects of binding site position and orientation on PBM signal. (PDF 629 kb)

Supplementary Fig. 5

Comparison of median signal intensities for 28 Zif268 variants for fixed versus variable position, orientation, and flanking sequence. (PDF 654 kb)

Supplementary Fig. 6

Correspondence between median signal intensities for 7-mers on distinct de Bruijn sequences. (PDF 296 kb)

Supplementary Fig. 7

Biacore measurements supporting interdependence between the first two positions of the Cbf1 binding site. (PDF 920 kb)

Supplementary Fig. 8

Minimum number of unique features on an array for different values of k. (PDF 652 kb)

Supplementary Fig. 9

Dependence of Cy3 dUTP incorporation upon sequence context. (PDF 609 kb)

Supplementary Methods (DOC 76 kb)

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Berger, M., Philippakis, A., Qureshi, A. et al. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat Biotechnol 24, 1429–1435 (2006). https://doi.org/10.1038/nbt1246

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