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Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays

Nature Genetics volume 36, pages 13311339 (2004) | Download Citation

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Abstract

We developed a new DNA microarray-based technology, called protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding–site sequence specificities of transcription factors in a single day. Using PBMs, we identified the DNA binding–site sequence specificities of the yeast transcription factors Abf1, Rap1 and Mig1. Comparison of these proteins' in vitro binding sites with their in vivo binding sites indicates that PBM-derived sequence specificities can accurately reflect in vivo DNA sequence specificities. In addition to previously identified targets, Abf1, Rap1 and Mig1 bound to 107, 90 and 75 putative new target intergenic regions, respectively, many of which were upstream of previously uncharacterized open reading frames. Comparative sequence analysis indicated that many of these newly identified sites are highly conserved across five sequenced sensu stricto yeast species and, therefore, are probably functional in vivo binding sites that may be used in a condition-specific manner. Similar PBM experiments should be useful in identifying new cis regulatory elements and transcriptional regulatory networks in various genomes.

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Acknowledgements

We thank T. Volkert and T. Lee for synthesis of the whole-genome yeast intergenic microarrays, B. Huber for assistance with motif searches, L. Campbell and M. Blayney for technical assistance and M. Chou and A. Philippakis for discussion. This work was supported by National Institutes of Health grants from the National Human Genome Research Institute to M.L.B. and to R.Y. and from the National Institute of General Medical Sciences to M.S. M.L.B. was also supported by a Pharmaceutical Research and Manufacturers of America Foundation Informatics Research Starter Grant and an HST Taplin Award. S.M. was supported in part by the MIT Class of 1973 Undergraduate Research Opportunities Program Fund and an MIT Bioengineering Undergraduate Research Award. M.F.B. was supported in part by a Graduate Research Fellowship from the National Science Foundation.

Author information

Author notes

    • Sonali Mukherjee
    •  & Michael F Berger

    These authors contributed equally to this work.

Affiliations

  1. Division of Genetics, Department of Medicine, Harvard Medical School; Boston; Massachusetts 02115; USA

    • Sonali Mukherjee
    • , Michael F Berger
    • , Xun S Wang
    • , Dale Muzzey
    •  & Martha L Bulyk
  2. Harvard/MIT Division of Health Sciences and Technology, Brigham and Women's Hospital and Harvard Medical School; Boston; Massachusetts 02115; USA

    • Sonali Mukherjee
    •  & Martha L Bulyk
  3. Harvard University Graduate Biophysics Program, Harvard Medical School; Boston; Massachusetts 02115; USA.

    • Michael F Berger
    • , Dale Muzzey
    •  & Martha L Bulyk
  4. Departments of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.

    • Ghil Jona
    •  & Michael Snyder
  5. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

    • Xun S Wang
    •  & Richard A Young
  6. Molecular Biophysics and Biochemistry, and Genetics, Yale University, New Haven, Connecticut 06520, USA.

    • Michael Snyder
  7. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

    • Richard A Young
  8. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Martha L Bulyk

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Martha L Bulyk.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    DNA microarray bound by CBP-FLAG-Rpn4.

  2. 2.

    Supplementary Fig. 2

    Negative control PBMs.

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    Supplementary Fig. 3

    Reproducibility of PBM data.

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    Supplementary Fig. 4

    Effect of less stringent P-value thresholds on the number of spots identified as bound.

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    Supplementary Fig. 5

    Binding site motifs for PBM data passing less stringent P-value thresholds.

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    Supplementary Fig. 6

    Comparison of bound intergenic regions derived from PBM versus ChIP-chip data at various P-value thresholds.

  7. 7.

    Supplementary Note

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    Supplementary Methods

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DOI

https://doi.org/10.1038/ng1473

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