Universal Protein-binding Microarrays for the Comprehensive Characterization of the DNA-binding Specificities of Transcription Factors

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2009 Mar 7

PMID 19265799

Citations 205

Authors

Michael F Berger

Martha L Bulyk

Affiliations

Soon will be listed here.

Abstract

Protein-binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA-binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding-site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF-binding specificities at high resolution using such 'all 10-mer' universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day.

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References

De Silva E, Gehrke A, Olszewski K, Leon I, Chahal J, Bulyk M . Specific DNA-binding by apicomplexan AP2 transcription factors. Proc Natl Acad Sci U S A. 2008; 105(24):8393-8. PMC: 2423414. DOI: 10.1073/pnas.0801993105. View

Iyer V, Horak C, Scafe C, Botstein D, Snyder M, Brown P . Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature. 2001; 409(6819):533-8. DOI: 10.1038/35054095. View

Wingender E, Chen X, Hehl R, Karas H, Liebich I, Matys V . TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res. 1999; 28(1):316-9. PMC: 102445. DOI: 10.1093/nar/28.1.316. View

Reece-Hoyes J, Deplancke B, Shingles J, Grove C, Hope I, Walhout A . A compendium of Caenorhabditis elegans regulatory transcription factors: a resource for mapping transcription regulatory networks. Genome Biol. 2006; 6(13):R110. PMC: 1414109. DOI: 10.1186/gb-2005-6-13-r110. View

Linnell J, Mott R, Field S, Kwiatkowski D, Ragoussis J, Udalova I . Quantitative high-throughput analysis of transcription factor binding specificities. Nucleic Acids Res. 2004; 32(4):e44. PMC: 390317. DOI: 10.1093/nar/gnh042. View

Wilson D, Desplan C . Structural basis of Hox specificity. Nat Struct Biol. 1999; 6(4):297-300. DOI: 10.1038/7524. View

Adryan B, Teichmann S . FlyTF: a systematic review of site-specific transcription factors in the fruit fly Drosophila melanogaster. Bioinformatics. 2006; 22(12):1532-3. DOI: 10.1093/bioinformatics/btl143. View

Chen X, Hughes T, Morris Q . RankMotif++: a motif-search algorithm that accounts for relative ranks of K-mers in binding transcription factors. Bioinformatics. 2007; 23(13):i72-9. DOI: 10.1093/bioinformatics/btm224. View

Bulyk M, Gentalen E, Lockhart D, Church G . Quantifying DNA-protein interactions by double-stranded DNA arrays. Nat Biotechnol. 1999; 17(6):573-7. DOI: 10.1038/9878. View

10.

Ho S, Jona G, Chen C, Johnston M, Snyder M . Linking DNA-binding proteins to their recognition sequences by using protein microarrays. Proc Natl Acad Sci U S A. 2006; 103(26):9940-5. PMC: 1502558. DOI: 10.1073/pnas.0509185103. View

11.

Berger M, Philippakis A, Qureshi A, He F, Estep 3rd P, Bulyk M . Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat Biotechnol. 2006; 24(11):1429-35. PMC: 4419707. DOI: 10.1038/nbt1246. View

12.

Stormo G . DNA binding sites: representation and discovery. Bioinformatics. 2000; 16(1):16-23. DOI: 10.1093/bioinformatics/16.1.16. View

13.

Sandelin A, Alkema W, Engstrom P, Wasserman W, Lenhard B . JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 2003; 32(Database issue):D91-4. PMC: 308747. DOI: 10.1093/nar/gkh012. View

14.

Bulyk M, Huang X, Choo Y, Church G . Exploring the DNA-binding specificities of zinc fingers with DNA microarrays. Proc Natl Acad Sci U S A. 2001; 98(13):7158-63. PMC: 34639. DOI: 10.1073/pnas.111163698. View

15.

Fried M, Crothers D . Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981; 9(23):6505-25. PMC: 327619. DOI: 10.1093/nar/9.23.6505. View

16.

Bulyk M . DNA microarray technologies for measuring protein-DNA interactions. Curr Opin Biotechnol. 2006; 17(4):422-30. PMC: 2727741. DOI: 10.1016/j.copbio.2006.06.015. View

17.

van Steensel B, Delrow J, Henikoff S . Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet. 2001; 27(3):304-8. DOI: 10.1038/85871. View

18.

Warner J, Philippakis A, Jaeger S, He F, Lin J, Bulyk M . Systematic identification of mammalian regulatory motifs' target genes and functions. Nat Methods. 2008; 5(4):347-53. PMC: 2708972. DOI: 10.1038/nmeth.1188. View

19.

Foat B, Morozov A, Bussemaker H . Statistical mechanical modeling of genome-wide transcription factor occupancy data by MatrixREDUCE. Bioinformatics. 2006; 22(14):e141-9. DOI: 10.1093/bioinformatics/btl223. View

20.

Bulyk M, Johnson P, Church G . Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors. Nucleic Acids Res. 2002; 30(5):1255-61. PMC: 101241. DOI: 10.1093/nar/30.5.1255. View