Title | Nanobody(R)-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites. |
Publication Type | Journal Article |
Year of Publication | 2013 |
Authors | Nguyen-Duc, T., E. Peeters, S. Muyldermans, D. Charlier, and G. Hassanzadeh-Ghassabeh |
Journal | Nucleic Acids Res |
Volume | 41 |
Issue | 5 |
Pagination | e59 |
Date Published | 2013 Mar 1 |
ISSN | 1362-4962 |
Keywords | Amino Acid Sequence, Animals, Antibody Specificity, Archaeal Proteins, Binding Sites, Camelids, New World, Chromatin Immunoprecipitation, DNA, Archaeal, Epitope Mapping, Genome, Archaeal, Immobilized Proteins, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Protein Binding, Sequence Analysis, DNA, Single-Domain Antibodies, Sulfolobus solfataricus, Surface Plasmon Resonance, Transcription Factors |
Abstract | Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid-protein and protein-protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA-protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation. |
DOI | 10.1093/nar/gks1342 |
Alternate Journal | Nucleic Acids Res. |
PubMed ID | 23275538 |
PubMed Central ID | PMC3597646 |
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