title: Nanobody(R)-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites.
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.
authors.affiliation: Research group of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium.
authors.forename: Trong
authors.initials: T
authors.lastname: Nguyen-Duc
authors.forename: Eveline
authors.initials: E
authors.lastname: Peeters
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Daniel
authors.initials: D
authors.lastname: Charlier
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Hassanzadeh-Ghassabeh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D019843:Archaeal Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002161:Camelids, New World; D047369:Chromatin Immunoprecipitation; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D018604:Epitope Mapping; D020745:Genome, Archaeal; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D061905:Single-Domain Antibodies / Q000737:chemistry*; D048229:Sulfolobus solfataricus / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D055767:Immobilized Proteins; D061905:Single-Domain Antibodies; D014157:Transcription Factors