Nanobody®-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 (ChIPchip) 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. © The Author(s) 2013.