Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation
Résumé
The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA ((I) under bar solation of (D) under bar NA (A) under bar ssociated (P) under bar roteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-kappa B model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved ((C) under bar hromatin-(o) under barf-(I) under bar nterest (F) under bar ragment (I) under bar solation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.