Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: 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-κB 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 (Chromatin-of-Interest Fragment Isolation: 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.

modified plasmids were incubated with streptavidin-coated magnetic beads in order to estimate the capture efficiency. Following various incubation times, beads are collected via a magnet. The supernatants fractions (i.e., the unbound fractions: UB) are analysed by agarose gel electrophoresis (Supplementary Figs. S1b and S1c) leading to the following conclusions: 1. In vitro biotinylation converts closed circular (CC) DNA to open circular (OC) and to an unidentified high molecular weight form in a dose dependent way; 2. CC plasmid is captured on beads more efficiently than OC plasmid; 3. Time dependency of capture efficiency strongly varies with the level of biotin modification. Taken together the data suggest that steric hindrance between plasmid and beads strongly limits capture efficiency.
Steric hindrance appears to be partially relieved by increasing the number of biotin residues per plasmid. The down side of heavy modification is the induction of plasmid relaxation and of the possible modification of the sequence-of-interest. As our main purpose, i.e. the isolation of potentially large protein complexes assembled on DNA, may be strongly limited by steric hindrance, we decided not to pursue this approach. We also attempted to introduce a biotin-containing DNA oligonucleotide into our plasmid. We found that the length of the spacer between the plasmid and the biotin moiety is crucial for efficient capture. In contrast to short oligonucleotides, whose capture is not limited by spacer length, circular plasmids (e.g., several kbp in size) require a spacer length in excess to 15 atoms.
Soluble fraction is separated from the insoluble fraction by centrifugation. The recovered supernatant is passed through streptavidin magnetic beads in order to remove proteins bound to beads. The resultant supernatant is used as an input for the IDAP methodology.

Reconstitution of mono-nucleosome
Human histones are purchased from NEB, and reconstitution of nucleosome follows the manufacture's instruction. Gel shift assay for confirming status of nucleosome is implemented on a 6% DNA retardation gel, and DNAs are detected by EtBr staining.

IDAP in mono-nucleosome
Cross-linked nucleosomes (1% formaldehyde for 10 min) derived from RN01 (10 ng (a) A representative pattern of genomic DNA prepared from an input sample to be used in CoIFI. This input sample is prepared from G69 cell line and used in the CoIFI experiment shown in Figure 6. (b) Relative abundance of target DNA (in red) versus bulk DNA. In G69 cell line, ≈ 160 copies of plasmid construct are stably integrated. As the target DNA fragments (in red) in input are present at a ratio of one in ≈ 10 5 non-target fragments, to be successful the CoIFI procedure requires ≥ 10 5 -fold enrichment. (c) Detection of histones as a way to monitor the actual enrichment during CoIFI: If an input sample is derived from G69 cells, the proportion of plasmid-derived DNA fragments to total gDNA is ≈ 0.002%. Therefore, if the signal-to-noise (s/n) ratio in CoIFI is < 10 5 , the signal intensity of histone (e.g., via WB) will be similar under all three conditions a, b and c as depicted in Figure 6c. On the other hand, if the s/n ratio in CoIFI is ≥ 10 5 , the signal intensity of histone (e.g., via WB) will be significantly higher under condition b compared to a and c.
10 Supplementary Figure S7. Standard curve used to quantify the recovery yield of DNA segment in CoIFI analysis: This curve is derived from the control DNA data shown in Figure   6d, and is used uniquely for quantification of band intensities in Figure 6d. When quantifying intensities of DNA bands of interests amplified by PCR (e.g., for test of reproducibility; in different assays), know amounts of reference DNA are also amplified in the same time and analysed in the same gel as well as data shown in Figure 6d.