Chromosome conformation capture (3C) assay

The chromosome conformation capture (3C) assay was performed as described previously with minor modifications (39). In brief, 2 × 10⁷ cells were cross-linked with 2% formaldehyde for 10 min and stopped by the addition of glycine at a final concentration of 0.125M. Cells were pelleted and washed twice with cold PBS. Cells were collected and washed with appropriate 1× restriction buffer and then resuspended in 1× restriction enzyme buffer containing 0.3% SDS at 37° with overnight shaking. Triton X-100 was then added to a final concentration of 2% to sequester SDS at 37°C for 1.5 h with shaking. Chromatin was than digested with 800U of BglII at 37°C overnight with shaking. Next day the reaction was stopped by adding SDS to a final concentration of 1.6% at 65°C for 20 min. The digested chromatin was then diluted in 1 ml of T4 DNA ligation buffer (NEB) containing 1% Triton X-100 and incubated at 37°C for 1.5 h with shaking. A total of 400 U of T4 DNA ligase (NEB) was added and the ligation was carried out at 16°C for 3 days followed by 1 h at room temperature. The ligated chromatin was then reverse crosslinked overnight by adding 200 μg of Proteinase K (Invitrogen) at 65°C followed by phenol chloroform extraction to purify 3C DNAs. Purified 3C ligated DNA was amplified using PCR and the products were cloned into pCR-TOPOII vector (Invitrogen) for sequencing. Relative crosslinking frequencies were calculated and plotted after normalization to the loading control (40).