DFF-ChIP: a method to detect and quantify complex interactions between RNA polymerase II, transcription factors, and chromatin

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provided the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs) and that DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking and to compare these results to those from standard ChIP-Seq that employs sonication and CUT&RUN which utilizes MNase to fragment the genomic DNA. The results presented indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions. Overall design: Nuclei from SUB-B15 were rapidly isolated following no treatment or treatment with 1 µM dexamethasone. After 1 hour of dexamethasone, both untreated and treated nuclei were either immediately isolated or crosslinked with 1% formaldehyde for 10 minutes followed by immediate quenching with 1M Tris. The resulting nuclei were then digested with DNA fragmentation Factor (DFF), an endonuclease with reduced intra-nucleosomal cutting as compared to Micrococcal nuclease (MNase), to generate primarily mono-nucleosomes. After release of chromatin from the nucleus following light sonication, transcription factors were then immunoprecipitated from the released chromatin. SUP-B15 DFF-ChIP was spiked in with 2% mouse nuclei (4T1) cells. HeLa nuclei were similarly prepared without treatment with dexamethasone or nuclei spike-in. Finally, SUP-B15 and B1 nuclei were prepared for ChIP-Seq as described in the methods section utilizing different crosslinking, sonication, and library prep conditions.