Deciphering the role of RNA in regulating CTCF's DNA binding affinity in leukemia cells [CTCF-ChIP]

CTCF, a highly studied transcription factor, is essential for chromatin interaction maintenance. Several independent studies have reported that CTCF interacts with RNAs in vitro and in cells. Yet continuous debates about the authenticity of the RNA-binding affinity of CTCF and its biological role remain in large part due to limited research techniques available, such as CLIP-seq. Here, we investigated the role RNA plays on CTCF's transcription factor function through its chromatin occupancy. To systematically investigate whether RNAs affect CTCF's ability to bind DNA, we perturbed CTCF-RNA interactions by three independent approaches and examined CTCF genome occupancy by ChIP-seq. Although RnaseA A and Triptolide treatment each affected a certain number of CTCF-binding peaks, few peaks overlapped between treatment groups indicating the effect of RNA in regulating CTCF's DNA binding affinity was modest and variable between loci. In addition, limited transcriptional or chromatin accessibility changes were observed between cells expressing wild-type CTCF or CTCF lacking the RNA binding region. Our data provide a complementary approach and in silico evidence to reconsider the significance of RNA affecting CTCF's DNA-binding affinity in a global manner. Overall design: For CTCF-ChIP samples, CTCFAID2/WT cells were treated with 1 mM Triptolide or DMSO for 8 h concurrent with 5-Ph-IAA and doxycycline treatment. For RnaseA A ChIP, RnaseA A treatment conditions were adapted from those previously reported (Saldana-Meyer et al. 2019, Beltran et al. 2016). Cells were permeabilized before fixation by 0.05% TWEEN-20 in PBS for 10 minutes on ice, washed once with PBS and resuspended in PBS plus 1mg/mL RnaseA A or mock treated and rotated for 45 minutes at room temperature. Each treatment group (+RnaseA A, -RnaseA A) was performed in triplicate. For both Triptolide and RnaseA A ChIP groups, chromatin was prepared as described above. Spike-in antibody and chromatin (Active Motif, 61686 and 53083) along with CTCF antibody (10 mgs, Diagenode, C15410210-50) were added to the chromatin and incubated at 4 °C overnight with gentle rotation. The next day, pre-washed Protein G beads (Dynabeads, Invitrogen, 10004D) were added and incubated 4 °C for 4 h with gentle rotation. On a magnetic stand, the beads were washed twice with wash buffer 1 (50 mM Tris HCL pH 7.4, 1 M NaCl, 1 mM EDTA, 1 % NP-40, 0.1 % SDS, 0.5 % Na Deoxycholate plus protease inhibitors) and once with wash buffer 2 (20 mM Tris HCL pH 7.4, 10 mM MgCl2, 0.2 % Tween-20 plus protease inhibitors). The beads in wash buffer 2 were transferred to a new 1.5 mL Eppendorf tube and placed on a magnetic stand to remove the wash buffer. Decrosslinking was carried out in 1X TE plus 1 % SDS, proteinase K, and 400 mM NaCl at 65 °C for 4 h, followed by phenol, chloroform, and isopropyl alcohol precipitation of the DNA. NEBNext Ultra II NEB Library Prep Kit and NEBNext Multiplex oligos for Illumina were used to construct libraries for sequencing.