Fig. 1
From: Deciphering the role of RNA in regulating CTCF’s DNA binding affinity in leukemia cells
Establish a cellular model to interfere with CTCF-RNA interactions to study the impact on CTCF’s DNA binding affinity. A Schematic diagram illustrating three techniques to disrupt CTCF-RNA interactions. On the left is an illustration of how the ectopic HA-tagged CTCF swap system works in combination with acute protein degradation of endogenous CTCF. The homozygous miniAID-mClover3 knockin SEM cell lines CTCFAID2/WT and CTCFAID2/dRBR were previously generated [11]. When added to cell culture, the 5-Ph-IAA auxin analog acts as a ligand to bind to the miniAID tag (fused to endogenous CTCF protein) and OsTIR1(F74G) protein to promote acute protein degradation through ubiquitination by the SCF complex. After 6 h of 5-Ph-IAA treatment, the CTCF HA-tagged WT or CTCF-HA-dRBR ectopic proteins were induced by doxycycline for a total of 18 h of doxycycline and 24 h of 5-Ph-IAA treatment. In the middle is an illustration of transcription inhibition by the natural product, triptolide. Triptolide was added to live cell culture to block the PolII activity and global nascent transcription. On the right is a diagram showing how RNase A was used during the ChIP-seq procedure, either added before (pre-fixation treatment) or after (post-fixation treatment) the chromatin fixation, to degrade global RNAs. B Immunoblot analysis of endogenous (CTCFAID2.0) and induced exogenous (HA-CTCF) expression of CTCF using an antibody for CTCF. CTCFAID2.0 expression can be seen in all untreated samples (−, −). After 6 h of 10 μM 5-Ph-IAA treatment, CTCFAID2 protein expression is degraded. Exogenous expression of HA-tagged CTCF wildtype and dRBR mutant is comparable to endogenous CTCF following 18 h of 1 μg/mL doxycycline with concurrent 10 μM 5-Ph-IAA treatment (+, +). GAPDH was included as a loading control. C Quality control of RNA inhibition upon triptolide and RNase A treatment. Total RNAs were collected after drug treatment, followed by reverse transcription. The cDNA was fragmented, amplified, and quantified by a bioanalyzer. Three replicates were included for each treatment