Fig. 1

The AID2 system promoted rapid CTCF degradation, reduced cellular toxicity, and genome-wide loss of CTCF binding and chromatin looping. a Diagram illustrating how the AID2 system works. The 5-Ph-IAA auxin analog binds to the miniAID tag and recruits the SCF complex by binding to the OsTIR1(F74G) protein. b Flow cytometry analysis was performed on CTCF^AID2 cells following 10 μM 5-Ph-IAA treatment over a time course from 0 to 24 h. RFP fluorescence represents exogenous expression from lentiviral integration of the pCDH-MND-OsTIR1(F74G)-P2A-EGFP^AID2-EF1a-RFP construct. Endogenous CTCF was expressed in frame with a miniAID tag and mClover3 (CTCF^AIDmClover3), and exogenous EGFP from the pCDH-MND-OsTIR1(F74G)-P2A-EGFP^AID2-EF1a-RFP construct was expressed in frame with a miniAID tag (EGFP^AID2). The fluorescence of both CTCF^AIDmClover3 and EGFP^AID2 was detected in the mCLover3 panel. c Immunoblot analysis from SEM^WT, SEM^OsTIR1(F74G), CTCF^AID1, and CTCF^AID2 cells following 24 h of DMSO or 1 μM 5-Ph-IAA treatment. The lower CTCF band represents untagged endogenous CTCF (CTCF-WT). The higher CTCF band represents CTCF^AIDmClover3 (CTCF-AID). GAPDH was included as a loading control. d Immunoblot analysis of CTCF^AID2 cells treated with AID2 conditions over a 4-day time course. CTCF antibody was used to detect CTCF^AIDmClover3. e Immunoblot analysis of CTCF^AID2 cells treated with AID1 conditions over a 4-day time course. CTCF antibody was used to detect CTCF^AIDmClover3. f Growth assay of CTCF^AID2 cells treated with AID2 conditions over 4 days. Samples were set up in triplicate and cell counts for each replicate were collected daily. Bars represent cell numbers (millions). g Growth assay of CTCF^AID2 cells treated with AID1 conditions over 4 days. Samples were set up in triplicate and cell counts for each replicate were collected daily. Bars represent cell numbers (millions). h Cell cycle analysis was performed on samples from the 24-h time points from f and g and plotted for cell fraction percentage according to cell cycle phase. i Cell cycle analysis was performed on samples from the 72-h time points from f and g and plotted for cell fraction percentage according to cell cycle phase. j Homer motif enrichment analysis illustrated the overall distribution of motifs identified in CTCF ChIP-seq of CTCF^AID2 cells without auxin treatment. Enriched CTCF motifs were shown as red circles. k Principal component analysis from CTCF ChIP-seq of CTCF^AID2 cells with (2 replicates) or without (3 replicates) 10 μM 5-Ph-IAA treatment for 24 h. l Genomic heatmap centered at reproducible CTCF peak summits from CTCF^AID2 cells with (2 replicates) or without (3 replicates) 10 μM 5-Ph-IAA treatment for 24 h. m Aggregate peak analysis (APA) plots from CTCF HiChIP of CTCF^AID2 cells with or without 10 μM 5-Ph-IAA treatment for 6 h (2 replicates each, merged after confirmation of reproducibility). 7220 loops were called in CTCF HiChIP without 5-Ph-IAA. The x/y-axis was centered at loop anchors spanning from − 10 to + 10 windows (window size 10 kb), and the z-axis was normalized aggregated contact frequency