Fig. 4
From: Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation
Disrupted chromatin binding of ZF1 and ZF10 mutants. a Design of the inducible CTCF HA-tagged constructs. b Immunoblot analysis of endogenous (CTCFAID2) and induced exogenous (HA-CTCF) expression of CTCF using an antibody for CTCF. CTCFAID2 expression can be seen in all untreated samples (− , −). After 6 h of 10 μM 5-Ph-IAA treatment, CTCFAID2 expression is depleted (+ , −). Exogenous HA-CTCF expression comparable to endogenous CTCF was only observed following 18 h of 1 μg/mL doxycycline (+ , +). c Pearson correlation scatterplot comparing the log2 mean RPM enrichment of mutant HA CTCF ChIP-seq peaks to the log2 mean RPM enrichment of WT CTCF ChIP-seq peaks. d Heatmap of normalized CTCF signal centered at differential CTCF peaks from 2 replicates of HA ChIP-seq of CTCFAID2/WT and CTCFAID2/dZF1 cells following 10 μM 5-Ph-IAA treatment for 24 h and 18 h of 1 μg/mL doxycycline. Differential ChIP-seq peak analysis respectively identified 582 and 1875 significantly enriched (up) or depleted (down) peaks (criteria: |log2(FC)|≥ 1 and FDR ≤ 0.05, using Limma-Voom analysis) in CTCFAID2/dZF1 samples. e Heatmap of normalized CTCF signal centered at differential CTCF peaks from 2 replicates of HA ChIP-seq of CTCFAID2/WT and CTCFAID2/dZF10 cells following 10 μM 5-Ph-IAA treatment for 24 h and 18 h of 1 μg/mL doxycycline. Differential ChIP-seq peaks analysis respectively identified 525 and 4084 significantly increased (up) or depleted (down) peaks (cutoff: |log2(FC)|≥ 1 and FDR ≤ 0.05, using Limma-Voom analysis) in CTCFAID2/dZF10 HA samples. f Heatmap of normalized CTCF signal centered at differential CTCF peaks from 2 replicates of HA ChIP-seq of CTCFAID2/WT and CTCFAID2/dRBR cells following 10 μM 5-Ph-IAA treatment for 24 h and 18 h of 1 μg/mL doxycycline. Differential ChIP-seq peaks analysis respectively identified 102 and 29 significantly increased (up) or depleted (down) peaks (cutoff: |log2(FC)|≥ 1 and FDR ≤ 0.05, using Limma-Voom analysis) in CTCFAID2/dRBR HA samples. g Venn diagram illustrating the differential binding profiles of CTCFAID2/dZF1, CTCFAID2/dRBR, and CTCFAID2/dZF10 had little overlap