Fig. 5

CTCF is also required for a subset of GATA1-mediated gene repression. a The top ten enriched TFs associated with the genes with decreased ATAC peaks and upregulated expression in IAA-treated cells were identified via the EnrichR database. The relative P value was calculated from the database. b Heatmaps showing the dynamic GATA1 binding signal centered at the GATA1 peak summit for decreased and increased GATA1 regions in the HUDEP-2 cell line without and with IAA treatment for 24 h. The GATA1 ChIP-seq results were merged with analyses from two independent replicates. c Screenshot of KIT, with ATAC-seq, CTCF ChIP-seq, and GATA1 ChIP-seq signals and annotated TAD domains and chromatin loops from the genome-wide Hi-C interaction map before and after IAA treatment in CTCF-AID HEL cells. The red arrow highlights the decreased CTCF ChIP-seq peaks in the enhancer and promoter regions. The black cycle indicates the loop position identified with no change before and after IAA treatment in CTCF-AID HEL cells. d Plot of fragments per kilobase of peaks per million reads mapped (FPKM) values and validated mRNA levels of KIT expression in CTCF-AID HUDEP-2 cells without and with IAA treatment in both the immature and mature states. The mRNA level was determined relative to that of β-actin from three replicates. ** P < 0.01, *** P < 0.001, unpaired Student’s t test. e Screenshot of GATA2, with ATAC-seq, CTCF ChIP-seq, and GATA1 ChIP-seq signals and annotated TAD domains and chromatin loops from the genome-wide Hi-C interaction map before and after IAA treatment in CTCF-AID HEL cells. The red arrow highlights the decreased ATAC-seq and CTCF ChIP-seq in the distal region. The black cycle indicates the loop position identified in CTCF-AID HEL cells. f Plot of fragments per kilobase of peaks per million reads mapped (FPKM) values and validated mRNA levels of GATA2 expression in CTCF-AID HEL cells without and with IAA treatment in both the immature and mature states. The mRNA level was determined relative to that of β-actin from three replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t test. g The cartoon illustrating the location of CBS1 (CTCF binding site 1) between GATA2 and its downstream neighbor gene RPN1, which is about 129 kb to the first exon of the GATA2 gene. h Measurement of GATA2 mRNA by quantitative real-time PCR (left panel) and GATA2 protein by Western blot (right panel) in the GATA2-CBS1-sgRNA targeted bulk population relative to non-targeting sgRNA control in HUDEP-2 cells. The mRNA expression levels were normalized to those of β-actin mRNA. The graph shows the results as the mean values ± SEMs from three replicates. *P < 0.05, unpaired Student’s t test. The β-actin was used as a loading control for immunoblots. i Measurement of GATA2 mRNA by quantitative real-time PCR in three individual GATA2-CBS1 knockout HUDEP-2 cell clones generated with Cas9 + CBS1-sgRNA. The expression levels were normalized to those of β-actin mRNA. The graph shows the results as the mean values ± SEMs from three replicates. **P < 0.01, **** P < 0.0001, unpaired Student’s t test