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. 2021 Jul 23;41(8):e00382-20. doi: 10.1128/MCB.00382-20

FIG 5.

FIG 5

CTCF assists in the recruitment or maintenance of YY1 binding to the promoter-proximal region of XIST. (A) ChIP-qPCR analysis showing a reduction of YY1 occupancy on the promoter-proximal site (+1.5 kb) upon siRNA-mediated knockdown of CTCF in HEK293T cells (siCTCF). The x axis represents the antibody used for ChIP, and the y axis represents the normalized fold enrichment over control siRNA. Each bar represents values from 3 independent experiments. Error bars represent ±SEM. Asterisks represent the significance over YY1 ChIP for vector control per Student’s t test (*, P value < 0.05). The x axis represents the transfected siRNA, and the y axis represents the normalized fold enrichment over control siRNA. (B) Immunoblotting to confirm knockdown of CTCF and overexpression of FLAG-tagged YY1 in HEK293T cells. γ-Tubulin serves as an equal loading control. (C) qRT-PCR for mature XIST upon knockdown of CTCF and/or overexpression of FLAG-YY1 in HEK293T cells. The x axis represents the DNA and siRNA transfected, and the y axis represents the normalized fold enrichment over 18S rRNA. Each bar represents values from 2 independent experiments. Error bars represent ±SEM. Asterisks represent the significance over control (first bar) per Student’s t test (***, P value < 0.001; ns, nonsignificant). (D) Schematic depicting dCas9-KRAB-based repression strategy. (E) qRT-PCR demonstrating reduction in XIST (both mature and premature) levels upon transfecting dCas9 + sgRNA in HEK293T cells. The x axis represents the mature or premature XIST, and the y axis represents the fold change normalized to dCas9-only control. Each point on the graph represents values from 6 independent experiments, and error bars represent ±SEM. Asterisks represent the significance over vector control per Student’s t test (****, P value < 0.0001). (F) ChIP-qPCR analysis showing enrichment of dCas9, CTCF, and H3K9me3 at cRE (+4.5 kb, as shown in the schematic above) on exon 1 in HEK293T cells transfected with just dCas9 or dCas9 + sgRNA. The x axis represents the antibodies used for ChIP, and the y axis represents the enrichment calculated as percent input. Each bar represents values from 3 independent experiments. Error bars represent ±SEM. Asterisks represent the significance over dCas9 control per Student’s t test (*, P value < 0.05). (G) ChIP-qPCR analysis showing enrichment of YY1 and H3K9me3 at promoter-proximal region (+1.5 kb, as shown in the schematic above) on exon 1 in HEK293T cells transfected with just dCas9 or dCas9 + sgRNA. The x axis represents the antibodies used for ChIP, and the y axis represents the enrichment calculated as percent input. Each bar represents values from 3 independent experiments. Error bars represent ±SEM. Asterisks represent the significance over dCas9 control per Student’s t test (****, P value < 0.0001; *, P value < 0.05).