Binding of CTCF to the VEGF promoter. (A) EMSA analysis of CTCF binding to the VEGF promoter in vitro. A radiolabeled 100-bp DNA fragment carrying the CTCF-binding site from the VEGF promoter was incubated with control lysate or purified CTCF protein, and the mixture was separated by native polyacrylamide gel electrophoresis. Arrow indicates the DNA–CTCF complex; * denotes the supershifted complex by an anti-CTCF antibody. (B) ChIP analysis of CTCF binding at the VEGF proximal promoter. MCF7 cells were cross-linked and sonicated. Sonicated chromatin fragments were precipitated with an anti-CTCF antibody or control IgG. The relative concentrations of DNA fragments at the VEGF locus in the immunoprecipitated fractions were determined by real-time quantitative PCR. The 3′-UTR served as a reference sequence. (C) ChIP analysis of cohesin occupancy at the VEGF locus with an anti-Rad21 antibody. The assay was performed similarly to B.

Enhancer-blocking activity of CTCF in reporter-based assays. The CTCF site from the VEGF promoter was tested in different positions relative to enhancers (i.e., HRE and ERE) in various luciferase constructs. Before transfection, all constructs were linearized to avoid bidirectional action of the enhancers due to the circular nature of plasmids. (A) Schematic organization of the 5′ region of the VEGF gene. The binding sites for ERα, HIF, and CTCF are shown. (B) CTCF blocks HRE function. The scheme of each reporter construct with the HREs and the CTCF site is shown at the Left of the histogram. HEK293 cells were transiently transfected and 1 d later, treated with DP (100 μM). Luciferase assay was performed 2 d after transfection. Histogram shows relative luciferase reporter activities. (C) CTCF blocks ERE function. The reporter constructs with EREs and the CTCF site are shown schematically. Similar to B, 1 d after transfection, HEK293 cells were treated with E2 (100 nM), and luciferase activities were determined the following day. (D) Enhancer-blocking activity is attributable to CTCF. HEK293 cells were transduced with lentiviral vector (pGIPZ) or shRNA targeting CTCF (shCTCF). The knockdown efficiency of CTCF was determined by immunoblotting (Left of the histogram). Subsequently, the transduced cells were transiently transfected with luciferase reporters. DP treatment and luciferase assay were carried out similarly to B.

CTCF restrains the induction of endogenous VEGF and angiogenesis. (A) Northern blotting analysis of VEGF and CTCF in vector control (pGIPZ) and CTCF-depleted MCF7 cells under estrogen E2 (100 nM) and DP (100 μM) treatment for indicated time. Ribosomal RNA (18S) served as a loading control. (B and C) Quantitative measurement of VEGF RNA levels in pGIPZ control and CTCF-depleted cells under hypoxia (1% O₂) and estrogen treatments, respectively. (D) VEGF protein concentrations in conditioned media from pGIPZ control and CTCF-depleted MCF7 cells after 24-h treatment with DP or estrogen. The assay was performed with a VEGF ELISA kit (Invitrogen) following the manufacturer's instructions.

CTCF negatively regulates tumor angiogenesis. (A) Intradermal angiogenesis assay of pGIPZ control and CTCF-depleted MCF7 cells. Mice were s.c. inoculated with control or shCTCF MCF7 cells. Half of the mice were daily fed with estrogen (E2). Two days later, tumor nodules were collected and microvessels around each tumor were counted. The number of capillary vessels per tumor nodule was shown (n = 4 per group). Error bars represent SD. (B) EMSA analysis of the binding activity of cancer-derived CTCF mutants to the VEGF promoter. Flag-tagged wild-type (WT) and various mutant CTCF proteins were immunoprecipitated from HEK293 cells with anti-Flag antibodies. These proteins were eluted and subjected to EMSA. The loading of CTCF proteins used in EMSA is shown at the Top. Arrow indicates the DNA–CTCF complexes. (C) Cancer-derived CTCF mutants are deficient in enhancer blocking of VEGF. MCF7 cells were first depleted of endogenous CTCF and subsequently transduced with lentivirus expressing exogenous WT and mutant CTCFs. Reconstituted cells were treated with DP, and VEGF transcript levels were determined by quantitative RT-PCR.

Depletion of CTCF causes abnormal angiogenesis in the developing retina. Neonatal mouse retinas were subjected to subretinal injection of shRNA plasmids (pGIPZ control or shCTCF) followed by electroporation. Cross-sections of P14 retina are shown. Endothelial cells were selectively stained with isolectin B4-Alexa 594 (red) and nuclei with DAPI (blue). Ectopic growth of blood vessels into the photoreceptor layer is indicated by arrowheads. “Vertical” vessels migrated along GFP⁺ track (arrows).