De-SUMOylation of CCCTC binding factor (CTCF) in hypoxic stress-induced human corneal epithelial cells

Abstract

Epigenetic factor CCCTC binding factor (CTCF) plays important roles in the genetic control of cell fate. Previous studies found that CTCF is positively and negatively regulated at the transcriptional level by epidermal growth factor (EGF) and ultraviolet (UV) stimulation, respectively. However, it is unknown whether other stresses modify the CTCF protein. Here, we report that regulation of CTCF by de-SUMOylation is dependent upon hypoxic and oxidative stresses. We found that stimulation of human corneal epithelial cells with hypoxic stress suppressed a high molecular mass form of CTCF (150 kDa), but not a lower molecular weight form of CTCF (130 kDa). Further investigation revealed that the hypoxic stress-suppressed 150-kDa CTCF was a small ubiquitin-related modifier (SUMO)ylated form of the protein. Hypoxic stress-induced de-SUMOylation of human CTCF was associated with lysine 74 and 689 residues, but not to the phosphorylation of CTCF. Overexpression of SENP1 induced de-SUMOylation of CTCF. However, knockdown of SENP1 could not rescue hypoxic stress-induced CTCF de-SUMOylation. Overexpression of SUMO1 and SUMO2 increased SUMOylation of CTCF and partially blocked hypoxic stress-induced CTCF de-SUMOylation, suggesting that free cellular SUMO proteins play roles in regulating hypoxia-induced CTCF de-SUMOylation. In addition, hypoxic stress significantly inhibited PAX6 mRNA and protein expressions by suppression of PAX6-P0 promoter activity. The result was further supported by data showing that knockdown of CTCF significantly enhanced expression of PAX6 and abolished hypoxia-induced suppression of PAX6. Thus, we conclude that hypoxic stress induces de-SUMOylation of CTCF to functionally regulate CTCF activity.

FIGURE 1.

Effects of stress stimulation on CTCF expression. A, CTCF expression in HCE cells induced by UV irradiation, hyperosmotic pressure, heat shock, H₂O₂, and hypoxic stress. B, time course of hypoxic stress-induced alteration of the 150-kDa CTCF expression. C, H₂O₂ oxidative stress-induced suppression of the 150-kDa CTCF following a time course. D, hypoxic stress-induced alterations of the 150-kDa CTCF in human telomerase-deficient immortalized corneal epithelial (HTCE), HEK-293, and α-TC-6 cells. Cells were synchronized by serum starvation for 24 h prior to stress treatments at the indicated time points. Endogenous CTCF was detected by Western analysis using anti-CTCF. Data were plotted as mean ± S.E. Asterisk indicates significant differences tested by analysis of variance and Student's t test (p < 0.05, n = 3).

FIGURE 2.

Effects of hypoxic stress on SUMOylation of CTCF. A, effects of NEM, protease inhibitor mixture (PIC), and SDS on SUMOylation of CTCF protein detected by Western analysis. Whole cell lysates were individually treated with 20 mm NEM, protease inhibitor mixture (PIC), containing 1 mm PMSF, 1 μg/ml of aprotinin, and 1 μg/ml of leupeptin) or 2% SDS. B, detection of CTCF SUMOylation in control and hypoxic stress-induced HCE cells by IP using anti-CTCF antibody and immunoblotting (IB) with anti-SUMO1 and anti-SUMO2/3 antibodies. C, effect of MG132 on de-SUMOylation of CTCF following a time course in HCE cells. CTCF in whole cell lysate was detected by Western analysis using antibody against CTCF. D, detection of CTCF SUMOylation in control and hypoxic stress-induced HCE cells by immunoprecipitation using anti-SUMO2/3 antibody and immunoblotting with anti-CTCF antibody. E, comparison of hypoxic stress-induced CTCF de-SUMOylation detected by Western blots of whole cell lysates and products immunoprecipitated with anti-SUMO2/3 antibody. F, effect of hypoxic stress on de-SUMOylation of CTCF by co-transfection of cells with FLAG-CTCF and HA-SUMO1 or HA-SUMO2. Co-transfected HEK-293 cells were treated by hypoxic stress for 4 h prior to harvesting. Tagged CTCF was pulled down by antibody against FLAG, and HA-SUMO groups were detected by anti-HA and anti-FLAG. All of data sets were repeated three times from three independent experiments, and the results were very consistent.

FIGURE 3.

Effects of overexpressing SENPs and CTCF-K74R/K689R mutant on CTCF SUMOylation. A, effects of overexpressing SENP1, SENP3, and SENP5 on CTCF SUMOylation. HEK-293 cells were transfected with GFP-SENP1, -3, and -5 for 48 h before they were harvested for Western analysis. Anti-CTCF was used to detect the endogenous CTCF. Anti-GFP was used to detect the expression level of exogenous SENPs. B, effect of co-transfecting FLAG-tagged wild type and CTCF K74R/K689R mutant with HA-SUMO1/2 on SUMOylation of CTCF, respectively. Immunoprecipitation experiments were performed by using anti-FLAG antibody and followed by immunoblotting using anti-HA and anti-FLAG antibodies. C, effect of co-transfecting wild type and K74R/K689R mutant of the human CTCF with HA-SUMO1 or HA-SUMO2 on the 150-kDa CTCF band detected by Western analysis using antibody against CTCF. D, localization of wild type CTCF and CTCF mutant in the nucleus. Exogenous CTCF and its mutant were localized in transfected 293T cells by immunostaining using anti-FLAG antibody. Data were plotted as mean ± S.E. Asterisk indicates a significant difference determined by analysis of variance (p < 0.05, n = 3).

FIGURE 4.

Effect of knocking down SENP1 mRNA on hypoxia-induced CTCF de-SUMOylation. A, detection of knocking down SENP1 mRNA with SENP1-specific siRNA by real-time PCR. B, effect of knocking down SENP1 mRNA with SENP1-specific siRNA on SENP1 protein expression. C, effect of knocking down SENP1 on hypoxic stress-induced de-SUMOylation of CTCF. HCE cells were transfected with siRNA against SENP1 or nonsilencing siRNA for 72 h. D, knockdown of SENP1 mRNA with SENP1-specific shRNA detected by real-time PCR. E, knockdown of SENP1 mRNA with SENP1-specific shRNA suppressed SENP1 protein expression. F, effect of knocking down SENP1 with SENP1-specific shRNA on hypoxic stress-induced CTCF de-SUMOylation. HCE cells were infected with CTCF-specific shRNA or nonsilencing shRNA in the lentiviral delivery system, and selected in the medium in the presence of 800 μg/ml of G418 for 2 weeks. HCE cells were stimulated by hypoxic stress for 1 h before the cells were harvested for Western analysis to detect the SUMOylation level of CTCF. Real-time PCR data were plotted as mean ± S.E. and the asterisk indicates significant difference determined by Student's t test (p < 0.05, n = 3).

FIGURE 5.

Regulation of CTCF de-SUMOylation by hypoxic stress. A, effect of mutating the phosphorylation site of CTCF (CTCF-ALA mutant) on SUMOylation of CTCF. The CTCF-ALA mutant was established by replacing four serine residues with glycine residues. HEK-293 cells were co-transfected with FLAG-wild type CTCF and FLAG-CTCF-ALA mutant with HA-SUMO1 or HA-SUMO2 for 48 h. B, effect of overexpressing SUMO1 or SUMO2 on hypoxic stress-induced de-SUMOylation of CTCF. HEK-293 cells were transfected with HA, HA-SUMO1, or HA-SUMO2 individually 48 h prior to exposure of cells to hypoxic stress for 2 h. Data were plotted as mean ± S.E. Asterisk indicates the significant difference between the marked groups determined by analysis of variance (p < 0.05, n = 3). C, detection of the global SUMOylation pattern in hypoxic stress-induced HCE cells. HCE cells were exposed to hypoxic stress for the indicated time points, and samples were subjects to Western analysis by using antibodies against SUMO1 (left panel) and SUMO2/3 (right panel). Arrows indicate SUMO-conjugates and free-SUMO. Stars indicate nonspecific bands.

FIGURE 6.

Effect of CTCF de-SUMOylation on PAX6 activity in HCE cells. A, effect of CTCF de-SUMOylation on PAX6-P0 promoter activity. HCE cells were co-transfected with FLAG-wild type CTCF and CTCF-K74R/K689R mutant with the pGL2-PAX6-P0 reporter, respectively. B, effect of CTCF deSUMOylation on CMV-promoter reporter. For the control experiments, pGL2-CMV vector was co-transfected with FLAG-wild type CTCF and CTCF-K74R/K689R mutant into HCE cells. C, effect of hypoxic stress on PAX6 mRNA expression. Expression of PAX6 mRNA was determined by real-time PCR and the β-glucuronidase (GUSB) mRNA level was also measured as an internal control. D, effect of hypoxic stress on PAX6 protein expression. E, suppression of CTCF expression by knocking down CTCF mRNA with CTCF-specific shRNA. F, effect of knocking down CTCF on hypoxia-induced PAX6 mRNA expression. G, effect of knocking down CTCF on hypoxia-induced PAX6 protein expression. CTCF-specific shRNA was introduced into HCE cells using the lentiviral delivery system to knockdown CTCF mRNA and nonsilencing (NS) shRNA served as controls. The infected cells were cultured and selected in the medium containing 800 μg/ml of G418 for 2 weeks. Expression of PAX6 protein was determined by Western analysis and the β-actin level was measured as a loading control. HCE cells were synchronized by serum starvation for 24 h prior to hypoxic stimulation. Asterisk represents the statistical significance determined by analysis of variance and Student's t test (p < 0.05, n = 3).