COUP-TFII is a major regulator of cell cycle and Notch signaling pathways

Abstract

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)II has been shown to play a major role in endothelial cell growth and regulation of the Notch signaling pathway to confer vein identity. However, the underlying mechanisms for COUP-TFII regulation in these pathways remain to be defined. Here we employed a genomic approach by using microarray analysis to identify downstream targets in human umbilical vein endothelial cells (HUVEC) cells and found the expression of many genes in the cell cycle pathway and Notch signaling pathway are significantly altered in the COUP-TFII-depleted cells. The expression of E2F transcription factor 1 (E2F1), a key transcription factor that regulates the expression of cell cycle regulators, is reduced in the absence of COUP-TFII. Using chromatin immunoprecipitation experiments, we showed that COUP-TFII directly regulates the expression of E2F1 through tethering to the Sp1 binding sites in the promoter of E2F1 to modulate cell proliferation. In addition, we also demonstrate that Foxc1 and Np-1, two upstream genes of Notch signaling and Hey2, a downstream effector of Notch signaling, are direct targets of COUP-TFII. Furthermore, COUP-TFII suppresses the expression of EphrinB2, an arterial marker, while enhancing the expression of ephrin receptor B4, a venous marker, supporting our in vivo findings that COUP-TFII regulates vein identity by suppressing the Notch signal pathway.

Fig. 1.

Up-regulation of members of the cell cycle signaling pathway in the COUP-TFII knockdown HUVEC cells. A, Heat map of microarray analysis of members of the cell cycle signaling pathway in HUVEC cells treated with COUP-TFII-specific siRNA or scrambled control siRNA. B, qRT-PCR analysis of CyclinA2, Cyclin B1, Cyclin B2, Cyclin E1, Cyclin E2, CDC6, CDC25A, SRC-3, E2F1, and COUP-TFII expression in control and COUP-TFII-depleted HUVEC cells. Error bars indicate sem. *, P < 0.05; **, P < 0.005; ***, P < 0.001.

Fig. 2.

COUP-TFII regulates E2F1 expression in HUVEC cells. A, ChIP analysis shows that COUP-TFII binds to the region containing Sp1 binding sites in the E2F1 promoter. The region in the downstream without a Sp1 binding site served as a negative control. B, ChIP and qPCR analysis of COUP-TFII binding to E2F1 promoter in control and COUP-TFII -depleted HUVEC cells. C, HUVEC cells were transfected with Sp1-specific siRNA to knock down Sp1 expression. ChIP assay was performed using anti-COUP-TFII or Sp1 antibody to test whether COUP-TFII was recruited to the E2F1 promoter in a Sp1-depedent manner. D, ChIP and qRT-PCR analysis shows that both COUP-TFII and Sp1 bind to the region containing a Sp1 binding site in the E2F1 promoter in PC3 cells. E, Relative luciferase activity of the E2F1 promoter-driven reporter with or without cotransfection of COUP-TFII. F, Western blotting analysis of E2F1 expression in control and COUP-TFII overexpression cells with or without Sp1 depletion. Neg, Negative. *, less than 0.05; **, less than 0.01.

Fig. 3.

Up-regulation of Notch signaling pathway genes in the COUP-TFII knockdown of HUVEC cells. A, Heat map of microarray analysis of members of the Notch pathway in HUVEC cells treated with COUP-TFII-specific siRNA or scrambled control siRNA. B, qRT-PCR analysis of Notch4, Foxc1, Hey2, Hes1, Hes2, PSENEN, NCSTN, EphrinB2, EphB4, CD44, Np-1, Np-2, and COUP-TFII expression in control and COUP-TFII-depleted HUVEC cells. Error bars indicate sd. *, P < 0.05; **, P <0.005; ***, P < 0.001.

Fig. 4.

qRT-PCR analysis of Hes1, Hes2, and Hey2 expression in control and COUP-TFII-depleted HUVEC cells after DAPT and DMSO treatment. Error bars indicate sd. **, P < 0.005; ***, P < 0.001.

Fig. 5.

COUP-TFII represses the transcription of Hey2, Foxc1, and Np-1 in HUVEC cells. A, Endogenous COUP-TFII in HUVEC cells binds to the region containing conserved DR3 binding site in the Hey2 promoter and DR2 binding sites in the Foxc1 and Np-1 promoters. Promoter region in the downstream without COUP-TFII binding site served as negative control. qPCR results of ChIP assay were shown in the bottom of the figure. B, Relative luciferase activity of Hey2 promoter-driven reporter (−2 kb, −480 bp, and –240 bp) with or without cotransfection of COUP-TFII. C, Relative luciferase activity of Foxc1 promoter-driven reporter (−2.5 kb, −1.1 kb, and –385 bp) with or without cotransfection of COUP-TFII. D, Western blotting analysis of Hey2, Foxc1, Np-1, and COUP-TFII expression in control and COUP-TFII-depleted HUVEC cells. E, Model depicts how COUP-TFII regulates genes in the Notch signaling pathway. COUP-TFII directly and negatively regulates Foxc1 and Np-1 expression. In the absence of COUP-TFII, an increase in Foxc1 and Np-1 expression will stimulate Dll4 expression and subsequently activate Notch signaling. In addition, COUP-TFII directly binds to the Hey2 promoter to repress Hey2 expression to ensure that Notch signaling is silent. Neg, Negative; *P < 0.05; **, P < 0.01.