Limits...
Dual regulatory roles of human AP-endonuclease (APE1/Ref-1) in CDKN1A/p21 expression.

Sengupta S, Mitra S, Bhakat KK - PLoS ONE (2013)

Bottom Line: Interestingly, APE1 and AP4 showed mutual dependence for p21 repression.Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression.These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53's DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1's stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53- HCT116 cells. Ectopic expression of p53 in the p53- cells abrogated this repression suggesting that APE1's negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1's another binding site in p21's proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression. These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.

Show MeSH

Related in: MedlinePlus

Mutual dependence of APE1 and AP4 for p21 repression.(A & B) Real Time RT-PCR analysis in HCT116p53 cells showing relative quantitation of (A) AP4 transcript level after AP4-depletion by siRNA transfection (72 hrs); Western analysis of AP4 level shown in the inset, (B) p21 transcript level in the same cells as in A; *: p value <0.05 (n = 3) calculated from control vs. AP4-depleted cells. (C) Effect of AP4-depletion in control vs. APE1-depleted HCT116p53 cells. Cells were first transfected with control siRNA or APE1 siRNA, the next day both cell types were re-transfected with control or AP4 siRNA and after 72 hours the cells were harvested; *: p value <0.05 (n = 2) calculated based on the effect of AP4 knockdown in control vs. APE1-depleted cells. (D) Effect of APE1-depletion in control vs. AP4-depleted HCT116p53 cells; same experiment was performed as in C but analyzed differently; *: p value <0.05 (n = 2) calculated based on the effect of APE1 knockdown in control vs. AP4-depleted cells. (E & F) ChIP Real Time PCR analysis showing relative enrichment of (E) APE1 and (F) AP4 in p21 proximal promoter in control vs. APE1-depleted HCT116p53 cells; cells were transfected with control or APE1 siRNA and ChIP assay was performed after 72–96 hours; *: p value <0.05 (n = 4) calculated based on relative enrichment of APE1 or AP4 in control vs. APE1-depleted cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3713036&req=5

pone-0068467-g005: Mutual dependence of APE1 and AP4 for p21 repression.(A & B) Real Time RT-PCR analysis in HCT116p53 cells showing relative quantitation of (A) AP4 transcript level after AP4-depletion by siRNA transfection (72 hrs); Western analysis of AP4 level shown in the inset, (B) p21 transcript level in the same cells as in A; *: p value <0.05 (n = 3) calculated from control vs. AP4-depleted cells. (C) Effect of AP4-depletion in control vs. APE1-depleted HCT116p53 cells. Cells were first transfected with control siRNA or APE1 siRNA, the next day both cell types were re-transfected with control or AP4 siRNA and after 72 hours the cells were harvested; *: p value <0.05 (n = 2) calculated based on the effect of AP4 knockdown in control vs. APE1-depleted cells. (D) Effect of APE1-depletion in control vs. AP4-depleted HCT116p53 cells; same experiment was performed as in C but analyzed differently; *: p value <0.05 (n = 2) calculated based on the effect of APE1 knockdown in control vs. AP4-depleted cells. (E & F) ChIP Real Time PCR analysis showing relative enrichment of (E) APE1 and (F) AP4 in p21 proximal promoter in control vs. APE1-depleted HCT116p53 cells; cells were transfected with control or APE1 siRNA and ChIP assay was performed after 72–96 hours; *: p value <0.05 (n = 4) calculated based on relative enrichment of APE1 or AP4 in control vs. APE1-depleted cells.

Mentions: We then explored APE1’s regulatory role in p21 repression. First, we confirmed AP4’s repressor role in p21 expression (Fig. 5B) in p53 cells after siRNA-mediated depletion of AP4 (Fig. 5A, Western analysis for AP4 level in these cells shown in the inset). Next, we analyzed the effect of AP4 depletion on p21 activation in control and APE1-downregulated cells. Depletion of endogenous AP4 activated p21 expression in control cells, and interestingly this AP4-knockdown mediated p21 activation was inhibited in APE1-depleted cells (Fig. 5C). The reference samples in both control and APE1-depleted cells are control siRNA transfected cells and the effect of AP4 depletion was measured. This suggests that APE1 is required for AP4-mediated repression of p21. Next, the same set of samples was analyzed differently to examine the effect of APE1 depletion in control and AP4 depleted cells. Again, as expected APE1 depletion activated p21 expression in control cells, and in AP4-depleted cells, APE1 depletion could not activate p21 expression to the same efficiency (Fig. 5D). The reference samples in both control and AP4-depleted cells are control siRNA transfected cells and the effect of APE1 depletion was measured. These results imply that APE1 and AP4 are mutually dependent for p21 repression.


Dual regulatory roles of human AP-endonuclease (APE1/Ref-1) in CDKN1A/p21 expression.

Sengupta S, Mitra S, Bhakat KK - PLoS ONE (2013)

Mutual dependence of APE1 and AP4 for p21 repression.(A & B) Real Time RT-PCR analysis in HCT116p53 cells showing relative quantitation of (A) AP4 transcript level after AP4-depletion by siRNA transfection (72 hrs); Western analysis of AP4 level shown in the inset, (B) p21 transcript level in the same cells as in A; *: p value <0.05 (n = 3) calculated from control vs. AP4-depleted cells. (C) Effect of AP4-depletion in control vs. APE1-depleted HCT116p53 cells. Cells were first transfected with control siRNA or APE1 siRNA, the next day both cell types were re-transfected with control or AP4 siRNA and after 72 hours the cells were harvested; *: p value <0.05 (n = 2) calculated based on the effect of AP4 knockdown in control vs. APE1-depleted cells. (D) Effect of APE1-depletion in control vs. AP4-depleted HCT116p53 cells; same experiment was performed as in C but analyzed differently; *: p value <0.05 (n = 2) calculated based on the effect of APE1 knockdown in control vs. AP4-depleted cells. (E & F) ChIP Real Time PCR analysis showing relative enrichment of (E) APE1 and (F) AP4 in p21 proximal promoter in control vs. APE1-depleted HCT116p53 cells; cells were transfected with control or APE1 siRNA and ChIP assay was performed after 72–96 hours; *: p value <0.05 (n = 4) calculated based on relative enrichment of APE1 or AP4 in control vs. APE1-depleted cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713036&req=5

pone-0068467-g005: Mutual dependence of APE1 and AP4 for p21 repression.(A & B) Real Time RT-PCR analysis in HCT116p53 cells showing relative quantitation of (A) AP4 transcript level after AP4-depletion by siRNA transfection (72 hrs); Western analysis of AP4 level shown in the inset, (B) p21 transcript level in the same cells as in A; *: p value <0.05 (n = 3) calculated from control vs. AP4-depleted cells. (C) Effect of AP4-depletion in control vs. APE1-depleted HCT116p53 cells. Cells were first transfected with control siRNA or APE1 siRNA, the next day both cell types were re-transfected with control or AP4 siRNA and after 72 hours the cells were harvested; *: p value <0.05 (n = 2) calculated based on the effect of AP4 knockdown in control vs. APE1-depleted cells. (D) Effect of APE1-depletion in control vs. AP4-depleted HCT116p53 cells; same experiment was performed as in C but analyzed differently; *: p value <0.05 (n = 2) calculated based on the effect of APE1 knockdown in control vs. AP4-depleted cells. (E & F) ChIP Real Time PCR analysis showing relative enrichment of (E) APE1 and (F) AP4 in p21 proximal promoter in control vs. APE1-depleted HCT116p53 cells; cells were transfected with control or APE1 siRNA and ChIP assay was performed after 72–96 hours; *: p value <0.05 (n = 4) calculated based on relative enrichment of APE1 or AP4 in control vs. APE1-depleted cells.
Mentions: We then explored APE1’s regulatory role in p21 repression. First, we confirmed AP4’s repressor role in p21 expression (Fig. 5B) in p53 cells after siRNA-mediated depletion of AP4 (Fig. 5A, Western analysis for AP4 level in these cells shown in the inset). Next, we analyzed the effect of AP4 depletion on p21 activation in control and APE1-downregulated cells. Depletion of endogenous AP4 activated p21 expression in control cells, and interestingly this AP4-knockdown mediated p21 activation was inhibited in APE1-depleted cells (Fig. 5C). The reference samples in both control and APE1-depleted cells are control siRNA transfected cells and the effect of AP4 depletion was measured. This suggests that APE1 is required for AP4-mediated repression of p21. Next, the same set of samples was analyzed differently to examine the effect of APE1 depletion in control and AP4 depleted cells. Again, as expected APE1 depletion activated p21 expression in control cells, and in AP4-depleted cells, APE1 depletion could not activate p21 expression to the same efficiency (Fig. 5D). The reference samples in both control and AP4-depleted cells are control siRNA transfected cells and the effect of APE1 depletion was measured. These results imply that APE1 and AP4 are mutually dependent for p21 repression.

Bottom Line: Interestingly, APE1 and AP4 showed mutual dependence for p21 repression.Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression.These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53's DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1's stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53- HCT116 cells. Ectopic expression of p53 in the p53- cells abrogated this repression suggesting that APE1's negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1's another binding site in p21's proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression. These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.

Show MeSH
Related in: MedlinePlus