ID1, inhibitor of differentiation/DNA binding, is an effector of the p53-dependent DNA damage response pathway

Abstract

ID1, inhibitor of differentiation/DNA binding, plays an important role in cell proliferation, differentiation, and tumorigenesis. It has been shown that ID1 is de-regulated in multiple cancers and up-regulation of ID1 is correlated with high grades and poor prognosis of human cancers. In contrast, the p53 tumor suppressor was found to be mutated or inactivated in most human cancers and loss of p53 results in early onset of multiple cancers. Although the biological functions of the ID1 oncogene and the p53 tumor suppressor have been intensively investigated, little is known about the upstream regulators of ID1 and the cross-talk between ID1 and p53. Here, we showed that ID1 is down-regulated in cells treated with various DNA damage agents in a p53-dependent manner. Interestingly, we found that DEC1, which was recently identified as a p53 target and mediates p53-dependent cell cycle arrest and senescence, is capable of inhibiting ID1 expression. Conversely, we found that knockdown of DEC1 attenuates DNA damage-induced ID1 repression. In addition, we identified several potential DEC1 responsive elements in the proximal promoter region of the ID1 gene. Moreover, we showed that overexpression of ID1 or ID1', an isoform of ID1, promotes cell proliferation potentially through inhibition of p21 expression. Finally, we found that the extent of DNA damage-induced premature senescence was substantially decreased by overexpression of ID1 or ID1'. Taken together, our study suggests that p53 trans-repressional activity can be mediated by its own target DEC1 and ID1 is an effector of the p53-dependent DNA damage response pathway.

FIGURE 1.

ID1 expression is inhibited by DNA damage in a p53-dependent manner. A and B, ID1 expression is down-regulated upon DNA damage. Western blots were prepared with extracts from HCT116 and U2OS cells that were untreated (–) or treated (+) with doxorubicin (Dox) or camptothecin (CPT) for 12, 16, 20, and 24 h, respectively. ID1, p53, p21, and actin were detected by their respective antibodies. C and D, knockdown of endogenous p53 partially alleviates the inhibition of ID1 expression upon DNA damage. Western blots were prepared with extracts from HCT116 cells that were transiently transfected with control scramble siRNA and p53 siRNA for 3 days, and then untreated (–) or treated (+) with doxorubicin (0.3 μg/ml) for 24 h or with camptothecin (100 nm) for 6 h. The blots were analyzed as in A.

FIGURE 2.

ID1 expression is down-regulated by DEC1. A, DEC1 is induced by DNA damage in a p53-dependent manner. Western blots were prepared with extracts from MCF7 and MCF7-p53KD cells that were untreated (–) or treated (+) with 0.35 μg/ml doxorubicin (Dox) for 24 h. B, ID1 is inhibited by DEC1. Northern blots were prepared with RNAs purified from MCF7 cells that were uninduced (–) or induced (+) to express DEC1. The blots were probed with cDNAs derived from the ID1 and GAPDH genes, respectively. C and D, ID1 expression is down-regulated by DEC1. Western blots were prepared with extracts from MCF7 cells that were uninduced (–) or induced (+) to express DEC1 or mutant DEC1-R58P and from U2OS cells that were uninduced (–) or induced (+) to express DEC1. DEC1, ID1, and actin were detected by their respective antibodies.

FIGURE 3.

Knockdown of endogenous DEC1 partially attenuates the inhibition of ID1 expression upon DNA damage. Western blots were prepared with extracts from HCT116 cells that were transiently transfected with control scramble siRNA and DEC1 siRNA for 3 days, and then untreated (–) or treated (+) with doxorubicin (Dox) (0.3 μg/ml) for 24 h. The blots were analyzed as described in the legend to Fig. 1A.

FIGURE 4.

ID1 is a direct target of DEC1. A, schematic presentation of the ID1 genomic structure with the location of the potential DEC1-REs (E-boxes). B, left panel, schematic presentation of three luciferase reporter constructs. See details in the text. Right panel, potential DEC1-REs in the ID1 gene are responsive to wild-type DEC1, but not mutant DEC1-M and DEC1-R58P. The lucifer-ase assay was performed as described under “Experimental Procedures.” C, generation of MCF7 cell lines that inducibly express HA-tagged DEC1. The levels of DEC1 were quantified with anti-HA. D, schematic presentation of the ID1, Survivin, and GAPDH promoters with the locations of potential DEC1-REs and PCR primers used for ChIP assays. E, DEC1 binds to the ID1 promoter in vivo. Upon induction or no induction of DEC1, MCF7 cells were cross-linked with formaldehyde followed by sonication. Chromatin was immunoprecipitated (IP) with anti-HA (HA-DEC1) or a control IgG. DEC1-responsive elements in the ID1 and Survivin genes were amplified by PCR. The binding of DEC1 to the GAPDH was quantified as a nonspecific binding control.

FIGURE 5.

Overexpression of ID1 or ID1′ promotes cell proliferation potentially through inhibition of p21 expression. A, generation of MCF7 cell lines that inducibly express ID1 or ID1′. The levels of ID1 and ID1′ were quantified with anti-ID1. B, ID1 and ID1′ promote colony formation in MCF7 cells. Colony formation assay was performed with MCF7 cells uninduced or induced to express ID1 or ID1′ for 14 days, as described under “Experimental Procedures.” C, quantification of the total number of colonies shown in B. The number of colonies was calculated in triplicate for each cell line. D, quantification of the percentage of colonies with a diameter of >1 mm. The percentage of colonies with a diameter of >1 mm was calculated in triplicate for each cell line. The average was plotted as the percentage of colonies (>1 mm) increased by ID1 or ID1′ expression. E, over-expression of ID1 and ID1′ inhibits p21 expression. Western blots were prepared using extracts from MCF7 cells uninduced (–) or induced (+) to express ID1 or ID1′ for 0, 24, 48, or 72 h.

FIGURE 6.

Overexpression of ID1 or ID1′ attenuates DNA damage-induced premature senescence. A, overexpression of ID1 or ID1′ diminishes DNA damage-induced premature senescence. MCF7 cells, which were uninduced (–) or induced (+) to express ID1 or ID1′ for 2 days and then untreated (–) or treated (+) with 0.03 μg/ml of doxorubicin for 2 days, were analyzed by SA-β-galactosidase staining assay as described in a previous study (34). To quantify SA-β-galactosidase positive colonies, 150–200 colonies were counted and colonies containing ≥50% SA-β-galactosidase positive cells were defined as senescent colonies. B, overexpression of ID1 or ID1′ diminishes DNA damage-induced up-regulation of hypophosphorylated p130 and pRb. Western blots were prepared with extracts from MCF7 cells that were uninduced (–) or induced (+) to express ID1 or ID1′ for 2 days and then untreated (–) or treated (+) with 0.03 μg/ml doxorubicin for 1 day.