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p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage
K F Macleod1, N Sherry, G Hannon
1Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge 02139, USA.
Abstract:
Expression of p21 has been shown to be up-regulated by the p53 tumor suppressor gene in vitro in response to DNA-damaging agents. However, p21 expression can be regulated independently of p53, and here we show that expression of p21 in various tissues during development and in the adult mouse occurs in the absence of p53 function. However, most tissues tested did require p53 for p21 induction following exposure of the whole animal to gamma irradiation. These results show that normal tissue expression of p21 to high levels is not dependent on p53 and confirm that induction of p21 by DNA-damaging agents does require p53. p21 is expressed upon differentiation of p53-deficient murine erythroleukemia (MEL) cells, and the kinetics of induction of p21 in this system suggest that it may be involved in the growth arrest that precedes terminal differentiation. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells. Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in p53-deficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels. p21 expression also appears to be regulated at the post-transcriptional level because moderate increases in mRNA expression, during differentiation of MEL cells and upon serum restimulation of fibroblasts, are followed by large increases in protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical p53-binding sites located 1.95 and 2.85 kb upstream from the transcriptional initiation site. The sequences mediating serum responsiveness of the promoter map to a region containing the proximal p53 site. p53 appears to play a critical role in p21 induction following DNA damage. Moreover, p21 can be regulated independently of p53 in several situations including during normal tissue development, following serum stimulation, and during cellular differentiation.
Insights
The p53 tumor suppressor gene often regulates p21 expression, but this study shows p21 can be expressed without p53 during development and differentiation. However, p53 is crucial for p21 induction after DNA damage.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The p53 tumor suppressor gene is known to up-regulate p21 expression in response to DNA damage.
- p21 expression can also be regulated independently of p53.
Purpose of the Study:
- To investigate the p53-independent regulation of p21 expression.
- To determine the role of p53 in p21 induction during development, differentiation, and in response to DNA damage.
Main Methods:
- Analysis of p21 expression in various mouse tissues during development and in adult mice.
- Studying p21 induction in p53-deficient murine erythroleukemia (MEL) cells and mouse fibroblasts.
- Investigating p21 promoter regulation using p53-binding sites and serum stimulation assays.
Main Results:
- p21 is expressed in various tissues during mouse development and in adults, independent of p53 function.
- p53 is required for p21 induction in most tissues following gamma irradiation.
- p21 is expressed during differentiation of p53-deficient MEL cells and upon serum restimulation of fibroblasts, with evidence of post-transcriptional regulation.
- p53-binding sites at 1.95 and 2.85 kb upstream regulate the mouse p21 promoter, with serum responsiveness linked to the proximal p53 site.
Conclusions:
- Normal tissue expression of p21 is largely independent of p53.
- p53 is critical for p21 induction by DNA-damaging agents.
- p21 exhibits p53-independent regulation during normal tissue development, serum stimulation, and cellular differentiation, potentially involving post-transcriptional mechanisms.