Studies Of P21～(WAF1/Cip1) Distal Promoter And Self-feeback Transcriptional Regulation

As a member of the Cip/Kip family of proteins, p21 was the first inhibitor of Cyclin dependent kinase identified in mammalian cells and the transcriptional target of p53. Induction of p21 is associated with many cellular processes, including checkpoint arrest in response to many different types of damage, cell proliferation, terminal differentiation and senescence. As the key protein, p21 regulates cell cycle G1/S or G2/M arrest, DNA repair and apoptosis. However, few mutation or allele loss of p21 was detected in several tumor tissue and almost a hundred of cell lines though it indeed plays important roles in tumor progress. Many indepth studies of transcriptional elements on p21 promoter have been shown. But most studies concentrated on the promoter region 0~ -2.5kb upstream of p21 transcription initiation site, and less works focused on the distal -2.5～-4.5kb upstream region. In addition to regulating the cell cycle, several studies revealed that p21 also associates with transcription factors and coactivators, such as C/EBPα, c-Myc, STAT3, E2F and CBP(CREB-binding protein)/p300, and modulate their function. Global changes in gene expression after p21 over-expression have recently been revealed. In particular, p21 is found may be activated by exogenous p21 protein. In our experiments, we found that the level of endogenous p21 was increased after over-expression of HA-p21. However, little is known about the molecular mechanism and biological significance of the self-regulation of p21 and to what extent it is linked to effects on the cell and cell cycle. In view of such puzzle of p21 function in tumor and the present situation in p21 study, we performed further investigations on the distal promoter, mechanism of p21 self-feedback regulation and protein modification.To analyze the p21 distal promoter, we identified a reverse repeat sequence in the -2664～-3338 bp region upstream of the transcription initiation site, which belongs to the Alu family but with several diversified bases. This region has potent inhibitory activity which is related to its specific secondary structure for only total reverse repeat sequence have the highest inhibitory activity comparing to upstream, downstream and middle region. Some inhibitory transcription fac- tor-binding elements may also exist in this region for separate parts of the reverse repeat region still have inhibitory activity. The inhibitory activity of reverse repeat region is ubiquitous in different cells and for different activators. Using EMSA assays, we show proteins binding activity in this region. With bioinfor-matics couple with experiments analysis, we identified several cisacting elements, including Sp1, C/EBPa and C/EBPβelements.In this study, we also show a new transcriptional self-feedback loop regulation mechanism of p21, that p21 is also regulated by its own protein product in the early period under cellular stress. We demonstrate that expression of exogenous p21 leads to increased endogenous p21 protein levels. The promoter of p21 fused to a luciferase reporter can be transactivated by p21 protein, indicating that p21 regulates its own promoter for transcriptional control of expression. Further regulatory mechanisms were then determined. In response to drug stress, p21 protein associates with and recruits Sp1, p53 and CBP but not p300 or E2F1, to the proximal Sp1 elements, and then they together with C/EBPαto the distal novel Sp1 and C/EBPαelements, thereby enhancing its own gene transcription. Using coimmunoprecipitation and Re-ChIP assays, we confirmed that p21, p53, Sp1 and CBP are involved in the same complex at proximal and distal sites during positive feedback regulation. Significantly, we found that p53 is not necessary but indeed participates in this regulation.Further investigation for physiological significance of p21 self-feedback regulation loop, we demonstrate that p21 self-feedback regulation exists in several different derivation cell lines and under different drugs treatment, suggesting that the self-feedback regulation of p21 may be ubiquitous in cells. Analysis of the promoter binding of proteins factors at different time points, we demonstrate that this feedback process is an early responsive mechanism under cellular stress. Through Flow cytometry assay, we revealed that pre-transfection with p21 promotes dys-growth HeLa cells into apoptosis and maintains cell cycle phases distribution in normal state under TSA-stress, suggesting that that the early transcriptional feedback enhancement of p21 may be an important self-protective mechanism for cells in response to cellular stress, and thus provide a useful reference for clinical cancer therapy. Take together, these data demonstrate that complex feedback regulation of p21 plays an important role in controlling itself transcription and in protecting cells from stress damage. These results reveal a previously undescribed mechanism for transcriptional regulation of p21 gene and demonstrate that p21 protein can also control its own gene transcription.After over-expression, we observed that p21 protein has been complicatedly modified, including ubiquitylation, phospholylation and acetylation, and acetyla-tion of p21 protein may be the main modification form in the early period of p21 expression. We demonstrate that p21 protein become more stable after acety-lated, which consistent with the fact that p21 feedback enhances its own gene transcription during the early period after transfection. The inhibition of the CBP HAT activity by curcumin greatly decreases the feedback activation, suggesting that the CBP HAT activity may correlate to the acetylation of p21 protein. Collectively, p21 may be acetylated by the recruited CBP and thereby increasing protein stability as well as recruits protein factors to feedback enhance its own gene transcription.In summary, our findings demonstrate a novel self-feedback regulation loop pathway of p21 transcription in the early period under cellular stress, in which p21 recruits protein factors to its own gene promoter to enhance transcription, thereby elevating the p21 protein level. A transcription inhibitory region and several cisacting binding elements are also identified. These results suggested that there exist more complex mechanism for p21 gene induction, and for the regulation in transcriptional and post-transcriptional level as well. These findings carry unexpected implications for the control of cell stress, proliferation, differentiation and cancer, and thus also provide a useful reference for clinical cancer therapy.