The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53 - PubMed (original) (raw)

The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53

K D Robertson et al. Mol Cell Biol. 1998 Nov.

Abstract

The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check.

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Figures

FIG. 1

(A) RT-PCR analysis of ARF (top) and p16 INK4a (bottom) expression patterns in the HCT15 cell line after treatment with 1.0 μM 5-aza-CdR for the times indicated. Also shown is a single time point (72 h) for the SW48, HCT116, and Raji cell lines. Hep 3B RNA was used as the positive (+) control. PCR products were probed with oligonucleotides specific for the unique first exon of each transcript, and RNA integrity was verified by amplification of transcripts for β-actin and PCNA (not shown). Note that a longer exposure is shown for the HCT15 5-aza-CdR time course experiment in order to emphasize differences at the earlier time points. (B) Quantitation of the results in panel A for the HCT15 cell line relative to the ubiquitously expressed transcript for PCNA.

FIG. 2

(A) Plots comparing the frequency of the CpG dinucleotide in the ARF promoter/exon 1β region (top) and the INK4a/exon 1α region (bottom) derived from GenBank accession no. AC000048. Bent arrows are the transcription start sites, and open boxes are transcribed regions. Analysis of the regions of each promoter denoted by the brackets in terms of their CpG contents yields the following: G/C content = 0.64, observed/expected for CpG = 0.85 for ARF and G/C content = 0.54, observed/expected for CpG = 0.68 for INK4a over approximately 2,400 bp. (B) Sequence of a portion of the ARF promoter region (GenBank accession no. AF082338). The previously mapped transcription start site (36) is indicated by a bent arrow above the italicized “G” and is defined here as position +1. The positions of several potential transcription factor binding sites are underlined, as is a region homologous to other known initiator elements (Inr). Potential E2F binding sites are denoted with a line above the sequence; (−) indicates that the consensus binding site is 5′ to 3′ on the bottom strand. Positions of restriction enzyme sites used in subsequent cloning steps for promoter deletion analysis are underlined, and their positions relative to the transcription start site are indicated. Downward arrows denote 5′ ends of deletion constructs generated by PCR. A subset of the repetitive elements described in the text (Alu and purine-pyrimidine [_Pur-Pyr_]) is also shown.

FIG. 3

ARF promoter deletion analysis. The extent of each of the 5′ deletions fused to the CAT reporter gene, as well as the extent of each construct relative to the transcription start site (indicated by the bent arrow), is indicated schematically at the left. Results are presented as the mean relative activity (percent acetylation/β-galactosidase activity) for triplicate transfections into the HCT116 (A), C-33A (B), and COS-7 (C) cell lines. Error bars indicate the standard deviations (SD) from the means.

FIG. 4

The ARF promoter is upregulated by E2F1 expression. Each of the ARF or INK4a promoter constructs denoted schematically at the left was cotransfected with an equal amount of an expression vector for E2F1 or empty parental expression vector. The results of duplicate transfections in the HCT116 cell line are presented as the mean fold activation with E2F1 cotransfection (activity with E2F1/activity with empty expression vector). Error bars indicate the standard deviations (SD) from the means.

FIG. 5

Methylation analysis of the ARF promoter. (A) Representative Southern blots after digestion of HCT116 and HCT15 genomic DNAs with the enzymes indicated and probing with the fragment shown in panels B and C. The presence of higher-molecular-weight bands in the HCT15 digests compared to the HCT116 digests is indicative of methylation. The low level of hybridization after digestion with enzymes like _Hpa_II and _Hha_I is a result of the large number of such sites, creating many small restriction fragments which hybridize poorly. (B) Schematic of the location of several of the rare-cutting methylation-sensitive restriction enzyme sites, a CpG plot of the entire sequenced region, and the transcription start site (+1). Brackets in the CpG plot indicate the boundaries used for the calculation of CpG island status. The location of the probe is indicated by the thick bar, and the methylation status at the CpG sites analyzed by restriction digest is indicated by the lollipops. (C) Blowup of the region immediately adjacent to the ARF promoter (−930 to +49) and summary of the methylation status of CpG sites in this region as determined from blots in panel A for the HCT15 cell line. Asterisks indicate that these particular CpG sites, while partially methylated in HCT15 cells, were completely methylated in the LoVo cell line (not shown). The sizes of the fragments are indicated below the lines. A lollipop displaced below a group of restriction enzyme sites indicates that the sites were too close together to accurately determine which site was digested. (D) Analysis of the methylation sensitivity of the p(−151)19ARF promoter CAT construct after in vitro methylation and transfection into HCT116 or COS-7 cells. Results after treatment with the various methylases are presented as the mean percent activity relative to the mock-methylated control (no methylase) for triplicate transfections. Error bars represent the standard deviations.

FIG. 5

FIG. 6

Comparison of activities of ARF and INK4a promoter-CAT constructs after transfection into ARF+ INK4a+ (A), ARF+ INK4a− (B), and ARF− INK4a− (C) cell lines. The map of each of the reporter constructs relative to the transcription start site of each promoter (for INK4a, the 5′-most initiation site was defined as +1) is indicated at the left and the mean relative activity (percent acetylation/β-galactosidase activity) for triplicate (duplicate for SW48 and HCT15) transfections is shown at the right. Error bars indicate the standard deviations (SD) from the means.

FIG. 7

Mapping p53-responsive regions of the ARF and INK4a promoters. Each of the reporter constructs indicated schematically at the left of each graph was cotransfected with 1.0 μg of wild-type p53 expression vector or empty parental expression vector into the C-33A cell line. Results are presented as the mean activity for triplicate transfections relative to the transfection containing no p53 expression vector, set at 100%. Error bars represent the standard deviations (SD).

FIG. 8

Effects of p53 on ARF and INK4a promoter-CAT constructs. (A) Dose-response cotransfection of wild-type p53 expression vector and p(−331)19ARF, p(−654)16INK4a, or p53proCAT promoter-CAT constructs into the HCT116 and C-33A cell lines. (B) The identical CAT reporter constructs cotransfected with increasing amounts of a mutant (Val-143→Ala) p53 expression vector. Results in panels A and B are presented as the mean activity for triplicate transfections relative to the transfection containing no p53 expression vector, set at 100%. Error bars represent the standard deviations. The total amount of expression vector was held constant by addition of parental vector containing no cDNA. (C) Representative results after transfection of a CAT reporter construct containing two copies of a consensus p53 binding site with increasing amounts of wild-type or mutant p53 expression vector into the HCT116 and C-33A cell lines. Results are presented as percent acetylation; independent experiments yielded similar results. Note that the endogenous p53 gene is wild type in HCT116 and mutant in C-33A.

FIG. 9

ARF and INK4a promoters are repressed after induction of p53 by DNA-damaging agents. The reporter constructs indicated below each graph were transfected into HCT116 (A) or C-33A (B) cells, after which cells were exposed to the indicated DNA-damaging agent (5 μM CMT or 50 μM Ara-C). After 48 h, cells were harvested for CAT assay and the protein concentration was determined for each. Results are presented as the mean relative activity (percent acetylation normalized to the protein concentration to account for small differences in toxicity) of triplicate transfections. Error bars are the standard deviations from the means. WT, wild-type; MT, mutant.

FIG. 10

Proposed regulatory cycle controlling cellular p53 levels mediated by p53, MDM2, and ARF. Interaction between MDM2 and ARF results in increased degradation of MDM2 (78) (shown by light stipple) and an increase in functional p53 (upward arrow) (48). This then represses ARF (×) and activates MDM2 (large arrow) transcription (1, 76). Elevated levels of MDM2 and decreased levels of ARF then promote degradation of p53 (shown by light stipple) and continue the cycle as shown. E2F levels may be one of the outside influences on the cycle.

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The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53 - PubMed (original) (raw)