E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies

E2F mediates induction of p19 in response to DNA damage or chromatin relaxation.

<p><b>A.</b> HEK-293 cells were transfected with 500 nM E2F decoy oligonucleotide. Twenty four hours later, cells were exposed to 40 J/m² UV or 50 ng/ml NCS and incubated in the presence or in the absence of 100 µM chloroquine. After 4 h, cells were harvested and subjected to northern blot analysis using a ³²P-labelled probe specific for human p19 mRNA and reprobed for E2F1 and β-tubulin mRNA. Figure shows a representative autoradiograph of three independent experiments with similar results. Densitometric analysis of p19 and E2F1 are represented in the lower panel. Bars represent the mean ± S.E of three experiments. Student’s <i>t</i>-test was used to compare treated and non-treated samples (* p<0.05, at least). <b>B.</b> HEK-293 cells transiently cotransfected with 4 µg of p19CAT, or equivalent amount of mutant plasmid, containing the 5′-flanking region of p19 gene and 5 µg pCEFL-β-galactosidase were exposed to 40 J/m² UV or incubated with 50 ng/ml NCS or 100 µM chloroquine or 200 nM TSA or hypotonic medium. After 24 h cells were harvested and CAT activity was determined as described. Results are expressed as relative CAT activity with respect to basal value of p19CAT which was set to 100. Bars represent the mean ± S.D. of three independent experiments performed in quadruplicate. Student’s <i>t</i>-test was used to compare treated with non treated samples (* p<0.01). <b>C.</b> HEK-293 cells, transiently cotransfected with 4 µg of pE2F4XCAT and 5 µg pCEFL-β-galactosidase and, when indicated, 4 µg of a vector expressing E2F1 cDNA, were treated with 100 µM chloroquine, or 200 nM TSA or subjected to hypotonic medium and incubated in the presence or in the absence of 10 µM Ku-55933 or 15 nM SB-218078 or 20 nM Chk2 inhibitor. After 24 h cells were harvested and CAT activity was determined as described. Results are expressed as relative CAT activity with respect to basal value of pE2F4XCAT which was set to 100. Bars represent the mean ± S.D. of three independent experiments performed in quadruplicate. Student’s <i>t</i>-test was used to compare treated with non treated samples (* p<0.01). Decoy E2F oligonucleotide (DecE2F), β-tubulin (β-tub), chloroquine (chlo), hypotonic medium (hypo), neocarzinostatin (NCS), Ku-55933 (Ku), SB-218078 (SB), Chk2 inhibitor (2I).</p

Chromatin-based defects as inducers of p19 protein.

<p>Chromatin structure defects can trigger ATM/ATR and Chk1/Chk2 activation leading to an increase in E2F1 protein levels as a result of transcriptional induction. E2F1 upregulation induces the transcription of p19 which, in turn, promotes DNA repair.</p

Chloroquine-mediated induction of p19 increases the ability of Neuro-2a cells to repair UV-damaged DNA.

<p><b>A.</b> Stably transfected p19 AS Neuro-2a cells were cultured in a serum free-medium during 24 h, and then incubated with 50 µM ZnSO₄ during 16 h. After this time, cells were treated with 100 µM chloroquine and, simultaneously (chlo+UV) or after 4 h (chlo → UV) irradiated with 40 J/m² UV, and incubated with 10 µCi/ml [³H]thymidine for 10 h. Cell lysates were tested for unscheduled DNA synthesis assay. Bars represent the mean ± S.D. of three different experiments performed in triplicate. Student’s <i>t</i>-test was used to compare Zn²⁺-treated with non treated samples (* p<0.05) and to compare chloroquine → UV-treated with chloroquine+UV-treated from stable transfectant samples (* p<0.05). <b>B.</b> Neuro-2a cells were assayed for the presence of CPD lesions. Stably transfected p19AS Neuro-2a cells were incubated in a free-Zn²⁺ medium and treated with 100 µM chloroquine and, simultaneously (Chlo+UV) or after 4 h (chlo → UV) irradiated with 40 J/m² UV. Immediately after UV irradiation, cells were harvested, DNA isolated and examined for the presence of CPD lesions by immuno-slot blot using and specific antibody. Ethidium bromide staining was used to ensure equal protein content. Figure shows a representative photograph of three independent experiments. Densitometric analysis of CPD lesions is represented in the right panel. Bars represent the mean ± S.D. of three experiments performed in triplicate. Student’s <i>t</i>-test was used to compare samples treated with choroquine → UV with samples treated with UV. (* p<0.05). Chloroquine (Chlo), EtBr (ethidium bromide).</p

Chromatin relaxation-mediated induction of p19 is specific.

<p><b>A.</b> HEK-293 cells, previously treated or not with 5 mM caffeine during 1 h, were incubated at 43°C for 1 h and then cultured at 37°C from 0 to 8 h. <b>B.</b> HEK-293 cells, previously treated with 5 mM caffeine for 1 h, were incubated with 100 µM chloroquine at the indicated times. In (<b>A</b>) and (<b>B</b>), cells were harvested and subjected to northern blot analysis using a ³²P-labelled probe specified at the right margin. Each figure shows a representative autoradiograph of three independent experiments with similar results. Densitometric analysis of p19, p21 and c-fos are represented in the lower panels. Bars represent the mean ± S.D. of three experiments. Student’s <i>t</i>-test was used to compare samples obtained at different times with samples obtained at zero time (* p<0.05, at least). β-tubulin (β-tub), heat shock (HS), caffeine (caff), chloroquine (chlo).</p

Absence of synergism of genotoxins and chromatin modifiers effects on p19 induction.

<p><b>A.</b> HEK-293 cells were exposed to 40 J/m² UV or 50 ng/ml neocarzinostatin and incubated in the presence or in the absence of 100 µM chloroquine, or 200 nM TSA or hypotonic medium. After 4 h, cells were harvested and subjected to northern blot analysis using a ³²P-labelled probe specific for human p19 mRNA and reprobed for E2F1 and β-tubulin mRNA. <b>B.</b> HEK-293 cells were transfected with increasing levels of an expression vector encoding E2F1 gene, harvested after 24 h, and p19 and E2F1 expression assessed by northern blot. In parallel cells were incubated with 100 µM chloroquine or 200 nM trichostatin A or hypotonic medium or exposed to 40 J/m² UV as indicated. After 4 h p19 and E2F1 expression was determined by northern blot. In (<b>A</b>) and (<b>B</b>) each figure shows a representative autoradiograph of three independent experiments with similar results. Densitometric analysis of p19 and E2F1 are represented in the lower panels. Bars represent the mean ± S.D. of three experiments. Student’s <i>t</i>-test was used to compare treated and non-treated samples (* p<0.05, at least). <b>C.</b> HEK-293 cells, transiently cotransfected with 4 µg of p19CAT and 5 µg pCEFL-β-galactosidase, were exposed to 40 J/m² UV or treated with 50 ng/ml NCS and incubated in the presence or in the absence of 100 µM chloroquine or 200 nM TSA or hypotonic medium. After 24 h cells were harvested and CAT activity was determined as described. Results are expressed as relative CAT activity with respect to basal value of p19CAT which was set to 100. Bars represent the mean ± S.D. of three independent experiments performed in quadruplicate. β-tubulin (β-tub), chloroquine (chlo), neocarzinostatin (NCS), hypotonic medium (hypo).</p

E2F1 sequentially induces cyclin E and p19 during the cell cycle.

<p><b>A</b>. WI-38 cells were synchronized by serum deprivation. Total RNA was extracted at the indicated times following serum restoration and subjected to northern blot analysis. <b>B</b>. EMSA was performed using oligonucleotides corresponding to the E2F-C site or the E2F consensus sequence (CS) as radiolabeled probes. WI-38 nuclear extracts (N.E.) were incubated with probes alone or in the presence of 20-, 50-, 100-, 200-, or 500-fold molar excess of the indicated unlabeled competitors. Relative quantification of DNA-protein complexes is shown (<i>bottom panel</i>). <b>C</b> and <b>D</b>. Synchronized BHK-ER-E2F1 cells were untreated (<b>C</b>) or treated with 4-OHT (<b>D</b>) for 3 h before cells were stimulated to re-enter the cycle. Total RNA was extracted at indicated time points and subjected to northern blot analysis. Results are representative of at least two independent experiments.</p

E2F1 increases the transcriptional activity of p19INK4d.

<p><b>A</b>. BHK-ER-E2F1 cells were treated with 4-OHT for 8 h and subjected to nuclear run-on assay. Transcription rates of the indicated genes were normalized to that of β-tubulin. Results are representative of two independent experiments. <b>B–D</b>. Indicated cells were cotransfected with p19CAT (4.4 µg) and pCEFL-β-galactosidase (5 µg) reporter plasmids. CAT activity was determined 48 h after transfection and normalized to β-galactosidase activity. BHK-21 cells were transfected with reporter plasmids and 1 or 3 µg of E2F1 expression vector and grown in medium containing 10% or 1% FBS (<b>B</b>). BHK-ER-E2F1 cells were transfected with reporter plasmids, grown in medium containing 10% or 1% FBS, and treated with 4-OHT as indicated (<b>C</b>). BHK-ER-E2F1 were transfected with wild-type or mutant pRB expression vectors (6 µg) or wild-type or mutant E2F DO (100 nM) for 24 h. Cells were transfected with reporter plasmids for another 24 h before 4-OHT treatment (<b>D</b>). In panels <b>B</b>, <b>C</b> and <b>D</b> values are the average ± SD of three independent experiments, each performed in triplicates.</p