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p16( INK4a) positively regulates cyclin D1 and E2F1 through negative control of AUF1.

Al-Khalaf HH, Colak D, Al-Saif M, Al-Bakheet A, Hendrayani SF, Al-Yousef N, Kaya N, Khabar KS, Aboussekhra A - PLoS ONE (2011)

Bottom Line: Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs.We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

ABSTRACT

Background: The cyclin-D/CDK4,6/p16(INK4a)/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined.

Methodology/principal findings: We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.

Conclusion/significance: These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

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Related in: MedlinePlus

Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (ARE regions 1 to 3), IL-8 3′UTR (ARE control), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.104 cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote p values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote p <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.104 cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.
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pone-0021111-g005: Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (ARE regions 1 to 3), IL-8 3′UTR (ARE control), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.104 cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote p values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote p <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.104 cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.

Mentions: In order to search for functional mRNA destability element in the E2F1 3′UTR, we analyzed the 3′UTR sequence, which is 1245 nt long, for the presence of conserved and AU-rich element clusters (Figure 5A). Both regions 2 and 3 contain two overlapping AUUUA pentameric repeats (Figure 5A, right panel). These regions were highly conserved among different species (Figure 5B). In order to evaluate the contribution of each of these regions in the E2F1 mRNA instability, we cloned each of these regions in a vector containing the stable bovine growth hormone (BGH) 3′UTR downstream of the EGFP reporter (Figure 5C, upper panel), and Huh-7 cells were transfected with the three constructs. The reporter activity fused to the 3′UTR with the E2F1 AU-rich regions 2 and 3 was significantly reduced (46% and 64%, respectively) as compared to the control 3′UTR that contains no ARE. On the other hand, the region 1 had no effect (Figure 5C, lower panel). As a positive control, the IL-8 ARE-3′UTR caused 78% reduction of the reporter activity. This EGFP reporter activity system correlated well with ARE- mRNA decay patterns in several cellular models [32], [34], [35]. These results suggest that the E2F1 3′UTR bears functional AREs.


p16( INK4a) positively regulates cyclin D1 and E2F1 through negative control of AUF1.

Al-Khalaf HH, Colak D, Al-Saif M, Al-Bakheet A, Hendrayani SF, Al-Yousef N, Kaya N, Khabar KS, Aboussekhra A - PLoS ONE (2011)

Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (ARE regions 1 to 3), IL-8 3′UTR (ARE control), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.104 cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote p values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote p <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.104 cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140473&req=5

pone-0021111-g005: Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (ARE regions 1 to 3), IL-8 3′UTR (ARE control), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.104 cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote p values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote p <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.104 cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.
Mentions: In order to search for functional mRNA destability element in the E2F1 3′UTR, we analyzed the 3′UTR sequence, which is 1245 nt long, for the presence of conserved and AU-rich element clusters (Figure 5A). Both regions 2 and 3 contain two overlapping AUUUA pentameric repeats (Figure 5A, right panel). These regions were highly conserved among different species (Figure 5B). In order to evaluate the contribution of each of these regions in the E2F1 mRNA instability, we cloned each of these regions in a vector containing the stable bovine growth hormone (BGH) 3′UTR downstream of the EGFP reporter (Figure 5C, upper panel), and Huh-7 cells were transfected with the three constructs. The reporter activity fused to the 3′UTR with the E2F1 AU-rich regions 2 and 3 was significantly reduced (46% and 64%, respectively) as compared to the control 3′UTR that contains no ARE. On the other hand, the region 1 had no effect (Figure 5C, lower panel). As a positive control, the IL-8 ARE-3′UTR caused 78% reduction of the reporter activity. This EGFP reporter activity system correlated well with ARE- mRNA decay patterns in several cellular models [32], [34], [35]. These results suggest that the E2F1 3′UTR bears functional AREs.

Bottom Line: Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs.We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

ABSTRACT

Background: The cyclin-D/CDK4,6/p16(INK4a)/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined.

Methodology/principal findings: We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.

Conclusion/significance: These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

Show MeSH
Related in: MedlinePlus