Limits...
Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

View Article: PubMed Central - PubMed

ABSTRACT

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and that is regulated by the Plk1 phosphorylation sites in FoxM1b. Our results show that the Plk1 phosphorylation sites in FoxM1b serve as a regulator for its repressor function, and they provide insights into how FoxM1b inhibits differentiation genes and activates proliferation genes during cancer progression.

No MeSH data available.


FoxM1b methylates and represses FoxA1 in MCF7 cells.(A and B) MCF7 cells and MDA-MB-453 cells were transfected with Control siRNA or two different FoxM1siRNA and 72hr post transfected cells were harvested and RNA was used to quantify the FoxA1 transcripts using qRT- PCR. Silencing of FoxM1 expression in two cell lines was verified by western blots (lower panel). (C) Cells were transfected with empty vector control, WT-T7-FoxM1, T7-FoxM1 (596 A), T7-FoxM1 (DD) and T7-FoxM1 (AA) expressing plasmids. RNA was extracted 48hr post transfection for FoxA1 transcripts and quantification was performed by qRT-PCR. (D) genomic-DNAs from the transfected cells were isolated and C/T conversion was performed to check the promoter methylation that was quantified using the real time PCR. Statistical analysis was done using Graph pad prism unpaired t test and p-values represented as *≤0.05; **≤0.01; ***≤0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384083&req=5

f7: FoxM1b methylates and represses FoxA1 in MCF7 cells.(A and B) MCF7 cells and MDA-MB-453 cells were transfected with Control siRNA or two different FoxM1siRNA and 72hr post transfected cells were harvested and RNA was used to quantify the FoxA1 transcripts using qRT- PCR. Silencing of FoxM1 expression in two cell lines was verified by western blots (lower panel). (C) Cells were transfected with empty vector control, WT-T7-FoxM1, T7-FoxM1 (596 A), T7-FoxM1 (DD) and T7-FoxM1 (AA) expressing plasmids. RNA was extracted 48hr post transfection for FoxA1 transcripts and quantification was performed by qRT-PCR. (D) genomic-DNAs from the transfected cells were isolated and C/T conversion was performed to check the promoter methylation that was quantified using the real time PCR. Statistical analysis was done using Graph pad prism unpaired t test and p-values represented as *≤0.05; **≤0.01; ***≤0.001.

Mentions: Interestingly, we observed that FoxM1 regulates expression of the luminal transcription factor FoxA1. Depletion of FoxM1, using two different FoxM1-siRNA in MCF7 or MDA-MB-453 cells, resulted in increased expression of FoxA1 (Fig. 7A and B). Moreover, expression of FoxM1 leads to repression of FoxA1. FoxM1 binds to the FoxA1 promoter (Fig. S5). We show that, as in the case of GATA3, the phospho-defective mutants and wild type FoxM1 repress FoxA1 expression whereas the Plk1-site phospho-mimetic mutant had no inhibitory effect instead we observed some stimulation of transcription (Fig. 7C)). Further, the phospho-mimetic mutant failed to increase methylation of the CpG-islands in the FoxA1 promoter (Fig. 7D). Together these observations suggest that the Plk1 phosphorylation sites in FoxM1 play critical roles in converting a repressor complex of FoxM1 to an activator complex.


Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein
FoxM1b methylates and represses FoxA1 in MCF7 cells.(A and B) MCF7 cells and MDA-MB-453 cells were transfected with Control siRNA or two different FoxM1siRNA and 72hr post transfected cells were harvested and RNA was used to quantify the FoxA1 transcripts using qRT- PCR. Silencing of FoxM1 expression in two cell lines was verified by western blots (lower panel). (C) Cells were transfected with empty vector control, WT-T7-FoxM1, T7-FoxM1 (596 A), T7-FoxM1 (DD) and T7-FoxM1 (AA) expressing plasmids. RNA was extracted 48hr post transfection for FoxA1 transcripts and quantification was performed by qRT-PCR. (D) genomic-DNAs from the transfected cells were isolated and C/T conversion was performed to check the promoter methylation that was quantified using the real time PCR. Statistical analysis was done using Graph pad prism unpaired t test and p-values represented as *≤0.05; **≤0.01; ***≤0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384083&req=5

f7: FoxM1b methylates and represses FoxA1 in MCF7 cells.(A and B) MCF7 cells and MDA-MB-453 cells were transfected with Control siRNA or two different FoxM1siRNA and 72hr post transfected cells were harvested and RNA was used to quantify the FoxA1 transcripts using qRT- PCR. Silencing of FoxM1 expression in two cell lines was verified by western blots (lower panel). (C) Cells were transfected with empty vector control, WT-T7-FoxM1, T7-FoxM1 (596 A), T7-FoxM1 (DD) and T7-FoxM1 (AA) expressing plasmids. RNA was extracted 48hr post transfection for FoxA1 transcripts and quantification was performed by qRT-PCR. (D) genomic-DNAs from the transfected cells were isolated and C/T conversion was performed to check the promoter methylation that was quantified using the real time PCR. Statistical analysis was done using Graph pad prism unpaired t test and p-values represented as *≤0.05; **≤0.01; ***≤0.001.
Mentions: Interestingly, we observed that FoxM1 regulates expression of the luminal transcription factor FoxA1. Depletion of FoxM1, using two different FoxM1-siRNA in MCF7 or MDA-MB-453 cells, resulted in increased expression of FoxA1 (Fig. 7A and B). Moreover, expression of FoxM1 leads to repression of FoxA1. FoxM1 binds to the FoxA1 promoter (Fig. S5). We show that, as in the case of GATA3, the phospho-defective mutants and wild type FoxM1 repress FoxA1 expression whereas the Plk1-site phospho-mimetic mutant had no inhibitory effect instead we observed some stimulation of transcription (Fig. 7C)). Further, the phospho-mimetic mutant failed to increase methylation of the CpG-islands in the FoxA1 promoter (Fig. 7D). Together these observations suggest that the Plk1 phosphorylation sites in FoxM1 play critical roles in converting a repressor complex of FoxM1 to an activator complex.

View Article: PubMed Central - PubMed

ABSTRACT

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and that is regulated by the Plk1 phosphorylation sites in FoxM1b. Our results show that the Plk1 phosphorylation sites in FoxM1b serve as a regulator for its repressor function, and they provide insights into how FoxM1b inhibits differentiation genes and activates proliferation genes during cancer progression.

No MeSH data available.