Ndy1/KDM2B immortalizes mouse embryonic fibroblasts by repressing the Ink4a/Arf locus

Abstract

The histone H3 demethylase Not dead yet-1 (Ndy1/KDM2B) is a physiological inhibitor of senescence. Here, we show that Ndy1 is down-regulated during senescence in mouse embryonic fibroblasts (MEFs) and that it represses the Ink4a/Arf locus. Ndy1 counteracts the senescence-associated down-regulation of Ezh2, a component of polycomb-repressive complex (PRC) 2, via a JmjC domain-dependent process leading to the global and Ink4a/Arf locus-specific up-regulation of histone H3K27 trimethylation. The latter promotes the Ink4a/Arf locus-specific binding of Bmi1, a component of PRC1. Ndy1, which interacts with Ezh2, also binds the Ink4a/Arf locus and demethylates the locus-associated histone H3K36me2 and histone H3K4me3. The combination of histone modifications driven by Ndy1 interferes with the binding of RNA Polymerase II, resulting in the transcriptional silencing of the Ink4a/Arf locus and contributing to the Ndy1 immortalization phenotype. Other studies show that, in addition to inhibiting replicative senescence, Ndy1 inhibits Ras oncogene-induced senescence via a similar molecular mechanism.

Fig. 1.

Ndy1 represses the senescence-associated up-regulation of p16^Ink4a and p19^Arf. (A) Total mRNA was isolated from MEFs at the indicated passage and analyzed by real-time PCR for the relative mRNA levels of Bmi1, Ezh2, Suz12, Eed, Ndy1, and Ndy2 (n = 2). *P < 0.05. (B) MEFs overexpressing Ndy1 were plated at a concentration of 10⁵ cells per 6-cm dish and serially passaged. In passage 5 (P5), P10, and P15, total mRNA was isolated and analyzed by real-time PCR for the relative mRNA levels of p16^Ink4a and p19^Arf. The graphs show the fold decrease of p16^Ink4a and p19^Arf mRNAs in MEFs that overexpress Ndy1 normalized to control empty-vector (EV)–transduced cells from 3 independent infections. (C) As in B, MEFs were serially passaged, and whole-cell lysates from the indicated passages were analyzed by Western blotting with the indicated antibodies. p19^Arf was undetectable at P2 and P5. (D) Passage 3 MEFs were transfected with siRNA, which specifically targets either the long or short form of Ndy1. Four days after transfection, cells were analyzed by Western blotting and total mRNA was collected and analyzed by real-time PCR with primers specific for p16^Ink4a and p19^Arf and primers specific for the long and short forms of Ndy1 (n = 3). *P < 0.05

Fig. 2.

Ndy1 counteracts the senescence-associated down-regulation of Ezh2 and induces global H3K27 trimethylation. (A) The same samples described in Fig. 1C were further analyzed by Western blotting with the indicated antibodies. (B) Passage 3 MEFs were transfected with siRNA against Ndy1. Four days after transfection, cells were collected and analyzed by Western blotting. The graph at the bottom of the figure shows the efficiency of Ndy1 knockdown. *Nonspecific band. (C) Western blotting analysis of whole-cell extracts from MEFs immortalized with the MigR1-Ndy1 LoxP retroviral construct before and after infection with a retroviral construct that expresses the Cre recombinase. (D) The graph shows the fold difference of different mRNAs in MigR1-Ndy1 LoxP-immortalized MEFs before and after infection with the Cre recombinase (n = 2). (E) ChIP analysis of H3K27me3 at the Ink4a/Arf locus in early- and late-passage MEFs that overexpress Ndy1. The graph shows the fold increase of H3K27me3 methylation at the Ink4/Arf locus in MEFs that overexpress Ndy1 normalized to control empty-vector (EV)–transduced cells. (F) ChIP analysis of Bmi1 binding at the Ink4a/Arf locus in passage 3–5 MEFs that overexpress Ndy1. The graph shows the fold increase of Bmi1 binding at the Ink4a/Arf locus in MEFs that overexpress Ndy1 normalized to control EV-transduced cells. *P < 0.05; #P < 0.01.

Fig. 3.

Ndy1 cooperates with Ezh2 and Bmi1 to repress the Ink4/Arf locus. (A) Passage 2 MEFs were transfected with the indicated siRNA for 4 days. Whole-cell lysates were analyzed by Western blotting with the indicated antibodies. (B) Ndy1-transduced MEFs were transfected with siRNA against Bmi1. Whole-cell lysates were analyzed by Western blotting with the indicated antibodies. (C) ChIP analysis of Ndy1 binding at the Ink4a/Arf locus in MEFs. The graph shows the fold enrichment of Myc-tagged Ndy1 at the Ink4a/Arf locus normalized to control empty-vector (EV)–transduced cells. *P < 0.05; #P < 0.01. (D) HEK293T cells were transfected with the EV and Myc-tagged Ndy1. Whole-cell lysates were immunoprecipitated with the Myc antibody and probed with an antibody against Ezh2 (Upper) and a mix of antibodies against Myc and tubulin (Lower).

Fig. 4.

Ndy1 functions in vitro and in vivo as H3K36me2 and H3K4me3 demethylase. (A) Representative Western blot analysis of an in vitro demethylation assay with native murine Ndy1 protein isolated from MEFs overexpressing Ndy1. (B) Fluorescence-coupled demethylation assay of bacterial purified fragments of Ndy1 using the indicated peptide substrates. (C) ChIP analysis of the H3K36me2 methylation status at the Ink4a/Arf locus in MEFs that overexpress Ndy1. The graph shows the fold decrease of H3K36me2 methylation at the Ink4a/Arf locus in MEFs that overexpress Ndy1 normalized to control empty-vector (EV)–transduced cells. (D) ChIP analysis of the H3K4me3 methylation status at the Ink4a/Arf locus in MEFs that overexpress Ndy1. The graph shows the fold difference of H3K4me3 methylation at the Ink4a/Arf locus in MEFs that overexpress Ndy1 normalized to control empty-vector–transduced cells. (E) ChIP analysis of RNA Pol II binding at the Ink4a/Arf locus in MEFs that overexpress Ndy1. The graph shows the fold difference of RNA Pol II binding at the Ink4a/Arf locus in MEFs that overexpress Ndy1 normalized to control EV-transduced cells. *P < 0.05; #P < 0.01.

Fig. 5.

Ndy1 up-regulates Ezh2 and represses p16^Ink4a in IMR90 cells. (A) Total mRNA was isolated from Ndy1- or empty-vector (EV)–transduced IMR90 cells at passage 20 (P20) and analyzed by real-time PCR for the relative mRNA levels of p16^Ink4a (n = 2). *P < 0.05. (B) Ndy1- or EV-transduced IMR90 cells were serially passaged and analyzed by Western blotting. (C) Data from ref. reanalyzed to show expression levels of Ndy1 in normal bone marrow, B- and T-cell acute lymphoblastic leukemias, and acute myeloid leukemia. (D) Data from ref. reanalyzed to show expression levels of Ndy1 in normal testis and seminomas.

Fig. 6.

The histone demethylase Ndy1 represses the Ink4a/Arf locus. Ndy1 represses the Ink4a/Arf locus by 2 distinct mechanisms: it up-regulates Ezh2 and promotes histone H3K27 trimethylation and Bmi1 binding within the Ink4a/Arf locus, and it binds to and demethylates H3K36me2 and H3K4me3 within the Ink4a/Arf locus. These histone modifications, when combined, interfere with the binding of RNA Pol II and contribute to the silencing of the Ink4a/Arf locus.