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. 2008 Feb 12;105(6):1907-12.
doi: 10.1073/pnas.0711865105. Epub 2008 Feb 4.

Members of a family of JmjC domain-containing oncoproteins immortalize embryonic fibroblasts via a JmjC domain-dependent process

Raymond Pfau  1 , Alexandros Tzatsos, Sotirios C Kampranis, Oksana B Serebrennikova, Susan E Bear, Philip N Tsichlis
Affiliations

Members of a family of JmjC domain-containing oncoproteins immortalize embryonic fibroblasts via a JmjC domain-dependent process

Raymond Pfau et al. Proc Natl Acad Sci U S A. .

Abstract

A common integration site, cloned from MoMuLV-induced rat T cell lymphomas, was mapped immediately upstream of Not dead yet-1 (Ndy1)/KDM2B, a gene expressed primarily in testis, spleen, and thymus, that is also known as FBXL10 or JHDM1B. Ndy1 encodes a nuclear, chromatin-associated protein that harbors Jumonji C (JmjC), CXXC, PHD, proline-rich, F-box, and leucine-rich repeat domains. Ndy1 and its homolog Ndy2/KDM2A (FBXL11 or JHDM1A), which is also a target of provirus integration in retrovirus-induced lymphomas, encode proteins that were recently shown to possess Jumonji C-dependent histone H3 K36 dimethyl-demethylase or histone H3 K4 trimethyl-demethylase activities. Here, we show that mouse embryo fibroblasts engineered to express Ndy1 or Ndy2 undergo immortalization in the absence of replicative senescence via a JmjC domain-dependent process that targets the Rb and p53 pathways. Knockdown of endogenous Ndy1 or expression of JmjC domain mutants of Ndy1 promote senescence, suggesting that Ndy1 is a physiological inhibitor of senescence in dividing cells and that inhibition of senescence depends on histone H3 demethylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Provirus insertion activates the Ndy1 gene. (A Upper) Sites and orientation of provirus integration at the 5′ end of the Ndy1 gene. The numbers above the arrows showing the sites of provirus integration identify the tumors in which the integrations were detected. (Lower) Domain structure of the Ndy1 protein. (B Upper) Site and orientation of provirus integration at the 5′ end of the Ndy2 gene in tumor #4. (Lower) Domain structure of the Ndy2 protein. (C) NIH 3T3 cells were infected with a MigR1 construct of Ndy1.HA. (Left) Two GFP-positive, infected cells. (Center) The same cells stained with an anti-HA antibody. (Right) Ndy1.HA-expressing cell stained with an anti-HA antibody and visualized by confocal microscopy. The darker spots correspond to nucleoli. (D Upper) Northern blot of total cell RNA derived from normal rat thymus and the indicated tumors (R6 to 5677) probed with a full-length rat Ndy1 cDNA probe. (Lower) Ethidium bromide staining of the gel (loading control). (E) Western blot of nuclear cell lysates from normal thymus and the indicated tumors probed with anti-Ndy1 and anti-CREB antibodies as indicated.
Fig. 2.
Fig. 2.
MEFs expressing Ndy1 or Ndy2 bypass replicative senescence and undergo immortalization. (A) β-Galactosidase staining of 11th-passage MEFs infected with the MigR1 retrovirus vector (Left) or with a MigR1-Ndy1construct (Right). (B and C) MEFs infected with the MigR1 vector or MigR1-Ndy1 and MigR1-Ndy2 constructs. The graph shows the cumulative number of cells in each culture at sequential passages.
Fig. 3.
Fig. 3.
Immortalization depends on the histone demethylase activities of Ndy1 and Ndy2. (A) Western blots of total cell lysates derived from MEFs infected with MigR1 or with wild-type or mutant MigR1-Ndy1.Myc. The blots were probed with anti-Ndy1 or with anti-GAPDH (loading control) antibodies as indicated. Ndy1-ΔPRR carries a deletion of the prolene-rich region, upstream of the F-box (see Fig. 1A). The LRR deletion mutant was also tested in separate experiments, and it was shown to immortalize MEFs as efficiently as the wild-type protein (data not shown). (B) MEFs shown in A were passaged in culture. Graphs show the cumulative number of cells at each passage. The Ndy1.H283Y mutant was also tested in separate experiments, and it was shown to have the same dominant-negative phenotype as the Ndy1.Y221A mutant (data not shown). (C) Western blots of cell lysates from cells infected with MigR1-Ndy1 or MigR1-ΔJmjC Ndy1 were probed with the anti-Ndy1 antibody. (D) MEFs infected with the indicated constructs were passaged in culture. Cumulative numbers of cells at each passage are indicated.
Fig. 4.
Fig. 4.
Endogenous Ndy1 physiologically inhibits replicative senescence in MEFs. (A) The Ndy1 and scrambled siRNAs were transfected into wild-type or MigR1-Ndy1.HA-infected MEFs. Western blots of transfected cell lysates were probed with the anti-Ndy1 antibody. (A1) Western blot of cell lysates harvested 48 h after the transfection. (A2) Time course of exogenous Ndy1 starting with cell lysates harvested 24 h after transfection. (B) Early-passage wild-type and MigR1-Ndy1.HA-infected MEFs were transfected with Ndy1 or control siRNAs, and they were passaged twice every 72 h. siRNA transfection was repeated after the first passage at the 72-h time point. Cells were counted, and the numbers were plotted as indicated. (C) Early-passage wild-type and MigR1-Ndy1.HA-infected MEFs were transfected with Ndy1 or control siRNA, and they were stained for β-galactosidase.
Fig. 5.
Fig. 5.
Ndy1 promotes immortalization by targeting the Rb and p53 pathways. (A) Western blots of MEFs infected with the indicated constructs were probed with the indicated antibodies. (B) Passaged MEFs infected with the indicated constructs express high levels of p53 and its target p21CIP1. Western blots of cell lysates harvested from passaged cells, probed with the indicated antibodies are shown. (C) MEFs infected with the indicated constructs were passaged in culture. Cumulative numbers of cells at each passage are indicated. (D) Western blots of passaged cells infected with the indicated constructs were probed with the indicated antibodies.
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