DNA damage signaling triggers degradation of histone methyltransferases through APC/C(Cdh1) in senescent cells.

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

Interplay between Homeobox proteins and Polycomb repressive complexes in p16INK4a regulation.

The INK4/ARF locus regulates senescence and is frequently altered in cancer. In normal cells, the INK4/ARF locus is found silenced by Polycomb repressive complexes (PRCs). Which are the mechanisms responsible for the recruitment of PRCs to INK4/ARF and their other target genes remains unclear. In a genetic screen for transcription factors regulating senescence, we identified the homeodomain-containing protein HLX1 (H2.0-like homeobox 1). Expression of HLX1 extends cellular lifespan and blunts oncogene-induced senescence. Using quantitative proteomics, we identified p16(INK4a) as the key target mediating the effects of HLX1 in senescence. HLX1 represses p16(INK4a) transcription by recruiting PRCs and HDAC1. This mechanism has broader implications, as HLX1 also regulates a subset of PRC targets besides p16(INK4a). Finally, sampling members of the Homeobox family, we identified multiple genes with ability to repress p16(INK4a). Among them, we found HOXA9 (Homeobox A9), a putative oncogene in leukaemia, which also recruits PRCs and HDAC1 to regulate p16(INK4a). Our results reveal an unexpected and conserved interplay between homeodomain-containing proteins and PRCs with implications in senescence, development and cancer.

Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.

Histone demethylase JMJD3 contributes to epigenetic control of INK4a/ARF by oncogenic RAS.

The INK4a/ARF tumor suppressor locus, a key executor of cellular senescence, is regulated by members of the Polycomb group (PcG) of transcriptional repressors. Here we show that signaling from oncogenic RAS overrides PcG-mediated repression of INK4a by activating the H3K27 demethylase JMJD3 and down-regulating the methyltransferase EZH2. In human fibroblasts, JMJD3 activates INK4a, but not ARF, and causes p16(INK4a)-dependent arrest. In mouse embryo fibroblasts, Jmjd3 activates both Ink4a and Arf and elicits a p53-dependent arrest, echoing the effects of RAS in this system. Our findings directly implicate JMJD3 in the regulation of INK4a/ARF during oncogene-induced senescence and suggest that JMJD3 has the capacity to act as a tumor suppressor.

Mitogen-activated protein kinase signaling mediates phosphorylation of polycomb ortholog Cbx7.

Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.

Ubiquitin-specific proteases 7 and 11 modulate Polycomb regulation of the INK4a tumour suppressor.

An important facet of transcriptional repression by Polycomb repressive complex 1 (PRC1) is the mono-ubiquitination of histone H2A by the combined action of the Posterior sex combs (Psc) and Sex combs extra (Sce) proteins. Here, we report that two ubiquitin-specific proteases, USP7 and USP11, co-purify with human PRC1-type complexes through direct interactions with the Psc orthologues MEL18 and BMI1, and with other PRC1 components. Ablation of either USP7 or USP11 in primary human fibroblasts results in de-repression of the INK4a tumour suppressor accompanied by loss of PRC1 binding at the locus and a senescence-like proliferative arrest. Mechanistically, USP7 and USP11 regulate the ubiquitination status of the Psc and Sce proteins themselves, thereby affecting their turnover and abundance. Our results point to a novel function for USPs in the regulation and function of Polycomb complexes.

Tumor suppressor p16INK4a determines sensitivity of human cells to transformation by cooperating cellular oncogenes.

The Ink4a/Arf locus encodes two distinct proteins, both of which may contribute to senescence and tumor suppression. We find that human diploid fibroblasts (HDFs) that are specifically deficient for p16INK4a achieve anchorage independence when transduced with retroviruses encoding telomerase (hTERT) and either Ras or Myc. Significantly, Ras and Myc together enable the cells to form tumors in nude mice but at a frequency that suggests additional genetic changes. All five tumors analyzed expressed high levels of Ras and retained functional p53, although two showed downregulation of Arf. Cytogenetic analyses identified clonal chromosomal alterations that may have contributed to tumorigenesis, but the tumor cells were essentially diploid.

CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms.

Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.

Functional, structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma-prone families or patients.

Germline mutations of the CDKN2A gene are found in melanoma-prone families and individuals with multiple sporadic melanomas. The encoded protein, p16(INK4A), comprises four ankyrin-type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16(INK4a) with its physiological targets, the cyclin-dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16(INK4A) structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16(INK4A) not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction.

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest.

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.

A CDKN2A mutation in familial melanoma that abrogates binding of p16INK4a to CDK4 but not CDK6.

The CDKN2A locus encodes two distinct proteins, p16INK4a and p14ARF, both of which are implicated in replicative senescence and tumor suppression in different contexts. Here, we describe the characterization of a novel strain of human diploid fibroblasts (designated Milan HDFs) from an individual who is homozygous for the R24P mutation in p16INK4a. As this mutation occurs in the first exon of INK4a (exon 1alpha), it has no effect on the primary sequence of p14(ARF). Based on both in vitro and in vivo analyses, the R24P variant is specifically defective for binding to CDK4 but remains able to associate with CDK6. Nevertheless, Milan HDFs behave as if they are p16INK4a deficient, in terms of sensitivity to spontaneous and oncogene-induced senescence, and the R24P variant has little effect on proliferation when ectopically expressed in normal fibroblasts. It can, however, impair the proliferation of U20S cells, presumably because they express more CDK6 than primary fibroblasts. These observations suggest that CDK4 and CDK6 are not functionally redundant and underscore the importance of CDK4 in the development of melanoma.

The role of the aryl hydrocarbon receptor-interacting protein gene in familial and sporadic pituitary adenomas.

CONTEXT: Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE: AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS: Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS: Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS: Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

Immortalisation and transformation revisited.

Access to cDNA encoding the catalytic subunit of telomerase and the consequent ability to immortalize human cells in culture has enabled researchers to 'transform' normal cells into malignant clones. However, there is a continuing debate over the number of genetic alterations required and clear differences in the way that mouse and human cells respond to these alterations.

Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts.

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.

Immortalization of primary human prostate epithelial cells by c-Myc.

A significant percentage of prostate tumors have amplifications of the c-Myc gene, but the precise role of c-Myc in prostate cancer is not fully understood. Immortalization of human epithelial cells involves both inactivation of the Rb/p16INK4a pathway and telomere maintenance, and it has been recapitulated in culture by expression of the catalytic subunit of telomerase, hTERT, in combination with viral oncoproteins. Here, we show the immortalization of human prostate epithelial cells (HPrEC) by a single genetic event, the expression of the c-Myc oncogene. Myc stabilizes telomere length in HPrEC through up-regulation of hTERT expression and overrides the accumulation of cell cycle inhibitory proteins, such as p16INK4a. Overall, HPrECs expressing c-Myc retain many characteristics of normal cells, such as the induction of a senescence-like growth arrest in response to oncogenic Ras, an intact p53 response, and an absence of gross karyotypic abnormalities. However, HPrECs expressing c-Myc lack a Rb/p16INK4a checkpoint and can be transformed without the need for additional genetic lesions in that pathway. These results give a partial explanation for the physiologic role of c-Myc overexpression in prostate cancer.

Glycolytic enzymes can modulate cellular life span.

An unbiased screen for genes that can immortalize mouse embryonic fibroblasts identified the glycolytic enzyme phosphoglycerate mutase (PGM). A 2-fold increase in PGM activity enhances glycolytic flux, allows indefinite proliferation, and renders cells resistant to ras-induced arrest. Glucosephosphate isomerase, another glycolytic enzyme, displays similar activity and, conversely, depletion of PGM or glucosephosphate isomerase with short interfering RNA triggers premature senescence. Immortalized mouse embryonic fibroblasts and mouse embryonic stem cells display higher glycolytic flux and more resistance to oxidative damage than senescent cells. Because wild-type p53 down-regulates PGM, mutation of p53 can facilitate immortalization via effects on PGM levels and glycolysis.

CBX7 controls the growth of normal and tumor-derived prostate cells by repressing the Ink4a/Arf locus.

Control of cell proliferation by Polycomb group proteins (PcG) is an important facet of cellular homeostasis and its disruption can promote tumorigenesis. We recently described CBX7 as a novel PcG protein controlling the growth of normal cells. In an attempt to identify a putative role of CBX7 in tumorigenesis, we analysed CBX7 expression in a panel of cancer cell lines and primary tissues. CBX7 was highly expressed in three different prostate cancer cell lines and present at elevated levels in normal prostate. Ablation of CBX7 expression using short hairpin RNAs (shRNA) resulted in upregulation of p16Ink4a and p14Arf in both LNCaP and PC-3 prostate cell lines. CBX7 knockdown caused an impairment of cell growth that was dependent on the status of the p14Arf/p53 and p16Ink4a/Rb pathways in both normal and cancer prostate cells. CBX7 overexpression in LNCaP cells resulted in a slight growth advantage in both androgen-dependent and -independent conditions. Moreover, CBX7 expression cooperated with c-Myc in rendering LNCaP cells insensitive to growth arrest by androgen receptor inhibition. Together, these data suggest that CBX7 represses p16Ink4a and p14Arf expression in normal and tumor-derived prostate cells, affecting their growth depending on the status of the p16Ink4a/Rb and the p14Arf/p53 pathways.

Multiple interacting domains contribute to p14ARF mediated inhibition of MDM2.

The small basic protein p14ARF, encoded by one of the alternative transcripts from the human INK4A/ARF locus, interferes with MDM2-mediated ubiquitination of the p53 tumour suppressor protein. The resultant stabilization of p53 leads to increased expression of p53-regulated genes, such as MDM2 itself and the cyclin-dependent kinase inhibitor p21(CIP1). Here we relate physical interactions between p14ARF and MDM2, as determined using synthetic peptides and systematic deletions of p14ARF, with consequential effects on p53 stabilization and transcriptional activity. The data imply that the amino terminal half of p14ARF, encoded by the alternative first exon (exon 1beta) contacts MDM2 through multiple domains that can independently impede MDM2-mediated degradation of p53, provided that they are localized in the cell nucleus. As well as identifying previously unrecognized functional domains, our findings offer an explanation for the relative paucity of missense mutations in exon 1beta in human tumours.