Despite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).

Inheritance of epigenetic information encoded by cytosine DNA methylation patterns is crucial for mammalian cell survival, in large part through the activity of the maintenance DNA methyltransferase (DNMT1). Here, we show that SET7, a known histone methyltransferase, is involved in the regulation of protein stability of DNMT1. SET7 colocalizes and directly interacts with DNMT1 and specifically monomethylates Lys-142 of DNMT1. Methylated DNMT1 peaks during the S and G 2 phases of the cell cycle and is prone to proteasome-mediated degradation. Overexpression of SET7 leads to decreased DNMT1 levels, and siRNA-mediated knockdown of SET7 stabilizes DNMT1. These results demonstrate that signaling through SET7 represents a means of DNMT1 enzyme turnover.DNA methyltransferase ͉ methylated lysine ͉ proteasome ͉ protein degradation M ammalian DNA methylation is essential for development and is controlled by a variety of factors including 3 active DNA cytosine methyltransferases (DNMT1, DNMT3A, and DNMT3B) and a methyltransferase-like protein, DNMT3L (1-4). DNMT1 encodes the maintenance DNA methyltransferase (DNMT) responsible for methylating hemimethylated CpG sites shortly after DNA replication, and it is assisted by an accessory factor capable of recognizing hemimethylated DNA called UHRF1 (5, 6). Aberrant DNA methylation of CpG islandcontaining promoters leads to permanent silencing of genes in both physiological and pathological contexts and specifically in cancer cells (7). In cancer cells, disruption of DNMT1 resulted in hemimethylation of a fifth of the CpG sites in the genome and activation of the G 2 /M checkpoint, leading to arrest in the G 2 phase of the cell cycle (8). Apart from DNA methylationmediated gene silencing, DNMT1 also binds to several transcriptional inhibitors and represses gene expression in a DNA methylation-independent manner (9-11). Pharmacological inhibitors of DNMT1 [5-azacytidine (5-aza-CR) and its deoxy analog, 5-aza-2Ј-deoxycytidine (5-aza-CdR)] get incorporated into newly-synthesized DNA (12, 13). Once incorporated into DNA, these compounds form covalent complexes with DNMTs, thereby depleting active enzymes (14, 15) and activating gene expression (16). Recently, 5-aza-CdR-induced depletion of DNMT1 was shown to be mediated by proteasomal pathways in mammalian nuclei (17). However, little is known about other factors regulating DNMT1 levels in cells. Here, we show that DNMT1 stability is regulated by protein methylation coupled to proteasome-mediated protein degradation through the protein methyltransferase activity of SET7. Results DNMT1 Colocalizes and Associates with and Is Methylated by SET7.We used gel filtration and Western blot analysis to analyze DNMT1 from nuclear extracts. Using a highly-specific antibody we observed a major species of DNMT1 at 185 kDa and a higher molecular mass minor species (Fig. 1A). It seemed likely that this minor species of DNMT1 may be posttranslationally modified (18). To test whether DNMT1 might be modified by protein methylation, recombinant DNMT1 wa...

Pituitary tumors are commonly encountered, and result from clonal expansion of a single mutated cell. Hypothalamic hormones, local growth factors and circulating sex steroid hormones promote pituitary tumor growth and expansion into large invasive tumors. Estrogen acting directly through its receptor and by stimulation of fibroblast growth factor regulates prolactin synthesis and secretion. Fibroblast growth factor-2 (bFGF) modulates angiogenesis, tumor formation and progression in many tissues, including the anterior pituitary. A pituitary tumor-derived transforming gene (PTTG) has been isolated, which is tumorigenic in vivo, regulates bFGF secretion, and inhibits chromatid separation. The human PTTG family consists of at least three homologous genes, of which PTTG1 is located on chromosome 5q33 and is expressed at low levels in most normal human tissues but is highly expressed in malignant human cell lines and in pituitary tumors. We report here that pituitary pttg is regulated in vivo and in vitro by estrogen. Maximal induction of rat pituitary pttg mRNA in vivo occurred early in pituitary transformation (normal cell to hypertrophic/hyperplastic cell), coincident with bFGF and vascular endothelial growth factor induction and pituitary angiogenesis. We also demonstrate that pttg expression is induced by bFGF, and show concordant pttg and bFGF expression in experimental and human pituitary adenomas. As bFGF and estrogen both induce pttg, and pttg expression coincides with the early lactotrophic hyperplastic response, angiogenesis and prolactinoma development, we propose a previously unknown paracrine growth factor-mediated mechanism for pituitary tumor pathogenesis and potentially other estrogen-regulated tumors.

Adenovirus e1a induces quiescent human cells to replicate. We found that e1a causes global relocalization of the RB (retinoblastoma) proteins (RB, p130, and p107) and p300/CBP histone acetyltransferases on promoters, the effect of which is to restrict the acetylation of histone 3 lysine-18 (H3K18ac) to a limited set of genes, thereby stimulating cell cycling and inhibiting antiviral responses and cellular differentiation. Soon after expression, e1a binds transiently to promoters of cell cycle and growth genes, causing enrichment of p300/CBP, PCAF (p300/CBP-associated factor), and H3K18ac; depletion of RB proteins; and transcriptional activation. e1a also associates transiently with promoters of antiviral genes, causing enrichment for RB, p130, and H4K16ac; increased nucleosome density; and transcriptional repression. At later times, e1a and p107 bind mainly to promoters of development and differentiation genes, repressing transcription. The temporal order of e1a binding requires its interactions with p300/CBP and RB proteins. Our data uncover a defined epigenetic reprogramming leading to cellular transformation.The adenovirus small e1a oncoprotein interacts with multiple cellular factors to induce cell cycling in G 0 -arrested cells to favor viral replication. Mutations of e1a regions that interact with the RB proteins or p300/CBP [cyclic adenosine monophosphate response element-binding protein (CREB)-binding protein] result in loss of e1a-transforming and mitogenic activities (1-3) (figs. S1 and S2). Binding of e1a to p300/CBP inhibits transcriptional activation by certain enhancers (4); however, it is unclear how this interaction promotes cell cycling and why it is required for e1a oncogenicity. The e1a-p300/CBP interaction causes a factor of ~3 reduction in total cellular histone 3 Lys 18 acetylation (H3K18ac) specifically (5). Therefore, we sought to determine how e1a affects the genome-wide distributions of its interacting cellular factors as well as histone modifications (including H3K18ac) to establish an oncogenic gene expression program. Using chromatin immunoprecipitation (ChIP) combined with microarrays (6), we examined the genome-wide binding of e1a at 2, 6, 12, and 24 hours (here and below, all times are postinfection) of confluent, contact-inhibited human IMR90 primary fibroblasts (ATCC CCL-186) in which e1a induces entry into S phase between 18 and 24 hours ( fig. S2). We used an Agilent microarray containing probes for ~17,000 promoters, tiling an 8-kb region, which we divided computationally into 16 fragments of 500 base pairs (bp) each, spanning -5.5 to +2.5 kb of the transcription start site (TSS). Cells were infected with Ad5 mutant dl1500, which expresses only the small e1a protein (7). Using unbiased partitional clustering, we grouped the genes primarily into three clusters that captured the main trends in the data. We calculated a Z score to indicate the degree of enrichment for a given factor in each cluster.During the 24-hour period after expression, at a cutoff of Z ≥ 2, e1a bou...

Adenovirus small early region 1a (e1a) protein drives cells into S phase by binding RB family proteins and the closely related histone acetyl transferases p300 and CBP. The interaction with RB proteins displaces them from DNA-bound E2F transcription factors, reversing their repression of cell cycle genes. However, it has been unclear how the e1a interaction with p300 and CBP promotes passage through the cell cycle. We show that this interaction causes a threefold reduction in total cellular histone H3 lysine 18 acetylation (H3K18ac). CBP and p300 are required for acetylation at this site because their knockdown causes specific hypoacetylation at H3K18. SV40 T antigen also induces H3K18 hypoacetylation. Because global hypoacetylation at this site is observed in prostate carcinomas with poor prognosis, this suggests that processes resulting in global H3K18 hypoacetylation may be linked to oncogenic transformation.The adenovirus small e1a protein drives contact-inhibited primary cells through the cell cycle dependent on three conserved regions (CRs) in e1a ( fig. S1) (1). e1a is not a DNA binding protein, but it binds several other proteins. e1a CR2 binds the retinoblastoma protein (RB) and its paralogs p107 (RBL1) and p130 (RBL2) with high affinity and, together with a lower affinity binding region within CR1, displaces RB proteins from E2F family transcription factors (heterodimers of E2F1 through E2F5 with DP1 or DP2) (1-3). Release of RB family proteins and their associated repressing chromatin modifying activities (4) results in de-repression of cell cycle genes (1). Although the N terminus of e1a is not as extensively conserved among primate adenoviruses as CR1 and CR2, it is nonetheless well conserved among these viruses (5) and is required to drive cells into the cell cycle (1). The N terminus and residues in CR1

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA–directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA–directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA–directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA–directed DNA methylation systems consist of a combination of ancestral and convergent features.

We recently cloned a novel pituitary tumor transforming gene (PTTG). Here we report PTTG expression in human pituitary adenomas and in normal pituitary tissue. In situ hybridization revealed PTTG expression in nonfunctioning and in GH-secreting adenomas but not in normal pituitary tissue. Using a more sensitive detection method, RT-PCR, low level PTTG expression was detected in normal pituitary. However, when expression levels in normal pituitary tissue were compared with those in 54 pituitary tumors using comparative reverse transcription polymerase chain reaction (RT-PCR), we found that most tumor samples expressed higher levels of PTTG. More than 50% PTTG increases were observed in 23 of 30 nonfunctioning pituitary tumors, all 13 GH-producing tumors, 9 of 10 prolactinomas, and 1 ACTH-secreting tumor, with more than 10-fold increases evident in some tumors. Furthermore, higher PTTG expression (P = 0.03) was observed in hormone-secreting tumors that had invaded the sphenoid bone (stages III and IV; 95% CI 3.118-9.715) compared with hormone-secreting tumors that were confined to the pituitary fossa (stages I and II; 95% CI 1.681-3.051). Therefore, PTTG abundance is a molecular marker for invasiveness in hormone-secreting pituitary tumors. The ubiquitous and prevalent expression of pituitary adenoma PTTG suggests that PTTG plays a role in pituitary tumorigenesis and invasiveness.

The pituitary transforming gene, PTTG, is abundantly expressed in endocrine neoplasms. PTTG has recently been recognized as a mammalian securin based on its biochemical homology to Pds1p. PTTG expression and intracellular localization were therefore studied during the cell cycle in human placental JEG-3 cells. PTTG mRNA and protein expressions were low at the G1/S border, gradually increased during S phase, and peaked at G2/M, but PTTG levels were attenuated as cells entered G1. In interphase cells, wild-type PTTG, an epitope-tagged PTTG, and a PTTG-EGFP conjugate all localized to both the nucleus and cytoplasm, but in mitotic cells, PTTG was not observed in the chromosome region. PTTG-EGFP colocalized with mitotic spindles in early mitosis and was degraded in anaphase. Intracellular fates of PTTG-EGFP and a conjugate of EGFP and a mutant inactivated PTTG devoid of an SH3-binding domain were observed by real-time visualization of the EGFP conjugates in live cells. The same cells were continuously observed as they progressed from G1/S border to S, G2/M, and G1. Most cells (67%) expressing PTTG-EGFP died by apoptosis, and few cells (4%) expressing PTTG-EGFP divided, whereas those expressing mutant PTTG-EGFP divided. PTTG-EGFP, as well as the mutant PTTG-EGFP, disappeared after cells divided. The results show that PTTG expression and localization are cell cycle-dependent and demonstrate that PTTG regulates endocrine tumor cell division and survival.