Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

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During 3T3-L1 preadipocyte differentiation induction, the insulin-stimulated insulin-like growth factor-1 (IGF-1) receptor signal is responsible for the induction of adipocyte differentiation. Treatment with inhibitors of phosphatidylinositol 3-kinase, LY294002 or wortmannin, leads to the complete blockade of adipocyte differentiation in 3T3-L1 preadipocytes. Of the three factors (1-methyl-3-isobutylxanthine, dexamethasone, and insulin) inducing 3T3-L1 preadipocyte differentiation, only insulin was able to activate the phosphatidylinositol 3-kinase-protein kinase B/Akt signal cascade. In 3T3-L1 preadipocytes, protein kinase B/Akt 1 RNA interference not only suppressed the expression of protein kinase B/Akt 1 but also blocked hormone-induced adipocyte differentiation. In these protein kinase B/Akt 1 RNA interference cells, the signal molecules upstream of protein kinase B/Akt 1, such as IGF-1 receptor and insulin receptor substrate-1, were normally activated by insulin stimulation, whereas insulin-stimulated phosphorylation of forkhead transcription factor (FKHR), which is a downstream molecule of PKB/Akt 1, was blocked. Thus, protein kinase B/Akt 1 is an important signal mediator in IGF-1 receptor signal cascade for inducing adipocyte differentiation.Most of our current understandings on the cellular mechanisms of adipocyte differentiation regulation have come from the studies on established in vitro preadipocyte cell lines (1-3). These in vitro preadipocyte cell lines, e.g. 3T3-L1, 3T3-F442A, and TA1, can be induced to differentiate into adipocytes by insulin or in combination with other hormones. Subsequent studies on 3T3-L1 adipocyte differentiation induction indicate that the authentic hormone to induce the adipocyte differentiation is insulin-like growth factor-1 (IGF-1), 1 and insulin acts through the IGF-1 receptor on the cell membrane to induce the differentiation (4). In post-confluent 3T3-L1 preadipocytes, IGF-1 receptor signal initiates the adipocyte differentiation process. Immediately after the hormonal stimulation, postconfluent G 0 3T3-L1 preadipocytes reenter the cell cycle and start the adipocyte differentiation program. It is clear that the IGF-1 receptor signal plays an irreplaceable role in inducing the adipocyte differentiation process in 3T3-L1 cells.The signaling pathways or networks involved in mediating IGF-1 receptor signal for adipocyte differentiation are not fully understood. Two kinase systems, MAP kinases and phosphatidylinositol 3-kinase-protein kinase B/Akt, in 3T3-L1 cells can be activated by IGF-1 receptor signaling. The function of the MAP kinase system in IGF-1 receptor signal-induced 3T3-L1 cell adipogenesis is not clear. It is further complicated by the different effects exhibited by p38 MAP kinase and ERK1/2 on 3T3-L1 cell adipogenesis (5-12). More molecular studies are required in order to understand the relationship between MAP kinase system and 3T3-L1 cell adipogenesis.PI 3-kinase-PKB/Akt as an important intracellular signal cascade has been involved in the regulation o...

Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germlineinactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1 þ/À knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1 þ/À mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1 þ/À mice than in WT animals (median survival, 43 weeks vs. 55weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1 þ/À mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1 þ/À mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1 þ/À mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1 þ/À mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1 þ/À mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure. Cancer Res; 74(16); 4388-97. Ó2014 AACR.

Individuals harboring inherited heterozygous germline mutations in BAP1 are predisposed to a range of benign and malignant tumor types, including malignant mesothelioma, melanoma, and kidney carcinoma. However, evidence to support a tumor suppressive role for BAP1 in cancer remains contradictory. To test experimentally whether BAP1 behaves as a tumor suppressor, we monitored spontaneous tumor development in three different mouse models with germline heterozygous mutations in Bap1, including two models in which the knock-in mutations are identical to those reported in human BAP1 cancer syndrome families. We observed spontaneous malignant tumors in 54 of 93 Bap1-mutant mice (58%) versus 4 of 43 (9%) wild-type littermates. All three Bap1-mutant models exhibited a high incidence and similar spectrum of neoplasms, including ovarian sex cord stromal tumors, lung and mammary carcinomas, and spindle cell tumors. Notably, we also observed malignant mesotheliomas in two Bap1-mutant mice, but not in any wild-type animals. We further confirmed that the remaining wild-type Bap1 allele was lost in both spontaneous ovarian tumors and mesotheliomas, resulting in the loss of Bap1 expression. Additional studies revealed that asbestos exposure induced a highly significant increase in the incidence of aggressive mesotheliomas in the two mouse models carrying clinically relevant Bap1 mutations compared with asbestos-exposed wild-type littermates. Collectively, these findings provide genetic evidence that Bap1 is a bona fide tumor suppressor gene, and offer key insights into the contribution of carcinogen exposure to enhanced cancer susceptibility.

Previously, we have found that lipid rafts/caveolae were essential for insulin-like growth factor-1 (IGF-1) receptor signaling during 3T3-L1 preadipocytes differentiation induction. However, it was not identified as to which of the membrane lipid-ordered microdomains mediates the receptor signal. Using small double-stranded RNA-mediated interference (RNAi), we successfully suppressed the caveolin-1 protein expression. In cells stably transfected with vector expressing small interfering RNA (siRNA) fragment, no caveolin-1 protein or caveola was detected. On the other hand, removal of caveolin-1 did not affect the caveolinless lipid rafts or the localization of IGF-1 receptor in lipid rafts on plasma membrane. IGF-1 receptor signal transduction and induced cellular differentiation were normal in RNAi cells with only lipid rafts. Furthermore, these IGF-1 receptor signaling events were still sensitive to the cholesterol-binding reagents. Thus, our results suggest that lipid rafts are sufficient for IGF-1 receptor signaling and the recruitment of signal molecules by caveolin-1 is not essential for IGF-1 receptor signaling.

The oncogene v-akt was isolated from a retrovirus that induced murine thymic lymphomas. Transgenic mice expressing a constitutively activated form of the cellular homologue Akt2 specifically in immature T cells develop spontaneous thymic lymphomas. We hypothesized that tumors from these mice might exhibit oncogenic chromosomal rearrangements that cooperate with activated Akt2 in lymphomagenesis. Cytogenetic analysis revealed a recurrent clonal inversion of chromosome 6, inv(6), in thymic lymphomas from multiple transgenic founder lines, including one line in which 15 of 15 primary tumors exhibited this same rearrangement. Combined fluorescence in situ hybridization, PCR, and DNA sequence analyses showed that the distal inv(6) breakpoint resides at the T-cell receptor B chain locus, Tcrb. The proximal breakpoint maps to a region near a locus comprising the linked homeobox/transcription factor genes Dlx5 and Dlx6. Expression analysis of genes translocated to the vicinity of the Tcrb enhancer revealed that Dlx5 and Dlx6 are overexpressed in tumors exhibiting the inv(6). Experimental overexpression of Dlx5 in mammalian cells resulted in enhanced cell proliferation and increased colony formation, and clonogenic assays revealed cooperativity when both Dlx5 and activated Akt2 were coexpressed. In addition, DLX5, but not DLX6, was found to be abundantly expressed in three of seven human T-cell lymphomas tested. These findings suggest that the Dlx5 can act as an oncogene by cooperating with Akt2 to promote lymphomagenesis.

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