Methylation-independent Manner Research Articles

SET8 is a novel negative regulator of TGF-β signaling in a methylation-independent manner

Transforming growth factor β (TGF-β) is a multifunctional cytokine that induces a diverse set of cellular processes principally through Smad-dependent transcription. Transcriptional responses induced by Smads are tightly regulated by Smad cofactors and histone modifications; however, the underlying mechanisms have not yet been elucidated in detail. We herein report lysine methyltransferase SET8 as a negative regulator of TGF-β signaling. SET8 physically associates with Smad2/3 and negatively affects transcriptional activation by TGF-β in a catalytic activity-independent manner. The depletion of SET8 results in an increase in TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) and p21 expression and enhances the antiproliferative effects of TGF-β. Mechanistically, SET8 occupies the PAI-1 and p21 promoters, and a treatment with TGF-β triggers the replacement of the suppressive binding of SET8 with p300 on these promoters, possibly to promote gene transcription. Collectively, the present results reveal a novel role for SET8 in the negative regulation of TGF-β signaling.

Dynamics of imprinted genes and their epigenetic mechanisms in castor bean seed with persistent endosperm.

Genomic imprinting refers to parent-of-origin-dependent gene expression and primarily occurs in the endosperm of flowering plants, but its functions and epigenetic mechanisms remain to be elucidated in eudicots. Castor bean, a eudicot with large and persistent endosperm, provides an excellent system for studying the imprinting. Here, we identified 131 imprinted genes in developing endosperms and endosperm at seed germination phase of castor bean, involving into the endosperm development, accumulation of storage compounds and specially seed germination. Our results showed that the transcriptional repression of maternal allele of DNA METHYLTRANSFERASE 1 (MET1) may be required for maternal genome demethylation in the endosperm. DNA methylation analysis showed that only a small fraction of imprinted genes was associated with allele-specific DNA methylation, and most of them were closely associated with constitutively unmethylated regions (UMRs), suggesting a limited role for DNA methylation in controlling genomic imprinting. Instead, histone modifications can be asymmetrically deposited in maternal and paternal genomes in a DNA methylation-independent manner to control expression of most imprinted genes. These results expanded our understanding of the occurrence and biological functions of imprinted genes and showed the evolutionary flexibility of the imprinting machinery and mechanisms in plants.

KIAA1429 promotes infantile hemangioma regression by facilitating the stemness of hemangioma endothelial cells.

Infantile hemangiomas are common vascular tumors with a specific natural history. The proliferation and regression mechanism of infantile hemangiomas may be related to the multilineage differentiation ability of hemangioma stem cells, but the specific mechanism is not well elucidated. KIAA1429 is an N6 -methyladenosine methylation-related protein that can also exert its role in a methylation-independent manner. This study aims to explore the function of KIAA1429 in infantile hemangiomas. qRT-PCR, western blotting, and immunostaining were performed to verify the expression of KIAA1429. The endothelial and fibroblast-like phenotypes of hemangioma endothelial cells were detected after KIAA1429 knockdown and overexpression. The stemness properties of hemangioma endothelial cells and the underlying mechanism of KIAA1429 in hemangiomas were also investigated. Nude mouse models of infantile hemangiomas were conducted to ascertain the effects of KIAA1429 in vivo. The results showed that KIAA1429 was highly expressed in infantile hemangiomas, particularly in involuting hemangiomas. In vitro experiments confirmed that KIAA1429 inhibited the endothelial phenotype, enhanced the differentiation ability, and promoted the fibroblast-like phenotype of hemangioma endothelial cells by inducing endothelial cell transition to facultative stem cells. However, the effect of KIAA1429 on the potential target was shown to be independent of N6 -methyladenosine methylation modification. Mouse models further revealed that KIAA1429 could inhibit the proliferation and promote the regression of hemangiomas. In conclusion, this study found that KIAA1429 played an important role in the regression of infantile hemangiomas by enhancing the stemness of hemangioma endothelial cells and could be a potential treatment target for infantile hemangiomas.

Methylation-dependent and -independent roles of EZH2 synergize in CDCA8 activation in prostate cancer.

Cell division cycle-associated 8 (CDCA8) is a component of chromosomal passenger complex (CPC) that participates in mitotic regulation. Although cancer-related CDCA8 hyperactivation has been widely observed, its molecular mechanism remains elusive. Here, we report that CDCA8 overexpression maintains tumorigenicity and is associated with poor clinical outcome in patients with prostate cancer (PCa). Notably, enhancer of zeste homolog 2 (EZH2) is identified to be responsible for CDCA8 activation in PCa. Genome-wide assays revealed that EZH2-induced H3K27 trimethylation represses let-7b expression and thus protects the let-7b-targeting CDCA8 transcripts. More importantly, EZH2 facilitates the self-activation of E2F1 by recruiting E2F1 to its own promoter region in a methylation-independent manner. The high level of E2F1 further promotes transcription of CDCA8 along with the other CPC subunits. Taken together, our study suggests that EZH2-mediated cell cycle regulation in PCa relies on both its methyltransferase and non-methyltransferase activities.

Dysregulation of schizophrenia-associated genes and genome-wide hypomethylation in neurons overexpressing DNMT1.

Aim: To study the effects of DNMT1 overexpression on transcript levels of genes dysregulated in schizophrenia and on genome-wide methylation patterns. Materials & methods: Transcriptome and DNA methylome comparisons were made between R1 (wild-type) and Dnmt1tet/tet mouse embryonic stem cells and neurons overexpressing DNMT1. Genes dysregulated in both Dnmt1tet/tet cells and schizophrenia patients were studied further. Results & conclusions: About 50% of dysregulated genes in patients also showed altered transcript levels in Tet/Tet neurons in a DNA methylation-independent manner. These neurons unexpectedly showed genome-wide hypomethylation, increased transcript levels of Tet1 and Apobec 1-3 genes andincreased activity and copy number of LINE-1 elements. The observed similarities between Tet/Tet neurons and schizophrenia brain samples reinforce DNMT1 overexpression as a risk factor.

Activation-induced cytidine deaminase is a possible regulator of cross-talk between oocytes and granulosa cells through GDF-9 and SCF feedback system

Activation-induced cytidine deaminase (AID, Aicda) is a master gene regulating class switching of immunoglobulin genes. In this study, we investigated the significance of AID expression in the ovary. Immunohistological study and RT-PCR showed that AID was expressed in murine granulosa cells and oocytes. However, using the Aicda-Cre/Rosa-tdRFP reporter mouse, its transcriptional history in oocytes was not detected, suggesting that AID mRNA in oocytes has an exogenous origin. Microarray and qPCR validation revealed that mRNA expressions of growth differentiation factor-9 (GDF-9) in oocytes and stem cell factor (SCF) in granulosa cells were significantly decreased in AID-knockout mice compared with wild-type mice. A 6-h incubation of primary granuloma cells markedly reduced AID expression, whereas it was maintained by recombinant GDF-9. In contrast, SCF expression was induced by more than threefold, whereas GDF-9 completely inhibited its increase. In the presence of GDF-9, knockdown of AID by siRNA further decreased SCF expression. However, in AID-suppressed granulosa cells and ovarian tissues of AID-knockout mice, there were no differences in the methylation of SCF and GDF-9. These findings suggest that AID is a novel candidate that regulates cross-talk between oocytes and granulosa cells through a GDF-9 and SCF feedback system, probably in a methylation-independent manner.

Decreases in different Dnmt3b activities drive distinct development of hematologic malignancies in mice

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/− mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/− mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/− mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/− lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.

The De Novo DNA Methyltransferases <i>DNMT3A</i> and <i>DNMT3B</i> Initiate Erosion of Dosage Compensation in Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell based therapies. However, silencing of XIST expression followed by inappropriate reactivation and biallelic expression of X chromosome linked genes regularly occurs in female hPSCs, placing them in a state observed in human tumors. This gradual erosion of dosage compensation also occludes cellular phenotypes while modeling X-linked diseases and interferes with directed differentiation, greatly limiting the utility of female hPSCs. Here, we investigate the mechanisms regulating erosion of dosage compensation and whether it can be reversed or prevented. We found that changes to the X chromosome during erosion of dosage compensation both repress XIST through DNA methylation and prevent its RNA from silencing the chromosome, even if XIST expression is induced using CRISPR/Cas9 activation. Encouragingly, we found that elimination of the DNMT3A and genes, but the similarity of global transcriptional status of DNMT3A/3B deleted cell to non41 eroded cell differs in cell lines. While DNMT3A/3B deletion after erosion of dosage compensation failed to restore it. Thus, our study revealed that de novo DNA methyltransferases are primary factors responsible for the initiation of erosion of dosage compensation in female hPSCs and once DNA methylation is imposed on XIST promoterregions, the status is stably maintained to repress XIST by de novo DNA methylation independent manner.

Tumor Suppressor Function of Dnmt3b in Hematologic Malignancies Critically Depends on Its Catalytic Activity

DNA methylation regulates gene transcription and it is involved in various physiological processes in mammals including development and hematopoiesis. It is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B that not only mediate methylation but also interact with a number of repressive proteins, thereby contributing to transcriptional repression in methylation-independent manner. Dnmt3b is critical gene in mouse embryogenesis whose loss results in embryonic lethality around E13.5. It also functions as a tumor suppressor in lymphoid and myeloid malignancies in mice. The extent to which Dnmt3b's catalytic activity is involved in these processes is unclear. To address the role of catalytic activity in embryogenesis and tumorigenesis, we generated mice carrying a catalytically inactive allele of Dnmt3b (Dnmt3bCI) by introducing a double amino acid substitution in enzyme active center (P656V; C657D) in endogenous Dnmt3b locus. By interbreeding Dnmt3b+/CI mice we found that homozygous mutant Dnmt3bCI/CImice survived embryonic development and had a long life span. Such results demonstrated that catalytic activity of Dnmt3b is, surprisingly, dispensable for both pre- and post-natal development. The presence of catalytically inactive Dnmt3b rescued most methylation changes observed in the absence of Dnmt3b in embryogenesis suggesting that Dnmt3b plays a role of accessory protein for methylation mediated by other Dnmts. In contrast, the absence of Dnmt3b's catalytic activity promoted MYC/Ras-induced transformation of mouse embryonic fibroblasts in vitro, and accelerated both MYC-induced T-cell lymphomagenesis and MLL-AF9-induced acute myeloid leukemia. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in aberrant activation of IL7-R and STAT3 and downregulation of putative tumor suppressor genes including Cdkn2c and Dusp6. Altogether, our data suggest that Dnmt3b's methyltransferase activity is dispensable for mouse development but critical to prevent hematologic malignancies by controlling events involved in cellular transformation. Disclosures No relevant conflicts of interest to declare.

Abstract 4339: Critical role of Dnmt3b catalytic activity in prevention of oncogene-induced leukemias and lymphomas

Abstract Cytosine methylation is involved in a variety of biological processes, including development, X-chromosome inactivation, imprinting, and suppressing unwanted transcription. DNA methylation in mammalian cells is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. In addition to DNA methylation catalysis, these enzymes interact with a number of repressive proteins including HDACs, thereby repressing transcription in methylation-independent manner. The extent to which methylation-dependent and independent activities play roles in biological processes remains poorly understood. Here we examined the role of methyltransferase (MT) activity of Dnmt3b in normal mouse embryogenesis and in prevention of hematologic malignancies. Loss of Dnmt3b in mice is embryonically lethal between 10.5-12.5 dpc. Dnmt3b was also identified as tumor suppressor in murine models of lymphoid and myeloid malignancies. To address whether Dnmt3b’s catalytic activity is important for mouse embryogenesis and prevention of hematologic malignancies, we generated mice bearing a conventional Dnmt3bCI allele using CRISPR/Cas9 system. This catalytically inactive allele of Dnmt3b (Dnmt3bCI) was generated by a double amino acid substitution in enzyme active center (P656V and C657D) abolishing its ability to perform transfer of methyl group onto cytosine. Surprisingly, Dnmt3bCI/CI mice were born at similar ratios as wild-type littermates and lived over 12 months suggesting that Dnmt3b is dispensable for both pre- and post-natal development. Importantly, MYC-induced T-cell lymphomagenesis was accelerated in the absence of Dnmt3b’s methyltransferase activity in MYC;Dnmt3bCI/CI mice. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in activation of Pak, Fgfr and Igf2 signaling and downregulation of tumor suppressors (e.g. Lrp12, Dusp6 and Marcks). The lack of Dnmt3b catalytic activity also accelerated a development of acute myeloid leukemia induced by MLL-AF9 overexpression in mice. Altogether, our data suggest that Dnmt3b’s MT activity is dispensable for mouse development but critical to prevent hematologic malignancies. Citation Format: Katarina Lopusna, Pawel Nowialis, Staci L. Haney, Ajay Abraham, Jana Opavska, Rene Opavsky. Critical role of Dnmt3b catalytic activity in prevention of oncogene-induced leukemias and lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4339.

EBV infection is associated with histone bivalent switch modifications in squamous epithelial cells

Epstein-Barr virus (EBV) induces histone modifications to regulate signaling pathways involved in EBV-driven tumorigenesis. To date, the regulatory mechanisms involved are poorly understood. In this study, we show that EBV infection of epithelial cells is associated with aberrant histone modification; specifically, aberrant histone bivalent switches by reducing the transcriptional activation histone mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) at the promoter regions of a panel of DNA damage repair members in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA damage repair family members in base excision repair (BER), homologous recombination, nonhomologous end-joining, and mismatch repair (MMR) pathways showed aberrant histone bivalent switches. Among this panel of DNA repair members, MLH1, involved in MMR, was significantly down-regulated in EBV-infected NPE cells through aberrant histone bivalent switches in a promoter hypermethylation-independent manner. Functionally, expression of MLH1 correlated closely with cisplatin sensitivity both in vitro and in vivo. Moreover, seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched in pathway analysis in a promoter hypermethylation-independent manner. This observation is further validated by their down-regulation in EBV-infected NPE cells. The in vitro comet and apurinic/apyrimidinic site assays further confirmed that EBV-infected NPE cells showed reduced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner.

Abstract 2383: Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation

Abstract Recent evidence indicates that suppression of protein phosphatase 2A (PP2A) activity plays an essential role in malignant transformation and cancer cell maintenance. Despite the importance of PP2A as a tumor suppressor in cancer, the mechanisms by which other enzymes regulate PP2A remain largely unknown. We recently identified Nicotinamide N-methyltransferase (NNMT) as a novel regulator of PP2A activity in glioblastoma (GBM). NNMT is a cytosolic N-methyl transferase which utilizes methyl groups from S-Adenosyl methionine (SAM), forming the by-product S-Adenosyl Homocysteine (SAH). PP2A becomes activated following the trimer formation of the three PP2A subunits, which requires the methylation of the catalytic subunit of PP2A (PP2A C) at L309.In cancer, NNMT has been reported to decrease the cellular methylation potential (SAM:SAH) and correspond with decreased PP2A activity. It was hypothesized that the decrease in methylation potential accounts for the decrease in PP2A activity by kinetically disfavoring the methylation of PP2A C. Interestingly, when we overexpressed NNMT in GBM cell lines and used LC-MS/MS (LC-MS Quadrupole Time-of-Flight and LC-MS Triple Quad) to monitor the methylation potential, we discovered that overexpression of NNMT increased methylation potential, however, still lead to a decrease in PP2A activity. As a result, we hypothesized that NNMT suppresses PP2A activity in a methylation-independent manner. In an effort to identify an alternative mechanism by which NNMT decreases PP2A activity, we found that NNMT expression was required for the phosphorylation of PP2A C at residue T307, which inhibits PP2A trimer formation and activation. Further, we found that NNMT expression leads to the constitutive activation of the PP2A target serine/threonine kinases (STKs): Akt, MAPK, and SAPK/JNK. NNMT silencing resulted in the deactivation of these STKs and radiation-induced cellular stress was not sufficient to rescue their activation. This suggests that NNMT-induced PP2A suppression is essential for the activation of these kinases. Overall, this study demonstrates a novel mechanism independent of methylation by which NNMT represses the activity of tumor suppressor PP2A. Citation Format: John R. Jacob, Arnab Chakravarti, Kamalakannan Palanichamy. Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2383. doi:10.1158/1538-7445.AM2017-2383

G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression.

GSK-3β is Dephosphorylated by PP2A in a Leu309 Methylation-Independent Manner.

Hyperphosphorylation of tau is pivotally involved in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. Glycogen synthase kinase-3β (GSK-3β) and protein phosphate 2A (PP2A) are crucial enzymes to regulate tau phosphorylation. GSK-3β activity is regulated by its inhibitory phosphorylation at Ser9. We previously reported the cross-talk between GSK-3β and PP2A signaling and showed that PP2A could dephosphorylate GSK-3β at Ser9. Here, we investigated the dephosphorylation of GSK-3β in brain extracts in the presence of phosphatase inhibitors and found that a PP2A-like phosphatase activity was required for dephosphorylation of GSK-3β at Ser9. PP2A interacted with GSK-3β and suppressed its Ser9 phosphorylation in vitro and in HEK-293FT cells. Activity of PP2A negatively correlated to the level of phosphorylated GSK-3β in kainic acid-induced excitotoxic mouse brain. Alteration of methylation of the catalytic subunit of PP2A (PP2Ac) at Leu309 did not affect GSK-3β phosphorylation. These findings suggest that Leu309 methylation is not required for PP2A to dephosphorylate GSK-3β at Ser9.

Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis.

DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3a(Δ/Δ) mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3a(Δ/Δ) lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3a(Δ/Δ) mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.

PRMT3 regulates hepatic lipogenesis through direct interaction with LXRα.

Arginine methylation is responsible for diverse biological functions and is mediated by protein arginine methyltransferases (PRMTs). Nonalcoholic fatty liver disease (NAFLD) is accompanied by excessive hepatic lipogenesis via liver X receptor α (LXRα). Thus we examined the pathophysiological role of PRMTs in NAFLD and their relationship with LXRα. In this study, palmitic acid (PA) treatment increased PRMT3, which is correlated with the elevation of hepatic lipogenic proteins. The expression of lipogenic proteins was increased by PRMT3 overexpression, but decreased by PRMT3 silencing and use of the PRMT3 knockout (KO) mouse embryonic fibroblast cell line. PRMT3 also increased the transcriptional activity of LXRα by directly binding with LXRα in a methylation-independent manner. In addition, PA treatment translocated PRMT3 to the nucleus. In animal models, a high-fat diet increased the LXRα and PRMT3 expressions and binding, which was not observed in LXRα KO mice. Furthermore, increased PRMT3 expression and its binding with LXRα were observed in NAFLD patients. Taken together, LXRα and PRMT3 expression was increased in cellular and mouse models of NAFLD and human patients, and PRMT3 translocated into the nucleus bound with LXRα as a transcriptional cofactor, which induced lipogenesis. In conclusion, PRMT3 translocation by PA is coupled to the binding of LXRα, which is responsible for the onset of fatty liver.

Involvement of p53 Mutation and Mismatch Repair Proteins Dysregulation in NNK-Induced Malignant Transformation of Human Bronchial Epithelial Cells

Genome integrity is essential for normal cellular functions and cell survival. Its instability can cause genetic aberrations and is considered as a hallmark of most cancers. To investigate the carcinogenesis process induced by tobacco-specific carcinogen NNK, we studied the dynamic changes of two important protectors of genome integrity, p53 and MMR system, in malignant transformation of human bronchial epithelial cells after NNK exposure. Our results showed that the expression of MLH1, one of the important MMR proteins, was decreased early and maintained the downregulation during the transformation in a histone modification involved and DNA methylation-independent manner. Another MMR protein PMS2 also displayed a declined expression while being in a later stage of transformation. Moreover, we conducted p53 mutation analysis and revealed a mutation at codon 273 which led to the replacement of arginine by histidine. With the mutation, DNA damage-induced activation of p53 was significantly impaired. We further reintroduced the wild-type p53 into the transformed cells, and the malignant proliferation can be abrogated by inducing cell cycle arrest and apoptosis. These findings indicate that p53 and MMR system play an important role in the initiation and progression of NNK-induced transformation, and p53 could be a potential therapeutic target for tobacco-related cancers.

Suppression of PTEN Transcription by UVA

Although ultraviolet A (UVA; 315-400 nm) has different physical and biological targets than ultraviolet B (UVB; 280-315 nm), the contribution of UVA to skin cancer susceptibility and its molecular basis remain largely unknown. Here we show that chronic UVA radiation suppresses phosphatase and tensin homolog (PTEN) expression at the mRNA level. Subchronic and acute UVA radiation also downregulated PTEN in normal human epidermal keratinocytes, skin culture, and mouse skin. At the molecular level, chronic UVA radiation decreased the transcriptional activity of the PTEN promoter in a methylation-independent manner, whereas it had no effect on the protein stability or mRNA stability of PTEN. In contrast, we found that UVA-induced activation of the Ras/ERK/AKT and NF-кB pathways plays an important role in UV-induced PTEN downregulation. Inhibiting extracellular signal-regulated kinases (ERK) or protein pinase B (AKT) increases PTEN expression. Our findings may provide unique insights into PTEN downregulation as a critical component of UVA's molecular impact during keratinocyte transformation.

DNA Methylation-Independent Reversion of Gemcitabine Resistance by Hydralazine in Cervical Cancer Cells

BackgroundDown regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.Methodology/Principal FindingsThe cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.Conclusions/SignificanceOur results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.

Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

BackgroundDNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear.ResultsHere we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation.ConclusionsTaken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.