DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation

The inverse correlation between DNA methylation and lineage-specific gene expression during T helper cell development is well documented. However, the specific functions of the de novo methyltransferases Dnmt3a and Dnmt3b in cytokine gene regulation have not been defined. We demonstrate that the expression of Dnmt3a and Dnmt3b are induced to a greater extent in T helper 2 (Th2) cells than in T helper 1 cells during polarization. Using conditional mutant mice, we determined that Dnmt3a, but not Dnmt3b, regulated expression of T helper cell cytokine genes, with the Il13 gene most prominently affected. Dnmt3a deficiency was accompanied by decreases in DNA methylation and changes in the H3K27 acetylation/methylation status at the Il13 locus. Dnmt3a-dependent regulation of Il13 also occurred in vivo because Dnmt3a(fl/fl)Cd4cre mice exhibited increased lung inflammation in a murine asthma model, compared with littermate controls. Based on these observations, we conclude that Dnmt3a is required for controlling normal Il13 gene expression and functions as a rate-limiting factor to restrict T helper 2-mediated inflammation.     

Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL)

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 oncogene (TCL1) in mouse B cells causes a CD5(+) leukemia similar to aggressive human CLL. To examine the mechanisms by which Tcl1 protein exerts its oncogenic activity in B cells, we performed proteomics experiments to identify its interacting partners. We found that Tcl1 physically interacts with de novo DNA methylthansferases Dnmt3A and Dnmt3B. We further investigated the effects of Tcl1 up-regulation on the enzymatic activity of Dnmt3A and found that Tcl1 overexpression drastically inhibits Dnmt3A function. In addition, B cells from TCL1 transgenic mice showed a significant decrease in DNA methylation compared with WT controls. Similarly, CLL samples with high Tcl1 expression showed a decrease in DNA methylation compared with CLL samples with low Tcl1 expression. Given the previous reports of inactivating mutations of DNMT3A in acute myelogenous leukemia and myelodysplastic syndrome, our results suggest that inhibition of de novo DNA methylation may be a common oncogenic mechanism in leukemogenesis.     

DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.     

DNA methyltransferase 1 regulates reelin mRNA expression in mouse primary cortical cultures

The polygenic nature of complex psychiatric disorders suggests a common pathway that may be involved in the down-regulation of multiple genes through an epigenetic mechanism. To investigate the role of methylation in down-regulating the expression of mRNAs that may be associated with the schizophrenia phenotype, we have adopted a cell-culture model amenable to this line of investigation. We have administered methionine (2 mM) to primary cultures of cortical neurons prepared from embryonic day 16 mice and show that this treatment down-regulated reelin and glutamic acid decarboxylase 67 (GAD67) mRNA expression but not that corresponding to neuron-specific enolase mRNA. Moreover, methionine increased methylation of the reelin promoter, suggesting a possible mechanism for the observed change. These cultures contain a mixed population of neurons and glia. Approximately 83% of the neurons are GABAergic based on GAD immunoreactivity, and these neurons coexpress high levels of reelin and DNA methyltransferase (Dnmt) 1 immunoreactivity. To examine whether Dnmt1 regulates reelin gene expression, we used an antisense approach to reduce (knock down) Dnmt1 expression. The reduced Dnmt1 mRNA and protein were accompanied by increased reelin mRNA expression. More importantly, the Dnmt1 knockdown blocked the methionine-induced reelin and GAD67 mRNA down-regulation. These data support the hypothesis that the reduced amounts of reelin and GAD67 mRNAs documented in postmortem schizophrenia brain may be the consequence of a Dnmt1-mediated hypermethylation of the corresponding promoters.     

Age-dependent decreases in DNA methyltransferase levels and low transmethylation micronutrient levels synergize to promote overexpression of genes implicated in autoimmunity and acute coronary syndromes

T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine (SAM) levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22–81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age ∼50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly.     

丝裂原激活蛋白激酶阻断剂与DNA甲基化酶抑制剂对人结肠癌细胞的协同影响

目的研究细胞外信号渊节激酶-丝裂原激活蛋白激酶途径（ERKMAPK）与DNA甲基化问的关系及对结肠癌细胞生物学行为的协同影响。方法培养人结肠癌细胞SW1116，分别以PBS、二甲基亚砜（DMSO）为对照组，PD 9805950μmol／L、5氮脱氧胞苷（5-aza—dC）5μmol／L、PD9805950μmol／L＋5-aza-dC5μmol／L进行药物干预．以定量RT-PCR检测DNA甲基化酶（Dnmt）1、3a和3b基因转录水平；流式细胞仪分析细胞周期；MTT测定细胞活力；光学显微镜下观察细胞形态学变化。结果ERK—MAPK途径阻断剂PD98059下调Dnmt 1和Dnmt 3b．Dnmt抑制剂5-aza-dC下调Dnmt 1、Dnmt 3a和Dnmt 3b，且5-aza-dC联合PD98059对Dnmt1及Dnmt 3a mRNA的表达下调更为显著。5-azo-dC明显降低G0／G1期细胞百分比（P〈0．05），G2／M期细胞百分比明显增加（P〈0．05）；PD98059使G0／G1期细胞百分比降低（P（0．05）．G2／M期增加（P〈0．05）。PD98059明显抑制细胞生长。PD98059促进细胞分化，呈上皮样改变，细胞变狭长，胞质减少，细胞排列开始出现相对整齐；5-azm-dC干预组细胞大小不一，出现较多多倍体细胞（多个核分裂相）。结论ERK-MAPK途径阻断剂及Dnmt抑制剂均能抑制结肠癌SW1116细胞分裂、增殖，并诱导细胞分化；两者有协同作用；ERK—MAPK信号转导途径能调控DNA甲基化水平。     

In vivo stabilization of the Dnmt1 (cytosine-5)- methyltransferase protein

The Dnmt1o form of the Dnmt1 (cytosine-5)-methyltransferase enzyme is synthesized and stored in the cytoplasm of the oocyte and is used after fertilization to maintain methylation patterns on imprinted genes. After implantation of the blastocyst, Dnmt1o is replaced by the Dnmt1 form, which has an additional 118 aa at its amino terminus. To investigate functional differences between Dnmt1o and Dnmt1, mice were generated with a mutant allele, Dnmt1(V), which synthesized Dnmt1o instead of Dnmt1 in all somatic cells. Homozygous Dnmt1(V) mice were phenotypically normal, and had normal levels of genomic methylation, indicating that Dnmt1o adopts the maintenance methyltransferase function of Dnmt1. Despite the apparent equivalence of Dnmt1o and Dnmt1 maintenance methyltransferase function in somatic cells, the Dnmt1o protein was found at high levels (with a corresponding high enzymatic activity) in Dnmt1(V) mice. In heterozygous Dnmt1(V)/+ embryonic stem cells and early embryos, equal steady-state levels of Dnmt1o and Dnmt1 proteins were produced from the Dnmt1(V) and the WT Dnmt1 alleles, respectively. However, in older embryos and adults, the Dnmt1(V) allele produced five times the steady-state level of protein of the WT Dnmt1 allele. The difference in Dnmt1o and Dnmt1 levels is due to a developmentally regulated mechanism that degrades the Dnmt1 protein. The intrinsic stability of the Dnmt1o protein is the most likely reason for its use as a maternal-effect protein; stable ooplasmic stores of Dnmt1o would be available to traffick into the nuclei of the eight-cell stage embryo and maintain methylation patterns on alleles of imprinted genes during the fourth embryonic S phase.     

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.     

Global DNA methylation, DNMT1, and MBD2 in patients with systemic lupus erythematosus

To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with systemic lupus erythematosus (SLE), 108 patients with SLE and 97 healthy controls were enrolled in this study. DNA and total RNA were extracted from the peripheral blood mononuclear cells of the SLE patients and the controls. The global methylation levels of DNA were measured in 63 patients with SLE and 68 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 108 SLE patients and 97 controls using the quantitative real-time polymerase chain reaction method. The global methylation level of DNA was significantly decreased in the SLE patients in comparison with that in the controls (p?相似文献   

DNA甲基转移酶1蛋白在人胃癌黏膜组织中的表达及其临床意义

背景：肿瘤组织存在DNA甲基化紊乱．包括与细胞增殖周期密切相关的癌基因低甲基化和抑癌基因高甲基化等。DNA甲基转移酶（Dnmt）参与甲基化的形成（主要是Dnmt3a和Dnmt3b）和维持（主要是Dnmt1），但目前对胃癌Dnmt蛋白的表达情况仍知之甚少，更缺乏其与肿瘤生物学行为和临床参数关系的研究。目的：探讨癌区、癌旁和外周正常黏膜组织中Dnmt1蛋白表达的差异及其临床意义。方法：收集38例胃癌患者标本，取癌区、癌旁和外周正常黏膜组织各1．2块，应用免疫组化SP法分析各黏膜组织中Dnmt1蛋白的表达．并探讨其与胃癌浸润和转移的关系。结果：在38例胃癌黏膜组织中，Dnmt1蛋白的表达阳性率为81．6％，显著高于相应癌旁（39．5％）和正常黏膜组织（10．5％）（P〈0．001）。阳性切片中可见Dnmt1蛋白表达弥散分布于肿瘤或腺体的细胞质和细胞核，染色较均匀。Dnmt1蛋白表达与胃癌患者的年龄、肿瘤分化程度和有无淋巴结转移无明显相关性（P〉0．05），与性别呈一定的相关性（P=0．007）。结论：Dnmtl蛋白过表达在人胃癌的发生和发展中起一定作用。     

Heterozygous CuZn superoxide dismutase deficiency produces a vascular phenotype with aging

The goal of this study was to test the hypothesis that loss of a single copy of the gene for CuZn superoxide dismutase (CuZnSOD) increases vascular superoxide levels and produces vascular dysfunction with aging. Responses of carotid arteries from young (7 months) and old (22 to 24 months of age) heterozygous CuZnSOD-deficient (CuZnSOD(+/-)) mice and their wild-type (CuZnSOD(+/+)) littermates were examined in vitro. Total superoxide dismutase activity in aorta was reduced by approximately 30% (P<0.05) in CuZnSOD(+/-) mice compared with wild-type mice. Responses to acetylcholine (an endothelium-dependent agonist) produced relaxation that was similar (P>0.05) in carotid arteries from young wild-type, young CuZnSOD(+/-), and old wild-type mice. In contrast, relaxation to acetylcholine was markedly impaired in old CuZnSOD(+/-) mice (eg, 100 micromol/L acetylcholine produced 51+/-5% and 96+/-5% relaxation in vessels from old CuZnSOD(+/-) and old wild-type mice, respectively). This effect was selective, because relaxation to nitroprusside (an endothelium-independent agonist) was not affected by either CuZnSOD genotype or aging. The impaired response to acetylcholine in old CuZnSOD(+/-) mice was restored toward normal with either tempol (a scavenger of superoxide; 1 mmol/L) or PJ34 (an inhibitor of poly-ADP-ribose polymerase; 3 micromol/L). Vascular superoxide levels were increased in aorta in old CuZnSOD(+/+) mice and increased further in CuZnSOD(+/-) mice with aging. These findings provide the first direct evidence that normal CuZnSOD expression protects endothelial function and that deficiency in a single copy of the gene that encodes CuZnSOD produces increases in superoxide and marked impairment of endothelial function with aging.     

Chronic resveratrol intake reverses pro-inflammatory cytokine profile and oxidative DNA damage in ageing hybrid mice

Thymic involution and shrinkage of secondary lymphoid organs are leading causes of the deterioration of the T-cell compartment with age. Inflamm-aging, a sustained inflammatory status has been associated with chronic diseases and shortened longevity. This is the first study to investigate the effect of treating aging hybrid mice with long-term, low-dose resveratrol (RSV) in drinking water by assessing multiple immunological markers and profiles in the immune system. We found that hybrid mice exhibited marked age-related changes in the CD3+CD4+, C3+CD8+, CD4+CD25+, CD4M and CD8M surface markers. RSV reversed surface phenotypes of old mice to that of young mice by maintaining the CD4+ and CD8+ population in splenocytes as well as reducing CD8+CD44+ (CD8M) cells in the aged. RSV also enhanced the CD4+CD25+ population in old mice. Interestingly, pro-inflammatory status in young mice was transiently elevated by RSV but it consequently mitigated the age-dependent increased pro-inflammatory cytokine profile while preserving the anti-inflammatory cytokine condition in the old mice. Age-dependent increase in 8OHdG, an oxidative DNA damage marker was ameliorated by RSV. Immunological-focused microarray gene expression analysis showed that only the CD72 gene was significantly downregulated in the 12-month RSV-treated mice compared to age-matched controls. Our study indicates that RSV even at low physiological relevant levels is able to affect the immune system without causing marked gene expression changes.     

Keratinocyte expression of B7-1 in transgenic mice amplifies the primary immune response to cutaneous antigens.

Resting epidermal keratinocytes do not express B7-1 and other known CD28 counterligands with costimulatory activity. The absence of these costimulators on keratinocytes correlates with their ability to preferentially induce T-cell anergy instead of T-cell activation. To test the hypothesis that keratinocytes expressing a CD28 counterligand would be more effective inducers of T-cell-mediated immune responses in skin, we prepared transgenic mice in which expression of the B7-1 costimulator was targeted to basal keratinocytes by using the human K14 promoter. Keratinocytes from the K14/B7-1 transgenic line expressed high levels of surface B7-1. No spontaneous inflammatory changes were seen in transgenic skin, but epicutaneous application of contact sensitizers to these mice elicited a stronger primary ear swelling response than in controls. Sites of initial hapten application in transgenic mice also responded much more strongly to reapplication of hapten to a remote cutaneous site. Epidermal cell suspensions from transgenic mice contained normal numbers of Langerhans cells and dendritic epidermal T cells when analyzed by flow cytometry. Systemic treatment of the transgenic mice with interferon gamma induced high levels of class II major histocompatibility complex expression on keratinocytes but was not sufficient to initiate an inflammatory response. We conclude that the constitutive expression of the B7-1 molecule in vivo on a nonprofessional antigen-presenting cell is not by itself sufficient to trigger inflammatory changes, but B7-1 expression amplifies the host immune responses after exposure to nonself antigens presented by B7-1-expressing cells.     

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L-/- germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L-/- meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.     

Participation of Akt, menin, and p21 in pregnancy-induced beta-cell proliferation

β-Cell mass increases during pregnancy to accommodate for insulin resistance. This increase is mainly due to β-cell proliferation, a process that requires intact prolactin receptor (Prlr) signaling. Signaling molecules that are known to regulate β-cell proliferation include Jak2, Akt, the tumor suppressor menin, and cell cycle proteins. Whether these pathways are involved in prolactin-mediated β-cell proliferation is unknown. Using the heterozygous prolactin receptor-null (Prlr(+/-)) mice, we isolated pancreatic islets from both Prlr(+/+) and Prlr(+/-) mice on d 0 and 15 of pregnancy and examined the expression levels of these signaling molecules. In the wild-type mice (Prlr(+/+)), both phospho-Jak2 and phospho-Akt expression in pancreatic islets increased during pregnancy, which were attenuated in the pregnant Prlr(+/-) mice. During pregnancy, menin expression was reduced by 50 and 20% in the Prlr(+/+) and the Prlr(+/-) mice, respectively, and the pregnant Prlr(+/-) mice had higher islet p18 levels than the Prlr(+/+) mice. Interestingly, between d 0 and 15 of pregnancy, expression of cyclin inhibitory protein p21(cip) was increased in the Prlr(+/+) mice, but this increase was blunted in the Prlr(+/-) mice. Lastly, we did not find any difference in the expression levels of cyclins D1, D2, and inhibitory kinases between the pregnant Prlr(+/+) and Prlr(+/-) mice. Therefore, we conclude that during pregnancy, placental hormones act through the prolactin receptor to increase β-cell mass by up regulating β-cell proliferation by engaging Jak2, Akt, menin/p18, and p21. Future studies will determine the relative contribution of these molecules in maintaining normal glucose homeostasis during pregnancy.