Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation

RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of protein-coding gene expression.     

DNA Methylation Alterations and Their Association with High Temperature Tolerance in Rice Anthesis

DNA methylation is an important epigenetic mechanism involved in gene regulation under environmental stresses in plants. However, little information is available regarding its responses to high temperature (HT) and association with HT tolerance in rice. In this study, fourteen rice genotypes were classified into the susceptible, moderate, and tolerant groups by the high temperature susceptibility index (HTSI) after HT treatment. The changes of DNA methylation in rice anthesis under normal and HT30 conditions were investigated using methylation-sensitive amplified polymorphism31 (MSAP). The MSAP results showed that the DNA methylation level significantly increased in the susceptible rice group and decreased in the tolerant rice group under HT treatment, while no significant difference was observed in the moderate rice group. More hypomethylation events were detected in the tolerant rice group, while more hypermethylation was detected in the susceptible rice group. Forty-four differentially methylated epiloci (DME) were generated under both control and HT conditions, which can clearly distinguish the susceptible, moderate, and tolerant genotypes via PCoA analysis. Approximately 43.18% of DMEs were determined to be tolerance-associated epiloci (TAEs). 63.15% TAEs were sequenced and annotated into 12 genes. Quantitative RT-PCR analysis showed that 12 TAE genes were mainly upregulated in 14 rice genotypes, and their expression levels were related to the HT tolerance of rice. Here, DEGs, generated from a number of genotypes, indicate higher probabilities for association with stress tolerance. Overall, these results suggest that DNA methylation regulation might play a key role in adaptation to HT stress in rice.

     

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.

Methodology/Principal Findings

We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.

Conclusions/Significance

Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.     

外源NO对转基因白桦外源基因表达及DNA甲基化的影响

为探讨外源NO诱导转基因白桦外源基因表达与基因组DNA甲基化之间的关系,本研究分析了NO供体硝普钠（sodium nitroprusside,SNP）对转基因白桦愈伤组织中外源基因BGT转录的影响,并对此过程中基因组DNA甲基化水平、甲基转移酶基因DRM、MET表达量及生理生化指标进行研究。结果表明：2 mmol·L^-1SNP处理后,转基因白桦防御酶活性、丙二醛（MDA）含量显著升高,表明高浓度NO对白桦细胞正常生命活动产生了伤害;甲基转移酶DRM和MET基因上调表达,基因组DNA甲基化水平由10.6%增加到16.5%,外源基因BGT表达量在6 h时显著增加,3 d时仅为对照的0.46倍,说明转基因白桦外源BGT基因的表达对高浓度NO响应明显且受基因组甲基化水平的影响。本研究揭示了转基因白桦外源BGT基因和甲基转移酶MET、DRM基因对高浓度NO的响应模式,分析了基因组甲基化水平及生理生化特征的变化,为转基因植物生长发育的表观遗传调控和外源基因表达影响机制的研究奠定基础。     

DNA甲基化作用与鼠肝细胞的老化

本文比较了不同年龄的鼠肝ＤＮＡ甲基化酶活力及ＤＮＡ甲基化水平,发现它们均与鼠龄呈反相关。又以不同年龄的鼠肝ＤＮＡ为模板,检验了其体外转录活力,发现其与鼠龄呈正相关。     

CBX4与HOXA5在慢性粒细胞白血病中的表达及相互作用研究

Polycomb group complex（PcG）作为发挥转录抑制作用的重要表观遗传调控复合物,参与发育、衰老以及肿瘤发生等重要病生理过程。PcG成员众多,分为PRC1与PRC2两种复合物,各组分间功能既协同,又不失特性。PRC1中的CBX4独特的结构域使其功能尤为特殊。近年发现,作为一类造血干细胞恶性克隆性疾病,白血病中常伴有PcG基因的异常表达或者突变。本研究通过qPCR发现,在慢性粒细胞白血病（chronic myeloid leukemia, CML）患者外周血白细胞中存在CBX4的表达明显下调,而PcG经典靶基因HOX家族中的HOXA5则表现为上调。给予伊马替尼（Imatinib）治疗后,二者均向相反方向恢复至正常人的表达水平,并且CBX4的表达水平与CML的经典分子标志物BCR ABL1融合基因有较好的相关性。上述结果提示,CBX4可以作为CML潜在的预后标志物。为了进一步揭示CBX4与HOXA5是否存在相互作用关系,本文通过双荧光素酶实验证实,CBX4能通过HOXA5的启动子而负调控其表达。本研究发现,CBX4与HOXA5在CML中存在负相关的异常表达,且证明CBX4可作为HOXA5的负调控因子。     

The Impact of EGCG and RG108 on SOCS1 Promoter DNA Methylation and Expression in U937 Leukemia Cells

Background:The available evidence has increasingly demonstrated that a combination of genetic and epigenetic factors, such as DNA methylation, could be considered as causing leukemia. Epigenetic changes and methylation of the suppressor of the cytokine signaling 1 promoter (SOCS1) CpG region silence SOCS1 expression in cancer. In the current study, we evaluated the impact of epigallocatechin gallate (EGCG) and RG108 on SOCS1 promoter methylation and expression in U937 cells.Methods:In the current study, U937 leukemic cells were treated with EGCG and RG108 for 12, 24, 48, and 72 h and SOCS1 promoter methylation and its expression were measured by methylation-specific PCR (MSP) and quantitative real-time PCR, respectively.Results:The outcomes indicated that the SOCS1 promoter is methylated in U937 cells, and treatment of these cells with either EGCG or RG108 reduced its methylation. Moreover, we observed that SOCS1 expression was significantly upregulated in a time-dependent manner by both EGCG and RG108 in U937 cells compared with control cells. In the RG108-treated group at 12, 24, 48, and 72 h, SOCS1 expression was upregulated by 1, 4.2, 16.6, and 32.6 -fold respectively, and in the EGCG-treated group, by 0.5, 3.2, 10.8, and 22.3 -fold, respectively. Conclusion:Treatment with either EGCG or RG108 reduced SOCS1 promoter methylation and increased SOCS1 expression in U937 cells in a time-dependent manner, which may play a role in leukemia therapy.Key Words: DNA Methylation, EGCG, Leukemia, RG108, SOCS1