DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

Wnt通路中蓬松蛋白DNA甲基化对骨质疏松患者骨髓间充质干细胞成骨分化影响

摘要 目的：探讨蓬松蛋白（DVL）DNA甲基化对骨质疏松（OP）患者骨髓间充质干细胞（BMSCs）成骨分化及Wnt通路的影响。方法：分离、培养OP患者的BMSCs,成骨诱导培养BMSCs 0、7、14、21天,观察BMSCs细胞形态变化,检测碱性磷酸酶（ALP）染色及活性,检测茜素红染色及钙结节形成,荧光定量聚合酶链式反应（RT-PCR）及免疫印迹检测远端缺失同源盒5（Dlx5）、核心结合蛋白因子2（Runx2）、成骨细胞特异性转录因子（OSX）、Ⅰ型胶原蛋白（Colla Ⅰ）表达。检测DVL1、Wnt、糖原合成酶激酶3（GSK3）、β连环蛋白（β-catenin）表达及DVL DNA甲基化水平。在成骨诱导培养基中加入甲基转移酶抑制剂5-Aza,将BMSCs分为对照组（Control组）、甲基转移酶抑制剂组（5-Aza组）、甲基转移酶抑制剂+si-NC组（5-Aza+si-NC组）、甲基转移酶抑制剂+si-Wnt组（5-Aza+si-Wnt组）,依次进行ALP活性测定,茜素红染色及钙结节形成测定。RT-PCR检测Dlx5、Runx2、OSX、Colla 1水平,免疫印迹检测Dlx5、Runx2、OSX、Colla Ⅰ、DVL1、Wnt、GSK3、β-catenin的蛋白表达量,并检测DVL DNA甲基化水平。结果：成骨诱导后BMSCs具有强的ALP活性和矿化结节生成能力,且随着培养时间的增长,BMSCs细胞ALP活性和矿化结节生成能力增强,Dlx5、Runx2、OSX、Colla Ⅰ mRNA水平和Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza组较Control组ALP染色加深,活性增强,钙结节形成增多（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza+si-Wnt组较5-Aza+si-NC组ALP染色变浅,活性降低,钙结节形成减少（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达降低,DVL DNA甲基化水平升高（P＜0.05）。结论：DVL DNA甲基化可以通过抑制Wnt/β-catenin信号通路抑制OP患者BMSCs成骨分化。     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Characterization of H3.3K36M as a tool to study H3K36 methylation in cancer cells

Recurrent mutations at key lysine residues in the histone variant H3.3 are thought to play an etiologic role in the development of distinct subsets of pediatric gliomas and bone and cartilage cancers. H3.3K36M is one such mutation that was originally identified in chondroblastomas, and its expression in these tumors contributes to oncogenic reprogramming by triggering global depletion of dimethylation and trimethylation at H3K36 with a concomitant increase in the levels of H3K27 trimethylation. H3.3K36M expression can also cause epigenomic changes in cell types beyond chondrocytic cells. Here we show that expression of H3.3K36M in HT1080 fibrosarcoma cancer cells severely impairs cellular proliferation, which contrasts its role in promoting transformation of chondrocytic cells. H3.3K36M-associated cellular toxicity phenocopies the specific depletion of H3K36me2, but not loss of H3K36me3. We further find that the H3K36me2-associated toxicity is largely independent of changes in H3K27me3. Together, our findings lend support to the argument that H3K36me2 has distinct roles in cancer cells independent of H3K36me3 and H3K27me3, and highlight the use of H3.3K36M as an epigenetic tool to study H3K36 and H3K27 methylation dynamics in diverse cell types.