cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.     

叶酸缺乏对小鼠胚胎干细胞Nespas DMR 区甲基化修饰的影响

目的：探讨叶酸缺乏对小鼠胚胎干细胞（ESCs）中Nespas 差异甲基化区域（Differentially Methylated Region, DMR）甲基化修 饰的影响以及叶酸浓度与甲基化水平的关系。方法：多种不同浓度叶酸处理小鼠ESCs,化学发光免疫分析法检测ESCs 细胞内叶 酸浓度。利用MassARRAY 技术平台检测三种不同叶酸浓度处理后的ESCs中Nespas DMR 启动子区,外显子区和内含子区甲基 化修饰状态,并且分析Nespas DMR 启动子区,外显子区和内含子区甲基化水平与叶酸浓度之间的关系。结果：无叶酸组（FF）小鼠 ESCs 细胞内叶酸浓度显著低于低叶酸组（FD）与正常叶酸组（FN）（P<0.05）。Nespas DMR 中启动子区、外显子区以及内含子区甲 基化水平在FF组显著低于FD 和FN 组（P<0.05）,并且Nespas DMR中启动子区以及内含子区甲基化水平与叶酸浓度存在显著 的正相关（P<0.05）。结论：叶酸缺乏影响小鼠ESCs 中Nespas DMR 区甲基化修饰的建立。     

Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains IG-DMR and Gtl2-DMR, whose methylation patterns are established in the germline and after fertilization, respectively. In this study, we determine that acquisition of DNA methylation at the paternal allele of the Gtl2-DMR is initiated after the blastocyst stage and completed by embryonic day 6.5, and that Gtl2 (approved symbol: Meg3) is monoallelically expressed from the maternal allele as early as the blastocyst. Therefore, DNA methylation at the Gtl2-DMR is not a prerequisite for the imprinted expression of Gtl2, which may be involved in the control of proliferation and differentiation of cells during early gestation. We also reveal that a subregion of the IG-DMR exhibits tissue-specific differences in allelic methylation patterns. These results add to the growing body of knowledge elucidating the mechanism whereby parent-of-origin-dependent DNA methylation at the IG-DMR leads to the imprinted expression of the Dlk1-Dio3 cluster.     

Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.     

Intragenic DNA methylation status down-regulates bovine IGF2 gene expression in different developmental stages

DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P < 0.05 or P < 0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P < 0.05 or P < 0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.     

叶酸缺乏对小鼠胚胎干细胞Nespas DMR区甲基化修饰的影响

目的：探讨叶酸缺乏对小鼠胚胎干细胞（ESCs）中Nespas差异甲基化区域（Differentially Methylated Region,DMR）甲基化修饰的影响以及叶酸浓度与甲基化水平的关系。方法：多种不同浓度叶酸处理小鼠ESCs,化学发光免疫分析法检测ESCs细胞内叶酸浓度。利用MassARRAY技术平台检测三种不同叶酸浓度处理后的ESCs中Nespas DMR启动子区,外显子区和内含子区甲基化修饰状态,并且分析Nespas DMR启动子区,外显子区和内含子区甲基化水平与叶酸浓度之间的关系。结果：无叶酸组（FF）小鼠ESCs细胞内叶酸浓度显著低于低叶酸组（FD）与正常叶酸组（FN）（P〈0.05）。Nespas DMR中启动子区、外显子区以及内含子区甲基化水平在FF组显著低于FD和FN组（P〈0.05）,并且Nespas DMR中启动子区以及内含子区甲基化水平与叶酸浓度存在显著的正相关（P〈0.05）。结论：叶酸缺乏影响小鼠ESCs中Nespas DMR区甲基化修饰的建立。     

哈尔滨和湛江雄性褐家鼠Gsk3β差异甲基化区域DNA序列多态性分析与调控功能鉴定

糖原合成酶激酶-3β（glycogen synthase kinase 3β, Gsk3β）基因能够参与多条细胞周期信号传导通路,从而通过调控细胞分裂过程,影响组织与器官的发育。本实验室前期研究发现,与湛江褐家鼠种群相比,哈尔滨褐家鼠种群在秋冬季存在睾丸发育受抑制的现象,并通过基因组与甲基化组联合分析,在Gsk3β基因内含子区域筛选到1个差异甲基化区域（differentially methylated region, DMR）。为分析该DMR 的DNA序列多态性及其在Gsk3β基因表达调控中的可能作用,本研究选取了哈尔滨（n=52）和湛江（n=39）共91个性成熟褐家鼠睾丸样品。采用PCR测序方法在群体水平上分析了该DMR的DNA序列多态性,在2个褐家鼠亚种的分化特征,并选取2个群体中具有代表性的单倍型,通过荧光素酶基因报告系统在293T细胞中检测该DMR不同单倍型的调控活性。结果表明,该DMR存在2个SNP位点,共组成3个单倍型、6个基因型。卡方检验表明,其频率在2个群体间发生显著分化（单倍型,P=1.13E-29;基因型,P=1.15E-14）。Gsk3β基因的-2 218/+238 bp区具有明显启动子活性（P<0.05）;哈尔滨和湛江2个单倍型都显示显著的沉默子活性（P<0.05）,同时表现显著的调控活性差异（P<0.05）,表明这2个单倍型可以在293T细胞中作为沉默子抑制Gsk3β基因的启动子活性。2个单倍型调控活性差异,可能与SNP导致的转录因子结合位点的获得/丢失,以及2个单倍型在不同种群中甲基化状态差异有关。本研究结果表明,该DMR可能通过调控褐家鼠Gsk3β基因表达,在睾丸发育过程中发挥作用。2个种群主要单倍型的调控活性差异,及其甲基化状态差异可能是2个种群睾丸发育表型差异的重要调控因素之一。     

Optical biosensor provides insights for bradykinin B(2) receptor signaling in A431 cells

The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B(2) receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B(2) specific antagonist HOE140, suggested that the optical signature is a direct result of B(2) receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B(2) receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B(2) signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B(2) receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B(2) signaling. The roles of endocytosis and cytoskeleton modulation in B(2) signaling were also demonstrated.