落叶松体细胞胚胎发生过程中DNA甲基化模式变化分析

通过MSAP技术检测落叶松体细胞胚胎发生过程中的DNA甲基化模式变化发现,体细胞胚胎发生的14个时期中,5'-CCGG-3'序列的胞嘧啶上发生重新甲基化事件最多在ABA诱导后的0.5～6h,最少在5～7d的早期单胚阶段;而去甲基化事件发生最多的是5～7d的早期单胚阶段,最少在14～21d的中期单胚到晚期单胚阶段.与原胚...     

Epigenetic reprogramming in mammalian development

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods-in germ cells and in preimplantation embryos-in which methylation patterns are reprogrammed genome wide, generating cells with a broad developmental potential. Epigenetic reprogramming in germ cells is critical for imprinting; reprogramming in early embryos also affects imprinting. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. A role of reprogramming in stem cell differentiation is also envisaged. DNA methylation is one of the best-studied epigenetic modifications of DNA in all unicellular and multicellular organisms. In mammals and other vertebrates, methylation occurs predominantly at the symmetrical dinucleotide CpG (1-4). Symmetrical methylation and the discovery of a DNA methyltransferase that prefers a hemimethylated substrate, Dnmt1 (4), suggested a mechanism by which specific patterns of methylation in the genome could be maintained. Patterns imposed on the genome at defined developmental time points in precursor cells could be maintained by Dnmt1, and would lead to predetermined programs of gene expression during development in descendants of the precursor cells (5, 6). This provided a means to explain how patterns of differentiation could be maintained by populations of cells. In addition, specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. Neat and convincing as this model is, it is still largely unsubstantiated. While effects of methylation on expression of specific genes, particularly imprinted ones (7) and some retrotransposons (8), have been demonstrated in vivo, it is still unclear whether or not methylation is involved in the control of gene expression during normal development (9-13). Although enzymes have been identified that can methylate DNA de novo (Dnmt3a and Dnmt3b) (14), it is unknown how specific patterns of methylation are established in the genome. Mechanisms for active demethylation have been suggested, but no enzymes have been identified that carry out this function in vivo (15-17). Genomewide alterations in methylation-brought about, for example, by knockouts of the methylase genes-result in embryo lethality or developmental defects, but the basis for abnormal development still remains to be discovered (7, 14). What is clear, however, is that in mammals there are developmental periods of genomewide reprogramming of methylation patterns in vivo. Typically, a substantial part of the genome is demethylated, and after some time remethylated, in a cell- or tissue-specific pattern. The developmental dynamics of these reprogramming events, as well as some of the enzymatic mechanisms involved and the biological purposes, are beginning to be understood. Here we look at what is known about reprogramming in mammals and discuss how it might relate to developmental potency and imprinting.     

盐胁迫下红花基因组DNA甲基化的MSAP分析

以红花幼苗为研究材料,经200mmol.L-1 NaCl溶液分别处理4、8、12h,以未经盐胁迫处理为对照(CK),对DNA甲基化变化特征进行MSAP(甲基化敏感扩增多态性)分析。选用13对选扩引物,共检测到3 140个基因位点。甲基化水平分析表明,CK,4、8、12h处理样本中总甲基化率分别为31.8%、27.1%、31.0%和31.3%。进一步对不同处理时间红花基因组DNA甲基化模式的变化特征进行分析表明,NaCl处理4h后DNA的去甲基化模式调整占优势,而处理8h及12h后DNA甲基化水平恢复到CK水平,甲基化及去甲基化模式调整基本一致。上述结果显示,盐胁迫条件下红花基因组DNA甲基化状态发生了一定变化,并且这种变化与盐胁迫程度相关,推测DNA甲基化修饰可能参与了红花的盐胁迫应答。     

The ligase PIAS1 restricts natural regulatory T cell differentiation by epigenetic repression

CD4(+)Foxp3(+) regulatory T (T(reg)) cells are important for maintaining immune tolerance. Understanding the molecular mechanism that regulates T(reg) differentiation will facilitate the development of effective therapeutic strategies against autoimmune diseases. We report here that the SUMO E3 ligase PIAS1 restricts the differentiation of natural T(reg) cells by maintaining a repressive chromatin state of the Foxp3 promoter. PIAS1 acts by binding to the Foxp3 promoter to recruit DNA methyltransferases and heterochromatin protein 1 for epigenetic modifications. Pias1 deletion caused promoter demethylation, reduced histone H3 methylation at Lys(9), and enhanced promoter accessibility. Consistently, Pias1(-/-) mice displayed an increased natural T(reg) cell population and were resistant to the development of experimental autoimmune encephalomyelitis. Our studies have identified an epigenetic mechanism that negatively regulates the differentiation of natural T(reg) cells.     

NaCl胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析

【目的】研究黄瓜种子萌发过程中DNA甲基化动态变化情况以及外源施加NaCl对种子萌发及种子DNA甲基化水平的影响,探索DNA甲基化在黄瓜种子萌发过程及NaCl胁迫反应中的作用。【方法】运用甲基化敏感扩增多态性（methylation se nsitive amplified polymorphism,MSAP）技术,分析0、150、200 mmol•L-1 NaCl处理下,黄瓜种子萌发过程（0、1、2、4、6、8 d）的DNA甲基化水平及动态变化情况。【结果】MSAP结果显示,黄瓜种子CCGG位点的甲基化水平约15.25%,以双链甲基化方式为主。种子中DNA甲基化水平在萌发1—2 d略有升高,而在整个萌发过程中呈下降趋势;NaCl处理加剧了甲基化的变化幅度,并使萌发末期的甲基化水平更低。萌发过程中甲基化升高和降低同时发生,分别在不同萌发时期占主导,不同变化类型中CG/CHG（H=A,T,C）位点胞嘧啶甲基化同时变化的类型占主导。甲基化变化同时发生在编码序列和非编码序列中。【结论】黄瓜种子萌发过程的DNA甲基化表现出复杂性,甲基化和去甲基化进程同时进行,并具有时空特异性。NaCl处理降低了种子基因组甲基化水平的稳定性,对种子萌发有抑制作用。     

Nuclear acidic protein changes during differentiation in Physarum polycephalum

A class of acidic nuclear phosphoproteins has been isolated throughout the mitotic cycle and at two points during differentiation in the slime mold Physarum polycephalum. The electrophoretic profiles of these proteins are reproducible and unchanging throughout the mitotic cycle, but reproducible changes occur during differentiation. The proteins are rapidly synthesized after mitosis, and their molecular weights range from 34,000 to 88,000. The proteins rapidly incorporate [(32)P]orthophosphate, and the content of alkali-labile phosphate increases 20 percent during the period after DNA synthesis. The proteins comprise 6.5 percent by dry weight of nuclear material while the DNA comprises about 5.5 percent. These acidic nuclear proteins may have a role in control of gene activity.     

红芽芋超低温疗法脱毒苗基因组DNA甲基化的MSAP分析

对红芽芋超低温疗法脱毒苗的基因组DNA甲基化进行MSAP分析。结果表明,红芽芋超低温疗法脱毒苗总甲基化率提高10.27%,全甲基化率提高12.98%,但半甲基化率下降2.71%。红芽芋超低温疗法脱毒苗去甲基化模式和甲基化模式并存,但去甲基化模式高于甲基化模式,说明较多基因被激活。     

冬性与春性白菜品种花芽分化前后生理代谢的比较

 研究了冬性植物油冬儿白菜和春性植物菜心花芽分化前后茎尖DNA甲基化水平、赤霉素 (GA)含量和蛋白质的变化。结果表明 ,在花芽分化过程中 ,DNA甲基化水平逐渐下降 ,蛋白质含量逐渐升高 ,GA含量在临界期下降 ,花芽分化开始后又上升 ,2种蔬菜变化趋势一致 ;菜心DNA甲基化水平和赤霉素含量始终高于普通白菜 ,而蛋白质含量低于普通白菜 ,其赤霉素含量的变化幅度则大于普通白菜。此外 ,在白菜中还检测到了花芽分化的特异蛋白     

氯化钠胁迫下的蒙古黄芪基因组MSAP分析

利用甲基化敏感扩增多态性(Methylation-sensitive amplified polymorphism,MSAP)技术研究高盐胁迫过程中蒙古黄芪(Astragalus membranaceus)基因组DNA甲基化变化.结果表明,扩增22对引物共检测出不同盐浓度间甲基化位点179个.总体趋势是高盐引起蒙古黄芪...     

Study on DNA Cytosine Methylation of Cotton （Gossypium hirsutum L.） Genome and Its Implication for Salt Tolerance

To study the relations between DNA methylation and abiotic stress responses in cotton （Gossypium hirsutum L.）,the methylation-sensitive amplified polymorphism （MSAP） method was used to investigate the differences in methylation level and the change of cytosine methylation patterns under salt （NaCl） stress in two different salt-tolerant cotton lines.The results showed that the number of the cytosine methylation of CCGG sites in high salt-tolerant cotton line was less than that in low salt-tolerant line.Under salt stress,extensive cytosine methylation alterations including hypermethylation and demethylation as well as the potential conversion of methylation types occurred in the salt-treated cotton line compared with the corresponding control.Interestingly,salt stress-induced demethylation loci that occurred in high salt- tolerant cotton line were greater than those in low salt-tolerant cotton line,however,salt stress-induced hypermethylation loci in the high salt-tolerant cotton line were less than those in low salttolerant cotton line.It suggested that the demethylation positively contributed to salt tolerance and the hypermethylation had negative effect on salt tolerance in cotton.     

重金属铅镉对濒危植物中华水韭(Isoetes sinensis)DNA甲基化的影响

为研究古老植物对重金属胁迫的分子水平的响应,探究重金属对濒危植物DNA甲基化影响的特点,选择两种重金属Pb和Cd对濒危植物中华水韭进行胁迫处理,每种重金属设置3个处理浓度,胁迫处理至第28 d选取叶片,采用MSAP(甲基化敏感扩增多态性)技术测定DNA甲基化程度。试验结果表明:重金属铅和镉均能够对中华水韭DNA甲基化产生影响,甲基化总体水平基本一致(对照、Pb处理和Cd处理分别为46.96%、48.23%和48.1%),但全甲基化水平(Pb处理是28.34%,Cd处理是20.25%)均低于对照(33.91%),而半甲基化水平(Pb处理是19.89%,Cd处理是27.85%)均高于对照(13.04%)。甲基化增强的变化,以无甲基化或内外侧胞嘧啶半甲基化向内外侧胞嘧啶全甲基化变化的模式为主。去甲基化的变化,以内外侧胞嘧啶全甲基化向无甲基化或内侧胞嘧啶半甲基化或全甲基化变化的模式为主。铅和镉胁迫在导致中华水韭DNA甲基化增强所占比率方面几乎相等(39.04%和39.71%),而在去甲基化所占比率方面镉(46.86%)高于铅(33.92%)。     

DNA去甲基化对马立克氏病病毒(MDV)基因表达的影响

以鸡马立克氏病脾脏淋巴瘤继代细胞系MDCC-MBI(MSB1)为研究对象，用southern和northern斑点分子杂交放射自显影等方法，证明了5-氮胞苷可以阻断MSB1细胞DNA的甲基化，且去甲基化的DNA转录区和部分非转录区有特异增幅的转录产物mRNA，说明这些区基因转录活性上升。用5-氮胞苷处理MSB1细胞使其去甲基化后，该细胞DNA的内切酶Barn HⅠ区域对DNaseⅠ敏感性增高，该片段区的基因表达活性也增高。以上结果提示．DNA的去甲基化可以提高MDV的基因表达，进一步说明MDV DNA的甲基化在抑制MDV基因表达，维持MDV在淋巴瘤细胞株中潜伏感染状态可能起着重要作用。     

Global Analysis of Cytosine Methylation and Proteome Under Cold Treatment in Brassica napus

Cytosine methylation/demethylation plays pivotal roles in regulating gene expression at a genome-wide level. However, limited reports are available to reveal correlating changes of cytosine methylation and proteomic expression in Brassica napus so far. Therefore, in the present study, global cytosine methylation and proteome were analysed in B. napus after cold treatment by methylation-sensitive amplified polymorphism （MSAP） and two-dimensional protein electrophoresis technology （2-DE）. The results showed that the lowered genome-wide DNA methylation status was revealed after cold treatment, and about 0.88% of discrepancy in DNA methylation was detected between the non-flowering and flowering plants after cold treatment. Moreover, the 52 significantly up-regulated proteins emerged in comparison with the 36 down-regulated proteins, as well as the 14 proteins exclusively detected in the flowering plants. Intriguingly the 8 specifically expressed proteins in the non-flowering plants disappeared in the flowering plants with cold treatment. Therefore, these present data proved that the correlating changes of cytosine methylation and proteomic expression were evidenced under cold treatment in B. napus.     

Demethylated CD8 gene in CD4+ T cells suggests that CD4+ cells develop from CD8+ precursors

Mature T cells and medullary thymocytes bear either the CD4 or CD8 differentiation antigen. Precursor cells in the thymus express neither CD4 nor CD8 (CD4-8-), but most cortical thymocytes are CD4+8+. Whether CD4+ and CD8+ mature T cells arise directly from CD4-8- precursors or from a CD4+8+ intermediate remains unresolved. In this study, methylation of the CD8 gene in murine T cells and thymocytes was examined. There was progressive demethylation of the CD8 gene in the thymus during the transition from CD4-8- to CD4+8+. A similar pattern of demethylation of the CD8 gene was seen in CD4+ mature T cells, suggesting previous expression of CD8 in the CD4+ lineage.     

NaCl胁迫下二倍体和同源四倍体西瓜幼苗DNA甲基化差异分析

【目的】研究二倍体和同源四倍体西瓜幼苗NaCl胁迫后形态学指标差异和DNA甲基化变化情况,分析不同倍性西瓜幼苗之间耐盐差异,结合形态学指标从表观遗传学角度解释二倍体和四倍体抗逆性差异机制。【方法】以3组不同倍性（二倍体、四倍体）西瓜幼苗为研究对象,待长至三叶一心时,用含有0、100、200、300和400 mmol·L^-1 NaCl的1/2 Hoagland营养液处理西瓜幼苗,NaCl处理8 d之后,鉴定西瓜幼苗受到的盐害指数,并运用甲基化敏感扩增多态性（methylation sensitive amplified polymorphism,MSAP）技术,分析NaCl处理8 d之后二倍体和四倍体西瓜幼苗叶片基因组DNA甲基化水平和模式的变化。【结果】NaCl处理后,形态学指标比较发现,随着NaCl浓度的增加,三组西瓜幼苗的受伤害程度都随着增加,在不同的西瓜品种之间受伤害程度有差异,但是同一品种内同一NaCl浓度下,西瓜幼苗受伤害程度二倍体大于四倍体;从甲基化水平看,西瓜幼苗基因组DNA全甲基化率、半甲基化率、总甲基化率都随着NaCl浓度的增加而降低,同一NaCl浓度处理下甲基化率二倍体大于四倍体,二倍体和四倍体西瓜甲基化水平与其受伤害程度呈正相关;从甲基化模式来看,NaCl胁迫后DNA的去甲基化比率降低,超甲基化比率升高,并且其变化幅度是四倍体大于二倍体,不同倍性西瓜幼苗的甲基化模式与其受伤害程度呈负相关。【结论】NaCl胁迫后西瓜幼苗基因组DNA的甲基化状态发生了变化,并且这种变化与NaCl胁迫程度高度线性相关,西瓜幼苗通过降低甲基化率和降低去甲基化比率来应对NaCl胁迫,四倍体较二倍体有更低的甲基化比率和去甲基化比率,抗逆性四倍体强于二倍体。     

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control

Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms.     

成肌细胞成脂过程中PPARγ、C/EBPα和Myogenin 基因启动子的甲基化变化

【目的】利用C2C12成肌细胞探讨肌源性干细胞成脂过程与调控成脂和成肌分化的关键转录因子PPARγ（peroxisome proliferator-activated receptor gamma）、C/EBPα（CCAAT/enhancer binding protein alpha）和Myogenin启动子区甲基化的关系。【方法】分别用2%马血清和三联诱导剂诱导C2C12细胞成肌和成脂分化,在马血清促进的成肌分化第0、1、3和5天收集细胞进行姬姆萨染色观察肌管形成情况;在三联诱导的成脂分化第0、2、4、6和10天收集细胞进行油红O染色观察脂滴形成情况;提取成肌诱导第0、1、3、5天和成脂诱导第0、2、4、6天的RNA和DNA,分别采用qRT-PCR检测成肌和成脂分化相关基因的表达,采用重亚硫酸盐测序的方法检测PPARγ、C/EBPα和Myogenin启动子区甲基化的变化,并分析基因表达与甲基化状态的相关关系。【结果】①C2C12细胞经马血清诱导形成了多核肌管,表达成肌相关基因,但不表达脂肪特异性基因;三联诱导使C2C12细胞自主分化的肌管中沉积了脂滴,同时表达成脂和成肌相关基因;②重亚硫酸盐测序结果表明,在未分化的成肌细胞中,PPARγ基因启动子的甲基化水平是61%,在三联诱导第2、4和6天,其甲基化程度依次为49%、39%和42%,呈逐渐去甲基化趋势,与PPARγ基因转录负相关;在马血清诱导第3和5天,甲基化水平为56%和48%,与未分化的成肌细胞相比差异不显著,同时PPARγ基因的表达水平也没有显著变化;③Myogenin基因在马血清促进的成肌过程中甲基化水平不断降低（49%、42%、35%和34%）,转录水平急剧增加;在三联诱导过程中,Myogenin启动子的甲基化水平由0天的49%下降为第1天的37%、第3天的41%和第5天的38%,但下降幅度弱于马血清诱导的成肌过程,这一结果与降低的Myogenin转录上调相吻合;④C/EBPα基因在未分化的C2C12细胞中呈低甲基化状态,甲基化程度仅为1.6%,在成肌分化和脂肪沉积过程中均未发生显著变化,与基因表达无显著关联。【结论】成脂和成肌关键转录因子PPARγ和Myogenin启动子区的DNA甲基化变化参与了成肌细胞的分化和脂肪沉积的调控。     

留兰香茎尖超低温保存及遗传变异分析

为了长久保存留兰香种质资源,以留兰香(Menthae spicatae L.)茎尖为材料,对其玻璃化超低温保存条件进行研究,并运用扩增片段长度多态性(AFLP)和甲基敏感扩增多态性(MSAP)对玻璃化超低温保存再生材料(处理组)的遗传与表观遗传稳定性进行分析。结果表明:留兰香试管苗于4℃低温锻炼28d,于添加2mol/L甘油的MS培养基中预培养4d,0℃下于PVS2中脱水50min,液氮保存1h或者更长时间,成活率最高,可达60%左右,再生植株分化正常;超低温处理后再生材料(处理组)与正常继代材料(对照组)之间未发现有DNA片段的差异;与对照相比,处理组的材料均发生了甲基化状态与水平的变化,全基因组DNA胞嘧啶甲基化水平降低,另外,甲基化与去甲基化位点比率分别为8.93%和12.3%,说明超低温处理能够引起DNA甲基化的变化。由此推测,DNA甲基化可能是植物适应超低温保存的机制之一。