一种微小染色质免疫共沉淀方法的建立

染色质免疫共沉淀(chromatin immunoprecipitation assay,ChIP)是目前研究蛋白与DNA相互作用的强有力的技术之一.然而传统的ChIP实验方法的最大缺陷是要求大量的细胞数,这限制了它应用于少量细胞数的样品.在传统ChIP实验的基础上综合国外文献建立了一种能在少量细胞样品中进行的染色质免疫共沉淀实验,称之为微小染色质免疫共沉淀(miniChIP).并通过对诱导前后的MEL细胞中表达的β珠蛋白基因簇组蛋白H4乙酰化(acH4)的研究,证实了其可靠性和特异性.结果显示:高敏位点HS2和活跃基因β-maj的启动子区域存在一定的组蛋白H4乙酰化水平,DMSO诱导后显著增加,而不活跃基因Ey的启动子区域则检测到极低水平的组蛋白H4乙酰化,且诱导后无明显变化.     

染色质免疫沉淀技术——一种研究基因表达调控的工具

染色质结构在基因表达调节中起着重要的调节作用。利用甲醛固定活细胞中的DNA与蛋白质,通过免疫沉淀分离复合物的染色质免疫沉淀法是研究体内DNA和蛋白质相互作用的一种新方法,它不仅可用来研究体内反式因子与DNA的相互作用,也可以用来研究组蛋白修饰与基因表达的关系,从而分析蛋白质及其体内的DNA结合序列。目前,该方法在染色质结构研究中获得了广泛的应用。     

Hi-C的干细胞染色质多重相互作用特征分析

染色质高级结构是基因转录调节的重要因素,染色质多重相互作用是高级结构中的一种,是多个(≥3)染色质片段在空间上相互接触而形成的紧凑结构。为了解染色质多重相互作用这类高级结构的特征及其在干细胞中分化中起到的作用,通过对Hi-C数据进行相关分析并计算基因的FPKM表达量,研究了染色质多重相互作用。分析发现:多重相互作用约占所有作用的30%,包含近70%的基因;此类作用区域的高表达基因多于低表达基因;且与组蛋白乙酰化相关性高。在分化过程中,多重作用位点数目和比例减少;位于多重作用区域的基因的表达略有降低;组蛋白乙酰化(H3K27ac和H3K23ac)在多重作用区域的减弱,而组蛋白甲基化(H3K4me3和H3K27me3)倾向于增强。结果表明,染色质多重相互作用是一种广泛存在的染色质高级结构,在干细胞分化中有重要作用,此类结构多具有H3K27ac修饰,调节基因的表达。总之,染色质多重相互作用是一种重要的基因转录调节因素,在细胞分化中具有调控作用。     

染色质修饰酶在记忆中的作用

学习记忆的形成依赖于转录机制.近年研究发现,染色质修饰在基因表达调节中起重要作用.组蛋白乙酰化和去乙酰化是染色质修饰中最为常见调节方式,参与基因的转录调控.乙酰化可以激活转录,促进记忆的形成.组蛋白去乙酰化酶抑制剂可以增强突触可塑性,改善记忆损伤.因此,对于染色质修饰的深入研究,不仅有助于阐明记忆形成的分子机制,而且对记忆相关疾病的治疗以及新药物研发也具有重要指导意义.本文主要就组蛋白乙酰转移酶调节基因转录以及组蛋白去乙酰化酶抑制剂促进记忆形成的作用机理进行综述.     

植物表观遗传修饰与病原菌胁迫应答研究进展

真核生物基因表达受到染色质结构的调控,组蛋白与DNA的共价修饰构成表观遗传标签,并在植物胁迫应答如防御病原菌侵染过程中起重要作用.病原菌侵染可引起基因组整体DNA甲基化模式变化及胁迫应答基因的位点特异性去甲基化,导致植物抗性基因表达上调或下调,并进一步调控植物对病原菌的胁迫应答;组蛋白去乙酰化酶HDAC通过茉莉酸途径增强植物对病原菌的胁迫应答;此外,染色质重塑复合物Swr1复合体通过识别DNA基元和组蛋白乙酰化修饰状态靶向基因启动子,负调控SA敏感基因.该文从DNA甲基化、组蛋白乙酰化、甲基化修饰,染色质重塑等方面着重阐述植物与病原菌互作过程中发生的主要事件的分子基础及其研究进展.     

染色质构象调控真核基因的表达

真核基因的表达受到各种顺式调控元件、反式作用因子、染色质DNA以及组蛋白表观遗传修饰等多因素、多层次的调控。染色质三维空间结构的变化在调控真核基因表达方面也发挥了至关重要的作用。染色质构象的变化一方面可以使增强子等调控元件与靶基因相互靠近,从而促进基因表达;同时也可能通过形成空间位阻结构阻碍调控元件作用于靶基因,抑制基因表达。虽然染色质结构变化调控真核基因表达的机制仍缺乏较为精确的分子模型,但在组蛋白修饰、核小体定位、染色体领域以及染色质间相互作用等表观遗传学研究中,已经发现有诸多证据支持染色质构象在真核基因表达调控中的重要地位。文章主要综述了染色质结构及其构象的变化等对真核基因表达调控的影响。     

生肌素引起组蛋白H3和H4高度乙酰化及染色质溶解性增加

为研究生肌素在染色质重建过程中的作用 ,采用生肌素真核表达载体转染C2C12肌细胞 ,细胞核经微球菌核酸酶消化后 ,提取DNA进行SDS PAGE分析 .生肌素转染的细胞 ,其核小体 (染色质 )在Mg2 + 溶液中的溶解性明显增加 ,提示染色质组蛋白乙酰化程度提高 .组蛋白经TAU SDS(2 D)双相凝胶电泳分析 ,发现在转染生肌素真核表达载体 2 4h后 ,组蛋白H4的乙酰化修饰程度最高 .采用抗乙酰化组蛋白H3和H4抗体进行的Western印迹分析进一步证明了乙酰化的发生 .上述变化与染色质的活跃程度相关 .RT PCR结果显示 ,生肌素的靶基因烟碱样乙酰胆碱受体 (nAChR)α亚基和肌酸激酶 (MCK)基因在转染后表达水平提高 .结果提示 ,强制性表达外源性生肌素可引起肌细胞核染色质重建 ,进而激活靶基因     

RNA干扰与染色质沉默——生物体内精密的网络调控机制

基因表达受不同层次的调控.RNA干扰通过产生双链小RNA诱导同源mRNA序列降解,从而在转录后抑制特定基因的表达.最新的研究成果显示：RNA干扰产生的双链小RNA可通过与染色质中的重复序列DNA及组蛋白甲基化酶相互作用,引起组蛋白H3 Lys9的甲基化,进一步与异染色质形成相关蛋白结合,导致染色质沉默.综述了RNA干扰,小RNA,组蛋白修饰,染色质沉默及基因表达调控之间存在着精密的网络调控机制.     

染色质重塑复合物SAGA及其同源物的功能

基因表达调控是生物体生长发育的一个重要环节.在这一过程中,染色质重塑复合物扮演了非常重要的作用.SAGA是一个至少由20个蛋白组成的不依赖ATP的多功能染色质重塑复合物,它通过对组蛋白H3和H2B氨基末端赖氨酸乙酰化修饰来松动染色质结构,从而促进基因转录的起始.目前,对SAGA及其同源物的研究表明,SAGA及其同源物参与了许多重要的生物学功能,如mRNA输出、DNA损伤修复、胚胎发育、细胞癌变等.     

芽殖酵母和裂殖酵母中异染色质形成机制

芽殖酵母(Saccharomyces cerevisiae)和裂殖酵母(Schizosaccharomyces pombe)是用来研究异染色质形成、细胞周期、DNA复制等重要细胞功能的理想单细胞真核生物.本文主要介绍这2种酵母中异染色质形成的机制.异染色质是一种抑制基因转录和DNA重组的特殊染色质结构.尽管在芽殖酵母和裂殖酵母中异染色质形成都需要组蛋白修饰,但异染色质建立的机制不同.在芽殖酵母中参与异染色质形成的主要蛋白是Sir1-4蛋白（其中Sir2为组蛋白H3去乙酰化酶）,而组蛋白H3赖氨酸9甲基化酶Clr4和异染色质蛋白Swi6在裂殖酵母异染色质形成中起关键的作用.在这两个酵母中,参与异染色质形成的组蛋白修饰蛋白由DNA结合蛋白招募到异染色质.此外,裂殖酵母也利用RNA干扰系统招募组蛋白修饰蛋白.     

Regulation of nuclear histone acetyltransferase by nucleic acids,histone · DNA complex,and chromatin

Nuclear histone acetyltransferase is found to be inhibited by various nucleic acids and components. Of the adenosine phosphates, the order of inhibitory potency is ATP>ADP>AMP. Among the nucleoside triphosphates, GTP seems to be the best inhibitor, followed by ATP, CTP, and UTP. Deoxymononucleotides have the same order of inhibition potential as their ribonucleotide counterparts, with inhibition constants in the low millimolar range. Oligonucleotides and polynucleotides are much better inhibitors than mononucleotides. The inhibition constants of the DNA molecules are size dependent. Molecules larger than 40 base pairs have inhibition constants less than 18 µg/ml, whereas molecules with decreasing numbers of base pairs have increasing magnitudes of inhibition constants. However, acetyltransferase has a lower affinity for free DNA molecules than for DNA · histone complexes as revealed by its interaction with DNA-Sepharose and histone · DNA-Sepharose columns. Furthermore, native chromatin depleted of endogenous histone acetyltransferase activity shows no inhibitory effect on the enzyme. Yet heated chromatin not only loses substrate activity but also becomes an inhibitor for the enzyme. Since unmodified sea urchin sperm chromatin has been shown to be a potent acetyltransferase inhibitor, it seems possible that DNA · histone complexes may be the true inhibitory species and that the conformational states of such complexes may serve as a regulatory mechanism in the control of the enzyme activity.     

Assembly of Polycomb complexes and silencing mechanisms

Polycomb complexes assemble at their target sites and silence neighboring genes when these are not actively transcribed. The action of these complexes and of Trithorax complexes bound to the Polycomb Response Element establish alternative silent or derepressed states that are remembered through cell division and maintained for the rest of development. Recent results that may help explain the properties of these states are reviewed.     

Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.     

Histone variants in mouse centromeric chromatin

Summary Highly purified centromeric heterochromatin was isolated from mouse liver nuclei and the pattern of core histone variants was analyzed. In comparison with total chromatin, the centromeric heterochromatin of young animals was characterized by (1) enrichment in the replication-dependent variants H2A₁, H2B₂ and H3₂, (2) reduced amount of the minor variant H2A_z and (3) absence of ubiquitinated molecules of H2A. This specific variant pattern changed upon ageing as a result of accumulation of replacement variants so that in adult animals both chromatin preparations exhibited similar pattern for H2A and H2B, while the difference in the profile of H3 variants was preserved.     

On your histone mark,SET, methylate!

Lysine methylation of histones and non-histone proteins has emerged in recent years as a posttranslational modification with wide-ranging cellular implications beyond epigenetic regulation. The molecular interactions between lysine methyltransferases and their substrates appear to be regulated by posttranslational modifications surrounding the lysine methyl acceptor. Two very interesting examples of this cross-talk between methyl-lysine sites are found in the SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain-containing lysine methyltransferases SET7 and SETDB1, whereby the histone H3 trimethylated on lysine 4 (H3K4^me3) modification prevents methylation by SETDB1 on H3 lysine 9 (H3K9) and the histone H3 trimethylated on lysine 9 (H3K9^me3) modification prevents methylation by SET7 on H3K4. A similar cross-talk between posttranslational modifications regulates the functions of non-histone proteins such as the tumor suppressor p53 and the DNA methyltransferase DNMT1. Herein, in cis effects of acetylation, phosphorylation, as well as arginine and lysine methylation on lysine methylation events will be discussed.     

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence

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