Degree of methylation of transgenes is dependent on gamete of origin

Data derived from both pronuclear transplantation experiments and classical genetic experiments indicate that the maternal and paternal genetic contributions to the mammalian zygote nucleus do not function equivalently during subsequent development. These observations have been interpreted as resulting from differential 'genome imprinting' during male and female gametogenesis. The molecular mechanism responsible for genome imprinting is unknown, but data gathered to date require that the mechanism fulfill at least four criteria: (1) the imprint must be physically linked to the pronucleus; (2) the imprint must persist through DNA replication and cell division; (3) the mechanism must be capable of affecting gene expression; and (4) the mechanism must be capable of switching the identity of the imprint from one sex to the other in successive generations. One molecular mechanism which could satisfy the first three criteria is differential DNA methylation during gametogenesis itself, or before formation of the zygote nucleus during embryogenesis. We present data indicating that the methylation patterns of exogenous DNA sequences in transgenic mice can be changed by switching their gamete of origin in successive generations. These data suggest that DNA methylation can also satisfy the fourth criterion for an imprinting mechanism.     

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.     

中国对虾精子发生及受精细胞学

中国对虾（ＰｅｎａｅｕｓｃｈｉｎｅｎｓｉｓＯｓｂｅｃｋ１７６５）属于节肢动物门,有鳃亚门,甲壳纲,软甲亚纲,十足目,游泳亚目,对虾科,对虾亚科,对虾属,是我国最重要的经济虾类,主要分布在我国渤海、黄海,东海北部也有少量分布。常与墨吉对虾混栖。到目前为...     

盐透析体外组装核小体及检测方法

核小体是构成真核生物染色质的基本结构单位,体内研究核小体及染色质结构受到诸多因素限制,体外重构核小体结构是研究与核小体及染色质结构相关课题的一种重要的方法手段.实验将ES1,CS1以及601DNA序列克隆到载体中,通过PCR大量扩增回收得到目的DNA条带,表达纯化了4种组蛋白且装配成组蛋白八聚体,在盐透析的条件下组装形成核小体结构,利用EB染色以及Biotin标记的方法分析检测了形成核小体的效率.结果显示,在盐透析的条件下,可以有效的组装形成核小体结构,而且随着组蛋白八聚体与DNA的比例增加,核小体的形成效率显著提高.本实验为核小体定位、染色质重塑及组蛋白变体等表观遗传学以及结构生物学领域的研究奠定一定的基础.     

Crystal structure of the human centromeric nucleosome containing CENP-A

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg?80 and Gly?81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.     

金鱼精子头在卵细胞质中转化的超微结构研究

用透射电镜研究了金鱼卵子受精后起始阶段精子头在卵细胞质中的转化过程，结果表明：这种转化同样必须经精子核膜的崩解、原来高度致密的精子染色质的解聚与分散以及新的核膜（雄原核核膜）的形成。     

Regions of variant histone His2AvD required for Drosophila development.

One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes. H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice (R. Faast et al., in preparation). To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, we have performed amino-acid swap experiments in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues. Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality. We show that the unique feature of His2AvD does not reside in its histone fold but in its carboxy-terminal domain. This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome core.     

斑马鱼受精过程中原核的时空规律

斑马鱼的成熟卵子处于第2次减数分裂中期,精子通过受精孔进入卵内,精子入卵后,卵子被激活;受精后3 min,精核位于卵质膜内侧边缘,处于凝集状态;受精后5 min精核开始解凝;受精后8 min,精核明显解凝,受精卵出现明显的精子星光.同时进入第2次减数分裂后期,之后排出第二极体;受精后10 min,雄原核形成,具有双星光,此时,雄原核已位于胚盘中部;受精后15 min雌原核已形成,雌、雄原核向胚盘中央靠近;受精后20 min,雌雄原核开始接触;受精后30 min,雌雄原核完全融合成为1个合子核;35 min时,受精卵开始第1次卵裂;45 min后分裂形成2个子核;50 min时分裂为2细胞,60 min时分裂为4细胞.     

Hira基因的过量表达对果蝇早期胚胎发育的影响

Hir／Hira基因产物是组蛋白基因表达的一种负调节因子,其在果蝇发育过程中的作用还没有得到确认．本研究将果蝇Hira基因（dHira）的cDNA克隆到载体UAsP中,运用UAS—Gal4系统使其在果蝇早期胚胎中大量表达．结果发现在胚胎发育早期,无论是在头部还是在全胚胎过量表达Hira,都引起果蝇胚胎大量死亡,而且随着转基因拷贝数的增加,胚胎的死亡率也显著增加,说明Hira过量表达对果蝇胚胎发育产生严重影响．由于Hira基因产物与核小体的组装、染色质结构等的调节有关,因此推测Hira过量表达可能是通过对组蛋白的抑制对果蝇胚胎发育造成影响的．     

羊草(Aneurolepidium chinense)的双受精作用与胚胎发育

本文以采用电镜制片的方法对羊草的双受精过程和胚胎发育进行了研究。羊草的双受精属有丝分裂前配子配合型。开花后约4h精子进入胚囊。两精子几乎同时进卵核和极核之一,精核分别在卵核和极核内松散,同时出现1到2个雄核仁。合子时期雌雄性核仁不融合,直到合子分裂前期仍为多核仁。羊草胚胎发育为紫菀型。一般合子第一次分裂为横分裂,但偶见有斜分裂。合子未见有极性,在二细胞原胚后期见有极性表现,超微结构显示,顶细胞和基细胞中圆球体大小和发育时期不同。胚发育经过原胚、椭圆形胚,再经胚分化期和成熟期发育为成熟胚。     

The RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

Nucleosome assembly

Histones and DNA can spontaneously associate to form the nucleosome subunits of eukaryotic chromatin, but two proteins which occur in the eukaryotic nucleus can facilitate nucleosome assembly and greatly extend the conditions which permit assembly to occur. Recently several laboratories have reported new experimental systems which help to analyse, reconsruct and exploit the cellular mechanisms of chromatin assembly.     

Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads approximately 40 kb in either direction from the DSB. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX is required for histone loss, which also depends on INO80, a nucleosome remodelling complex. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage.     

雄性生殖单位的研究进展

介绍了雄性生殖单位概念的建立、精子异型性的确认及其内涵，以及近年来的研究进展。在花粉生物学和精细胞的研究中，应用三维重构和细胞定量分析技术，发现被子植物花粉和花粉管中，互相联系的两个精细胞或生殖细胞与营养核在生殖过程中联系在一起，形成一个配子传递，识别、与雌性细胞融合的功能单位，这种雄细胞成员的联合体称谓雄性生殖单位。同时发现姐妹精细胞间在大小，形态以及细胞器含量，甚至细胞核的大不是不同的。由此建     

Chromatin dynamics during epigenetic reprogramming in the mouse germ line

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.     

Crucial function of histone lysine methylation in plant reproduction

Unlike animals, plants do not set aside germ cells early in development. In angiosperm species, reproduction occurs in the adult plant upon flowering. The multicellular male and female gametophytes differentiate from meiotic products within reproductive floral organs. Double fertilization is another remarkable feature of most angiosperm species. The zygote derived from fertilization of the egg cell by one of the sperm cells and the endosperm from fertilization of the central cell by the second sperm cell develop in a coordinated manner together and enclosed in the sporophytic maternal integuments, forming the seed. Understanding plant reproduction is biologically pertinent and agronomically and ecologically important. Here, we describe the known functions of histone lysine methylations in various steps of reproduction in the reference plant Arabidopsis thaliana. It is emerging that histone lysine methylation is key for understanding epigenetic regulation networks of genome function.