银鲫HIRA多克隆抗体的制备及组织特异性表达分析

Hir/Hira基因家族的成员广泛存在于多种生物体中,但有关其在生物体发育过程中的具体功能还不甚清楚.对果蝇的研究表明,dHira基因产物可能在受精时精核的解凝过程和雄性原核的正常形成过程中起重要作用.本研究组前期已经分别克隆出雌核发育银鲫和两性生殖彩鲫的Hira基因(cagHira 和caHira),本实验在银鲫cagHira基因的特异区域,设计一对引物,以银鲫成熟卵母细胞总RNA逆转录出的cDNA为模板,扩增出cagHira的特异片段.再将该片段克隆到原核表达载体pET-32a上,转化BL21(DE3)菌株,经诱导后表达出融合蛋白.分析表明,该融合蛋白主要以包涵体形式表达.以纯化的融合蛋白作为抗原去免疫小鼠,制备多克隆抗血清,经蛋白质印迹分析检测,该抗血清(稀释到1:2000)与包涵体蛋白识别反应良好,确定获得了具有高效价的特异性银鲫CAGHIRA多克隆抗体,为进一步研究HIRA在鱼类发育和雌核生殖过程中的作用奠定了基础.对银鲫HIRA蛋白的组织特异性表达分析发现,该蛋白仅在成熟卵巢组织中特异表达,故表明HIRA可能对鱼类卵子发生和/或早期胚胎发育具有重要作用.     

组蛋白伴侣在发育过程中的功能

组蛋白伴侣能够协助组蛋白参与染色质的解凝和组装, 从而调控基因的表达, 对动植物的配子发生、受精、胚胎发育以及生长、衰老等发育过程都具有重要作用。文章主要对目前研究较多的组蛋白伴侣 - 核质蛋白、CAF-1、HIRA、ASF1/CIA及NAP1在发育过程中的相关功能作一综述。     

彩鲫hira基因片段的克隆及表达分析

hir／hira基因最先作为组蛋白基因表达的一种负调节因子从酵母中被鉴定出来。现已证实,HIRA包含一组保守的蛋白家族,广泛存在于低等真核生物、无脊椎动物和脊椎动物等多种生物体当中,为生命发育所必需。关于hira基因在动物发育过程中的具体作用研究还不是很多,为了进一步探讨hira基因在鱼类发育过程中的作用,作者根据已知hira基因保守序列设计一对简并引物,分别以彩鲫基因组DNA和卵巢cDNA为模板克隆了彩鲫hira基因（Cahira）片段,该片段基因组序列为2181bp,基因组片段含有6个内含子,长度分别为118bp、275bp、372bp、84bp、472bp、86bp;cDNA序列为759bp,编码253个氨基酸,具有5个WD结构域。对其氨基酸序列进行比较,结果表明,彩鲫hira基因的氨基酸序列与河鲍、爪蟾、鸡、小鼠、人的hira基因氨基酸序列具有非常高的同源性,分别高达92％、89％、89％、87％和88％。组织特异性表达分析表明,所检测的彩鲫组织除了精巢和肌肉中未检测到hira基因表达外,其余组织均有表达。其中卵巢和肝脏中表达很强,而在脑、心、脾、肾中表达较弱,说明该基因可能在维持卵巢和肝脏组织的功能方面起一定作用。     

组蛋白去乙酰化酶(HDACs)及其在植物中的作用

组蛋白去乙酰化酶(HDACs)是一个在真核生物(包括酵母、哺乳动物和植物)中广泛存在的超基因家族。HDACs与组蛋白乙酰基转移酶(HATs)共同作用来调节组蛋白乙酰化的状态,从而影响染色体的结构和功能,调节基因的转录和细胞的多种功能。目前,人们对植物HDACs的研究逐渐增多,很多HDAC基因在不同植物中得到鉴定和研究。综述了HDACs的分类以及在植物生长发育和胁迫反应中的作用,旨在为进一步研究HDAC在植物中的表观遗传调控机理以及培育抗逆新品种奠定理论基础。     

Hira基因产物在银鲫和彩鲫卵子发生过程中的动态变化

为进一步研究Hira基因在卵子发生和雌核发育过程中的作用,通过原位杂交和免疫荧光定位的方法检测了Hira mRNA和蛋白质在雌核发育银鲫和两性生殖彩鲫卵子发生过程中的动态变化。结果表明,银鲫和彩鲫卵子发生过程中Hira基因转录产物的变化基本一致,在Ⅰ期卵母细胞的细胞核中大量表达,至Ⅱ期卵母细胞时转至细胞质中均匀分布,在Ⅲ期卵母细胞中,杂交信号逐渐移向细胞的周边,到Ⅳ期时随着卵黄物质大量积累,杂交信号几乎不见。HIRA蛋白在银鲫和彩鲫卵子发生过程中的变化略有差别。HIRA蛋白在银鲫Ⅰ期卵母细胞中没有表达,在Ⅱ期卵母细胞的细胞质中有弱表达,在Ⅲ期早期卵母细胞的周边有强烈表达;而在彩鲫Ⅰ期卵母细胞中就有HIRA蛋白的弱表达,至Ⅱ期时HIRA蛋白在细胞质中大量表达,Ⅲ期早期卵母细胞的细胞质中有弱表达。在银鲫和彩鲫Ⅲ期末和Ⅳ期卵母细胞中都很难观察到荧光信号。Hira mRNA和蛋白质在银鲫和彩鲫早期卵母细胞中有较强表达,且在银鲫和彩鲫卵子发生过程中没有显著差异,说明其可能对于脊椎动物卵子发生和减数分裂没有显著影响,而是在受精和/或胚胎发育过程中起作用。       

异染色质及基因关闭

异染色质普遍存在于真核生物的染色质中,和细胞分裂、生存竞争等有密切关系,尤其在调节基因的活性上有重要作用。组蛋白尾的修饰,决定着异染色质的形成和解聚,从而控制基因的启闭,这一机制被称为组蛋白密码。本文以裂殖酵母的交配型区为例介绍了异染色质的的形成及维持机理。组蛋白密码可能是DNA遗传密码外生命的又一调节机制,而对异染色质形成和结构功能的研究,将成为破译组蛋白密码的钥匙。     

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.     

《自然》《基因与发育》:科学家破译"组蛋白密码"识别新机制

<正>清华大学医学院基础医学系和结构生物学中心李海涛课题组利用上海光源生物大分子晶体学线站,从结构生物学角度解析组蛋白甲基化修饰识别新机制,进一步揭开了错综复杂的表观遗传调控的神秘面纱。相关成果近期分别在线发表于《自然》和《基因与发育》杂志。据了解,真核生物基因表达调控不仅依赖于特定的DNA序列元件,还受到特定组蛋白翻译后修饰的精密调节。组蛋白变体和组蛋白修饰赋     

酵母Sirtuins在细胞寿命中的作用

酿酒酵母(以下简略为酵母)作为寿命分析模型广泛应用于寿命研究领域。酵母寿命分析方法有两种,分别是复制型酵母寿命分析法和时序型酵母寿命分析法。目前,通过酵母寿命分析模型已识别出包括SIR2在内的多个寿命调节基因。SIR2是目前较好的被确立起来的寿命调节基因,具有NAD依赖型脱乙酰化酶的活性,从原核生物到真核生物都有良好的保守性。Sirtuins (Sir2蛋白家族的总称)在细胞内具有功能上的多样性,其中包括对于压力耐受的调节、基因转录的调节、代谢通路的调节以及寿命调节作用等。Sir2是Sirtuins家族最早发现的成员,其功能是参与异染色质结构域转录的沉默调节,同时还参与复制型酵母寿命的调节。已证明,SIR2的缺失会缩短酵母的寿命,基因表达的增高会延长寿命。Sir2的高等真核生物的同源蛋白也被证实参与衰老相关疾病的调节。本文中,我们将阐述Sir2以及Sir2的酵母同源蛋白Hst1-Hst4的功能,以及由它们调节的酵母寿命。     

酿酒酵母组蛋白乙酰化修饰及其对基因表达的调控

真核生物核小体组蛋白修饰引起染色质重塑(Chromatin remodeling)是表观遗传的重要调控机制.乙酰化修饰(Acetylation modification)是其中一种重要的方式.组蛋白乙酰化修饰位点集中在各种组蛋白N末端赖氨酸残基上.细胞内存在功能拮抗的多种乙酰基转移酶和去乙酰化酶,二者相互竞争,共同调节组蛋白的乙酰化状态,通过影响核小体结构的致密性,并在多种效应分子的参与下,实现对基因的表达调控.以真核模式生物酿酒酵母(Saccharomyces cerevisiae)为对象,综述乙酰基转移酶和去乙酰化酶的种类、作用特点以及其基因调控的分子机制等方面的最新研究进展.     

Human CABIN1 is a functional member of the human HIRA/UBN1/ASF1a histone H3.3 chaperone complex

The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regulatory events from gene activation to heterochromatinization. In yeast, the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p.     

Human UBN1 Is an Ortholog of Yeast Hpc2p and Has an Essential Role in the HIRA/ASF1a Chromatin-Remodeling Pathway in Senescent Cells

Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.     

HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2 substrate whose expression blocks S-phase progression

Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1). Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.     

HIRA directly targets the enhancers of selected cardiac transcription factors during in vitro differentiation of mouse embryonic stem cells

Molecular Biology Reports - HIRA is a histone chaperone known to modulate&nbsp;gene expression through the deposition of H3.3. Conditional knockout of Hira in embryonic mouse hearts leads to...