组蛋白变体的功能与识别机制

组蛋白变体(histone variant)是常规组蛋白的变异体,在染色质的特定位置或特定生物学事件中替换常规组蛋白,调控染色质结构以及相关生物学过程。组蛋白伴侣(histone chaperone)是指可以结合组蛋白,运送组蛋白参与染色质组装和去组装等重要功能的蛋白质。综述了几种主要组蛋白变体在真核生物染色质高级结构的形成及维持、细胞编程与重编程的表观遗传机制等生命进程中发挥的重要作用,以及这些组蛋白变体与其特征伴侣之间特异识别的分子机制。     

Hira基因与发育：从酵母到人类

Hir/Hira基因产物HIR/HIRA为组蛋白的伴侣蛋白,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。Hir/Hira基因功能突变对酵母以及高等真核生物的发育都有非常严重的影响。结合研究组的工作,综述了组蛋白调节基因Hir/Hira在不同生物体发育过程中的作用,以及该领域的研究方向之一 ¾ HIRA作用机理的最新进展。     

综述: 组蛋白变体的染色质组装

真核细胞的染色质组装是组蛋白和DNA有序地形成核小体和染色质的过程．通过调节DNA的开放或折叠状态,染色质组装不但影响遗传信息的编码和存储,也决定了遗传信息的提取和解读．作为染色质组装的重要调控因子,组蛋白变体和组蛋白伴侣在与DNA相关的生命活动进程中发挥着至关重要的作用．本文综述了组蛋白变体H2A.Z以及CENP-A进行染色质组装的研究进展,并着重讨论了组蛋白变体和组蛋白伴侣在染色质组装中的重要作用．     

WT1在哺乳动物性别发育过程中的调控作用

哺乳动物的性别发育大致可分为性别决定和性别分化两步,是一个由WT1/Wt1、SRY/Sry和MIS/Mis等多基因参与的级联过程,但目前对于这些基因之间的相互作用尚不清楚。在性别发育过程中持续表达的WT1/Wt1与多种伴有性别发育异常的疾病相关,其重要性表现为对多个性别发育关键基因在转录水平和转录后水平的调控。简要概述了WT1/Wt1的复杂性及其对多基因的调控作用,以期为阐明性别发育机制和基因间的相互关系提供参考。     

黑腹果蝇dCAF-1-p55突变引起果蝇发育迟缓和染色体不稳定性

在真核生物中高度保守的染色质装配因子1(chromatin assembly factor 1,CAF-1)是染色质装配过程中的组蛋白分子伴侣之一.dCAF-1-p55是果蝇中CAF-1复合物中的最小亚基,它与另外两个亚基dCAF-1-p180及dCAF-1-p105一起负责将组蛋白H3/H4组装到新合成的DNA上.除了CAF-1复合物,dCAF-1-p55还参与其他多个复合物的形成,如NURF、PRC2及Sin3-HDAC1.dCAF-1-p55的这一广泛参与性提示了其功能的多样性和重要性.为了研究dCAF-1-p55的体内功能,我们利用基因靶向敲除技术制备了果蝇dCAF-1-p55突变体.实验结果表明,dCAF-1-p55的缺失导致果蝇发育迟缓并且最终致死.进一步研究发现,在dCAF-1-p55突变细胞中,中期染色体较为松散,姐妹染色单体连接异常,后期染色体不能正常分离.这些缺陷都是与癌症发生密切相关的染色体不稳定性(chromosome instability,CIN)的典型特征.综上所述,我们的研究表明了dCAF-1-p55在果蝇发育过程及维持染色体稳定性方面的重要作用,同时提示该基因具有保护细胞免遭CIN和癌变的潜在功能.     

组蛋白变体的染色质组装

真核细胞的染色质组装是组蛋白和DNA有序地形成核小体和染色质的过程.通过调节DNA的开放或折叠状态,染色质组装不但影响遗传信息的编码和存储,也决定了遗传信息的提取和解读.作为染色质组装的重要调控因子,组蛋白变体和组蛋白伴侣在与DNA相关的生命活动进程中发挥着至关重要的作用.本文综述了组蛋白变体H2A.Z以及CENP-A进行染色质组装的研究进展,并着重讨论了组蛋白变体和组蛋白伴侣在染色质组装中的重要作用.     

KAP-1:转录调控中的一个桥梁分子

KAP-1(又称TIF1b, TRIM28等)是一种转录中介因子, 在诸多转录调控复合体中起桥梁作用。它通过其N端RBCC结构域与含KRAB结构域的锌指蛋白、MDM2、MM1、C/EBPb等相互作用; 通过C端的PHD及BrD结构域与SETDB1、Mi-2a等分子相互作用, 参与形成具有组蛋白甲基化酶或组蛋白去乙酰化酶活性的复合体; 通过中间的HP1BD区域与HP1蛋白相互作用, 进而与组蛋白相结合。大量研究表明, KAP-1作为一个桥梁分子, 主要以共抑制因子形式参与转录抑制复合体的形成, 在某些复合体中也可作为共激活因子发挥作用。KAP-1参与形成的复合体在精细胞发育、胚胎早期发育等生理过程中发挥重要的调控作用, 这种调控属于表观遗传调控范畴。     

植物组蛋白乙酰基转移酶的研究进展

组蛋白乙酰化是一个动态的、可逆的过程,包括组蛋白乙酰化和去乙酰化两个过程。组蛋白乙酰基转移酶催化组蛋白乙酰化,与组蛋白去乙酰化酶共同作用来调节组蛋白乙酰化状态。组蛋白乙酰化状态影响染色质的结构,进而影响基因的转录,在植物的生长、发育和胁迫反应过程中具有十分重要的调节作用。对组蛋白乙酰基转移酶的细胞内分布、分类、底物特异性以及在发育和胁迫反应中的功能进行了综述。     

质谱法分析大鼠精子发生过程中组蛋白H1变异体的多样性

目的为探究连接组蛋白H1在精子发生过程染色体重构中的功能,了解一共有多少种连接组蛋白H1参与各期生精细胞的染色体的构建。方法分离高纯度的SD大鼠的各期生精细胞,提取组蛋白,应用SDS-PAGE分离组蛋白的各组分,组蛋白（H1）经过蛋白酶（Glu-c和Arg-c）酶切,应用质谱进行检测。结果鉴定了组蛋白H1的体细胞亚型（H1.1-H1.5）和睾丸特异的连接组蛋白亚型（H1t）。组蛋白H1t分别表达在精原细胞,精母细胞和圆形精子细胞中。结论大鼠精子发生过程中,其主要连接组蛋白H1的种类是：H1.1-H1.5和H1t。     

综述: H2A-H2B类型组蛋白及其伴侣蛋白的研究进展

染色质是真核细胞中遗传物质DNA的载体,染色质结构动态变化与DNA复制、转录、重组、修复等重要生物学事件密切相关.组蛋白是染色质结构的基本组成元件之一,组蛋白变体和组蛋白修饰是两类基本的染色质结构调控因子.在构成核小体的四种核心组蛋白(H2A、H2B、H3、H4)当中,H2A拥有最多的变体类型并在染色质结构调控中发挥重要作用.H2A组蛋白伴侣对H2A组蛋白及其变体的特异识别对于后者的折叠、修饰、传递、转运、组装、移除等生物学功能至关重要.本文着重探讨了组蛋白伴侣特异识别H2A组蛋白的分子机理,二者调控染色质结构的作用机制以及相应的生物学意义.     

Human CAF-1-dependent nucleosome assembly in a defined system

Replication-coupled nucleosome assembly is a critical step in packaging newly synthesized DNA into chromatin. Previous studies have defined the importance of the histone chaperones CAF-1 and ASF1A, the replicative clamp PCNA, and the clamp loader RFC for the assembly of nucleosomes during DNA replication. Despite significant progress in the field, replication-coupled nucleosome assembly is not well understood. One of the complications in elucidating the mechanisms of replication-coupled nucleosome assembly is the lack of a defined system that faithfully recapitulates this important biological process in vitro. We describe here a defined system that assembles nucleosomal arrays in a manner dependent on the presence of CAF-1, ASF1A-H3-H4, H2A-H2B, PCNA, RFC, NAP1L1, ATP, and strand breaks. The loss of CAF-1 p48 subunit causes a strong defect in packaging DNA into nucleosomes by this system. We also show that the defined system forms nucleosomes on nascent DNA synthesized by the replicative polymerase δ. Thus, the developed system reproduces several key features of replication-coupled nucleosome assembly.     

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.     

Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli

Human fibroblast growth factor receptor (FGFR) is responsible for multifunctional signaling that regulates developmental processes. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) include the determinants of ligand binding and specificity for fibroblast growth factor and heparan sulfate. D1 and the D1-D2 linker with a contiguous stretch of acidic amino acids are known to be involved in auto-inhibitory regulation. In an effort to gain a better understanding of the role of D1 and the linker in FGFR regulation, we have subcloned, overexpressed, and purified the extracellular fragments, D1-D2 and D1-D3, of FGFR1 in Escherichia coli. The recombinant proteins were produced in an insoluble form and were renatured using a dropwise or on-column refolding method. In addition, D2-D3 was coexpressed with chaperones to test the possibility that the presence of chaperones might enhance refolding efficiencies. A combination of immobilized nickel and heparin affinity chromatography and size-exclusion chromatography resulted in the purification of recombinant ectodomain proteins D1-D2 and D1-D3 of high purity for structural studies.     

Heat shock during the development of brain structures of Drosophila: the memory development in the l(1)ts403 mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophila and humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.     

Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibitors

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.