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.     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。     

乙酰转移酶MYST家族的生物学功能

组蛋白乙酰化是表观遗传修饰的重要方式,主要受到组蛋白乙酰转移酶（histone acetyltransferases, HATs)和组蛋白去乙酰化酶（histone deacetylase, HDACs）催化. MYST是人类HATs的4大家族之一,包括MOF(males absent on the first),TIP60 (tat interacting protein 60 kD),结合ORC1的组蛋白乙酰转移酶（histone acetyltransferase binding to ORC1, HBO1),单核细胞白血病锌指蛋白(monocytic leukemia zinc finger protein, MOZ)和MOZ相关蛋白（MOZ related factor, MORF）等,均具有典型的MYST结构域.MYST介导的乙酰化是重要的翻译后修饰,其催化底物包括组蛋白和非组蛋白,如组蛋白H3, H4, H2A, H2A突变体,以及许多参与DNA代谢、细胞增殖和发育调控的蛋白因子. MYST蛋白家族参与许多细胞的生理过程,本文主要综述其在调节基因转录、DNA损伤修复和肿瘤发生发展等方面的生物学功能.     

DNA损伤和修复中的H2AX磷酸化及其在生殖相关细胞中的作用

H2AX是组蛋白H2A家族常见的变体之一，H2AX磷酸化是指哺乳动物细胞中的组蛋白H2AX在其C端第139位丝氨酸上发生磷酸化修饰形成磷酸化组蛋白H2AX(histone H2AX phosphorylation,γH2AX)的过程。目前，γH2AX的检测方法主要有免疫荧光法、流式细胞术、免疫印迹法。γH2AX是DNA损伤尤其是DNA双链断裂(DNA double-strand breaks,DSB)或DNA修复的标志物，已经被应用到医学相关领域，如放射性DNA损伤的检测、癌症的辅助诊断以及预后监测。此外，γH2AX在检测生殖细胞中的DNA损伤及修复和维持胚胎干细胞的自我更新中有重要意义。检测γH2AX水平已成为评价生殖细胞和干细胞质量的重要方法。本文将从H2AX及其家族的生物学特征、γH2AX的检测方法、DNA损伤与修复中的H2AX磷酸化及其在生殖相关细胞中的应用等角度对γH2AX研究进展进行总结和探讨。     

纳米二氧化锆对人永生化角质形成细胞组蛋白H3修饰的影响

目的 阐明纳米二氧化锆暴露对人永生化角质形成细胞Ha Ca T组蛋白H3常见修饰位点的影响,探讨组蛋白H3修饰变化的潜在机制,为纳米材料的进一步安全应用提供理论基础。方法 在利用扫描电子显微镜、激光粒度仪、X射线衍射仪等技术对纳米二氧化锆进行详细表征的基础上,通过蛋白质免疫印迹及流式细胞术等方法评价纳米二氧化锆暴露对细胞生存率、细胞内蓄积量以及组蛋白H3修饰等的影响。结果 在分散介质中纳米二氧化锆明显团聚,比表面积减少,二次粒径增大,其短时间内（1 h）即诱导了组蛋白H3第10位丝氨酸的磷酸化、第9及14位赖氨酸的乙酰化、第4及27位赖氨酸的三甲基化修饰水平的升高。进一步分析发现,纳米二氧化锆的细胞内蓄积量及其引起的DNA损伤水平,与纳米二氧化锆诱导的组蛋白H3修饰水平均呈线性相关。结论 纳米二氧化锆暴露后诱导了Ha Ca T细胞组蛋白H3常见修饰位点的变化,其细胞内的蓄积是诱导组蛋白H3修饰变化的关键因素之一,且组蛋白H3修饰的调控机制可能涉及DNA损伤修复途径。     

CHD4在染色体重塑中的作用

染色质作为真核细胞遗传信息,体内外各种因素的作用致使不断的产生损伤,但是细胞仍能保持正常的生长、分裂和繁殖,这与基因组稳定性和完整性保持,并且通过自身的损伤修复有着密切的联系。ATP依赖的染色质重塑是染色质重塑的最重要的方式之一,主要是利用ATP水解释放的能量,将凝聚的异染色质打开,协调损伤修复蛋白与DNA损伤位点的作用,通过对组蛋白的共价键修饰或ATP依赖的染色质重塑复合物开启了DNA的损伤修复的大门。CHD4/Mi-2β的类SWI2/SNF2 ATP酶/解螺旋酶域结构域保守性最强,这一结构域存在与多种依赖于ATP的核小体重构复合物。Mi-2蛋白复合物称为核小体重塑及去乙酰化酶NuRd(nucleoside remodeling and deacetylase,NuRD),是个多亚基蛋白复合物,Mi2β/CHD4是该复合物的核心成员。近来的研究发现,CHD4具有染色质重塑功能,并且参与DNA损伤修复的调控。CHD4羧基端的PHD通过乙酰化或甲基化识别组蛋白H3氨基端Lys9(H3K9ac和H3K9me),并且通过Lys4甲基化(H3K4me)或Ala1乙酰化(H3A Lac)抑制与H3、H4的结合,为染色质重塑提供了保障。Mi-2β/CHD4参与DNA损伤反应,定位于DNA损伤γ-H2AX的foci。沉默Mi-2β/CHD4基因,细胞自发性DNA损伤增多和辐射敏感性增强。表明CHD4在染色质重塑中具有重要的作用。     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

组蛋白H2B单泛素化参与DNA损伤修复的调控机制

作为一种重要的组蛋白修饰形式,H2B的单泛素化(uH2B)广泛地参与DNA复制、基因的表达与转录、DNA损伤修复及异染色质维持等生物学事件.在裂殖酵母中,H2B的单泛素化发生在其羧基端的119位赖氨酸(K119),并依赖于Rhp6/Bre1泛素连接酶复合体.研究表明,uH2B通过破坏H2A/H2B二聚体的结构促进mRNA在转录过程中的延伸,同时促进H3K4的三甲基化激活基因的表达及参与DNA损伤修复.本研究发现,Rhp6能够对核糖核苷酸还原酶抑制基因(Spd1)位点进行活跃的染色质修饰,促进H2B的单泛素化并抑制基因表达,从而促进dNTP的合成并调控DNA复制及损伤修复.重要的是,本研究发现,该过程不依赖于H3K4而决定于H3K9的三甲基化.同时uH2B直接在DNA双链断裂位点富集,通过改变染色质的结构参与DNA损伤修复,该过程中可能存在其他更为复杂的分子机制.     

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.     

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.     

Focus on histone variant H2AX: To be or not to be

Phosphorylation of histone variant H2AX at serine 139, named γH2AX, has been widely used as a sensitive marker for DNA double-strand breaks (DSBs). γH2AX is required for the accumulation of many DNA damage response (DDR) proteins at DSBs. Thus it is believed to be the principal signaling protein involved in DDR and to play an important role in DNA repair. However, only mild defects in DNA damage signaling and DNA repair were observed in H2AX-deficient cells and animals. Such findings prompted us and others to explore H2AX-independent mechanisms in DNA damage response. Here, we will review recent advances in our understanding of H2AX-dependent and independent DNA damage signaling and repair pathways in mammalian cells.     

Chromatin modulation and the DNA damage response

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.     

Deregulation of DNA damage response pathway by intercellular contact

Deregulation of the DNA damage response (DDR) pathway could compromise genomic integrity in normal cells and reduce cancer cell sensitivity to anticancer treatments. We found that intercellular contact stabilizes histone H2AX and γH2AX (H2AX phosphorylated on Ser-139) by up-regulating N/E-cadherin and γ-catenin. γ-catenin and its DNA-binding partner LEF-1 indirectly increase levels of H2AX by suppressing the promoter of the RNF8 ubiquitin ligase, which decreases levels of H2AX protein under conditions of low intercellular contact. Hyperphosphorylation of DDR proteins is induced by up-regulated H2AX. Constitutive apoptosis is caused in confluent cells but is not further induced by DNA damage. This is conceivably due to insufficient p53 activation because ChIP assay shows that its DNA binding ability is not induced in those cells. Together, our results illustrate a novel mechanism of the regulation of DDR proteins by the cadherin-catenin pathway.     

SET8 methyltransferase activity during the DNA double-strand break response is required for recruitment of 53BP1

DNA double-strand breaks (DSBs) activate a signaling pathway known as the DNA damage response (DDR) which via protein–protein interactions and post-translational modifications recruit signaling proteins, such as 53BP1, to chromatin flanking the lesion. Depletion of the SET8 methyltransferase prevents accumulation of 53BP1 at DSBs; however, this phenotype has been attributed to the role of SET8 in generating H4K20 methylation across the genome, which is required for 53BP1 binding to chromatin, prior to DNA damage. Here, we report that SET8 acts directly at DSBs during the DNA damage response (DDR). SET8 accumulates at DSBs and is enzymatically active at DSBs. Depletion of SET8 just prior to the induction of DNA damage abrogates 53BP1’s accumulation at DSBs, suggesting that SET8 acts during DDR. SET8’s occupancy at DSBs is regulated by histone deacetylases (HDACs). Finally, SET8 is functionally required for efficient repair of DSBs specifically via the non-homologous end-joining pathway (NHEJ). Our findings reveal that SET8’s active role during DDR at DSBs is required for 53BP1’s accumulation.     

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations¹. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in²). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones^3,4, forming foci within 5-15 minutes⁵. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair⁶. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins^7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs⁶. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis¹⁰.In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1''s behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs¹¹. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis¹². Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.     

Kdm4b Histone Demethylase Is a DNA Damage Response Protein and Confers a Survival Advantage following γ-Irradiation

DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double-strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and dimethylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-enhanced GFP (EGFP) and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly(ADP-ribose) polymerase 1 activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at γ-irradiation-induced γH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of γH2AX foci 6 h after irradiation as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points after 2 gray of γ-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.