组蛋白甲基转移酶SETD2研究进展

SETD2基因是调控哺乳动物表观遗传的一个重要基因,其编码的SETD2蛋白主要参与组蛋白甲基化修饰,与RNA聚合酶II作用介导转录延长及错配修复。小鼠SETD2结构异常通过下调H3K36me3而引起血管构建功能缺陷。在许多人类肿瘤(乳腺癌、肾癌及膀胱肿瘤等)中都发现了SETD2基因改变。近年来的研究表明,SETD2基因表达下调可促进白血病干细胞的自我更新,从而促进白血病的发生和发展。SETD2-H3K36me3信号通路可认为是肿瘤中常见的一种抑癌机制,这也为临床疾病诊断和治疗提供了新策略。但是,关于SETD2异常引起肿瘤的具体作用机制需要进一步研究。该文就SETD2基因的结构、功能及其在胚胎发育及成体肿瘤尤其是白血病中的作用机制作一综述。     

组蛋白去甲基化酶KDM5A调控H3K4me3参与神经管畸形发生的分子机制

神经管畸形(NTDs)的病因与防治是出生缺陷领域研究的重点,叶酸可以预防神经管畸形但其机制不明。本文借助低叶酸细胞模型和低叶酸NTDs小鼠模型通过染色质免疫共沉淀、Cut&Tag等技术,探讨了组蛋白去甲基化酶lysine demethylase 5A(KDM5A)及其调控的下游组蛋白H3K4me3修饰在叶酸缺乏导致的NTDs发生中的潜在分子机制。结果显示,低叶酸的细胞模型中,qRT-PCR、Western印迹结果显示,KDM5A分子表达明显下降(P<0.05)。作为组蛋白H3K4me3调控的上游关键酶,进一步通过染色质免疫共沉淀ChIP、ChIP-qPCR实验证实,叶酸缺乏下组蛋白H3K4me3在神经发育基因Axin2和Atoh1基因启动子区富集增加(P<0.05)。通过构建KDM5A基因敲除细胞模型,借助Cut&Tag试验证实,KDM5A基因敲除后H3K4me3主要富集在神经发育基因上。最后在低叶酸导致的NTDs小鼠模型的脑组织中,RT-qPCR、Western印迹以及ChIP-qPCR实验显示,E9.5 d的NTDs胎鼠脑组织中KDM5A表达下降(P&l...     

过表达组蛋白H3K9me3去甲基化酶对猪克隆胚胎发育的影响

组蛋白异常修饰是克隆胚胎发育的重要制约因素，组蛋白H3K9me3去甲基化酶KDM4家族的过表达可以有效提高克隆胚胎的发育效率。为探究过表达H3K9me3去甲基化酶对猪克隆胚胎发育的影响，本研究在猪克隆胚胎1-细胞期和2-细胞期分别注射KDM4A mRNA和KDM4D mRNA检测胚胎的囊胚率；收集1-细胞期注射KDM4A mRNA和胚胎注射水(对照组)的2-细胞期克隆胚胎检测H3K9me3表达水平；此外，收集1-细胞期注射KDM4A mRNA和胚胎注射水的4-细胞期克隆胚胎进行单细胞转录组测序，并对测序数据进行GO与KEGG富集分析。结果显示：在1-细胞期注射KDM4A mRNA的猪克隆胚胎囊胚率显著高于对照组(25.32±0.74%vs14.78±0.87%)，注射KDM4D mRNA对猪克隆胚胎囊胚率无明显作用(16.27±0.77%vs 14.78±0.87%)；在2-细胞期注射KDM4A mRNA和KDM4D mRNA的克隆胚胎囊胚率与对照组相比均无显著差异(32.18±1.67%、30.04±0.91%vs 31.22±1.40%)。在1-细胞期注射KDM4A mRNA的克隆...     

KDM3A全长及截短质粒构建及其在细胞中的定位探究

赖氨酸去甲基化酶3A(lysine demethylase 3A,KDM3A)是组蛋白去甲基化酶家族成员之一,通过特异性对组蛋白H3K9甲基化进行去甲基化的作用调控基因转录。目前研究证实KDM3A在肝癌、雌激素受体(estrogen receptorα,ERα)阳性乳腺癌及前列腺癌等肿瘤中发挥促癌作用,但其具体作用机制仍有待更深层次的研究。为深入解析KDM3A在各种肿瘤中的作用及其分子机制,本研究利用分子生物学方法构建了KDM3A基因的全长和截短基因表达质粒,酶切鉴定及测序证实KDM3A全长及截短基因的表达质粒构建成功。在此基础上,再将构建成功的上述多种表达质粒分别转染到HEK293细胞和MCF7细胞中,进行Western blotting和免疫荧光共聚焦实验,进一步对各重组表达质粒在培养的哺乳动物细胞中的蛋白表达及蛋白细胞定位进行初步探究。Western blotting结果显示KDM3A全长基因表达的蛋白约147 kD,其他KDM3A截短基因表达的蛋白均对应其特异性的蛋白条带;免疫荧光共聚焦实验结果显示KDM3A全长质粒以及pN1、pN2、p N3、pΔ截短质粒表达的蛋白分布在细胞核,pC1、pC2截短质粒表达的蛋白分布在细胞质,pC3截短质粒表达的蛋白分别分布于细胞核与细胞质。本实验为后续深入研究KDM3A在肿瘤中的作用及分子机制提供了实验基础。     

异染色质相关蛋白TRIM28调控锌指蛋白和原钙黏蛋白家族基因的转录

目的 TRIM28是一种异染色质相关蛋白,通过和SETDB1、HP1相互作用参与H3K9me3修饰的建立,本文旨在更深入地研究TRIM28的相关功能。方法 本文利用CRISPR/Cas9技术、染色质免疫共沉淀技术、免疫印迹技术和实时荧光定量PCR技术,建立HEK293F Trim28基因敲除细胞系,分析一系列实验数据结果。结果 Trim28主要抑制内源表达水平较低的基因转录,进一步分析发现Trim28调控锌指蛋白家族基因和原钙黏蛋白β家族基因的转录。在Trim28敲除细胞系中,锌指蛋白家族基因H3K27ac修饰、H3K4me1修饰和H3K4me3修饰都显著上升,H3K9me3修饰下降。原钙黏蛋白β家族基因的H3K4me3修饰显著上升,H3K9me3修饰下降。结论 这些结果提示TRIM28通过改变染色质的开放程度调控锌指蛋白和原钙黏蛋白β家族基因的转录,为更深入研究TRIM28的功能提供了新的思路。     

组蛋白去甲基酶UTX在发育和疾病中的作用

UTX(ubiquitously transcribed tetratricopeptide repeat,X chromosome)是抑制性组蛋白H3K27me3的特异性去甲基化酶,和甲基转移酶PRC2共同调控H3K27me3。此外,UTX也是组蛋白H3K4甲基转移酶MLL3/MLL4的组成部分。UTX参与胚胎发育、HOX基因的表达和重编程等生命过程。在歌舞伎综合征中,UTX突变是关键的致病因素。同时,UTX作为肿瘤抑制因子参与多种实体肿瘤和血液肿瘤的产生。该文总结了UTX在正常发育和疾病发生中的作用及近期研究的重大突破,并结合我们的研究探讨了UTX对体细胞重编程的影响。     

组蛋白共价修饰——组蛋白H3第4赖氨酸甲基化

染色体组蛋白的共价修饰在调节染色体结构,控制基因的转录等方面发挥重要的作用。组蛋白H3第4赖氨酸的甲基化作为共价修饰的方式之一,可以调控基因的转录激活。随着对组蛋白甲基化转移酶及相关作用蛋白研究的深入,人们对组蛋白H3第4赖氨酸的甲基化的功能也有了更深的了解。目前研究发现它与癌症也有很密切的关系。     

PI3K/AKT信号通路调控Myogenin和MCK基因的表达

骨骼肌分化过程受多个信号通路调控, PI3K/AKT信号通路是其中最重要的信号转导通路之一。PI3K/AKT信号通路可以调控骨骼肌分化, 但在染色质水平上的调控机制还不是很清楚。文章以小鼠成肌细胞(C2C12)为研究材料, 采用免疫印迹、染色质免疫共沉淀(Chromatin immunoprecipitation, ChIP)、定量PCR (Q-PCR)的方法研究PI3K/AKT信号通路调控Myogenin和MCK基因的表达。研究发现, C2C12细胞分化过程中添加PI3K/AKT信号通路激活剂处理24 h, Myogenin和MCK蛋白表达水平显著升高, 组蛋白H3K27me3去甲基化酶UTX的表达也升高, H3K27me3在Myogenin基因启动子区和MCK基因启动子及增强子区的富集与对照组相比显著降低。用PI3K/AKT信号通路抑制剂处理, 结果相反。因此, PI3K/AKT信号通路可能通过调控组蛋白去甲基化酶UTX的表达活性改变靶基因的H3K27me3的富集进而调控骨骼肌分化。     

组蛋白H3K36甲基化与疾病

组蛋白H3K36位点可以发生甲基化修饰,其修饰状态受到H3K36甲基转移酶和去甲基化酶的动态调控。H3K36的甲基化修饰可引起多种生物学效应,如参与基因的转录激活或抑制、剂量补偿以及基因的选择性剪接等。H3K36甲基化修饰状态的异常与很多疾病相关,因此全面了解H3K36甲基化对于该类疾病的诊断和治疗具有重要意义。     

Bcl-2家族蛋白及其在细胞凋亡中的作用

Bcl-2家族蛋白是目前已知的细胞凋亡中最重要的调控因子,在细胞凋亡通路中起着重要的调节作用．对其作用机制的研究将有助于对肿瘤,自身免疫性和神经变性等疾病的治疗。该文介绍Bcl-2家族中主要的几种蛋白在凋亡中的作用,以及对线粒体膜通透性的调控作用。     

Oxygen tension affects histone remodeling of in vitro–produced embryos in a bovine model

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O₂; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.     

Histone 3 modifications and blood pressure in the Beijing Truck Driver Air Pollution Study

Context: Histone modifications regulate gene expression; dysregulation has been linked with cardiovascular diseases. Associations between histone modification levels and blood pressure in humans are unclear.

Objective: We examine the relationship between global histone concentrations and various markers of blood pressure.

Materials and methods: Using the Beijing Truck Driver Air Pollution Study, we investigated global peripheral white blood cell histone modifications (H3K9ac, H3K9me3, H3K27me3, and H3K36me3) associations with pre- and post-work measurements of systolic (SBP) and diastolic (DBP) blood pressure, mean arterial pressure (MAP), and pulse pressure (PP) using multivariable mixed-effect models.

Results: H3K9ac was negatively associated with pre-work SBP and MAP; H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP; and H3K27me3 was negatively associated with pre-work SBP. Among office workers, H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP. Among truck drivers, H3K9ac and H3K27me were negatively associated with pre-work SBP, and H3K27me3 was positively associated with post-work PP.

Discussion and conclusion: Epigenome-wide H3K9ac, H3K9me3, and H3K27me3 were negatively associated with multiple pre-work blood pressure measures. These associations substantially changed during the day, suggesting an influence of daily activities. Blood-based histone modification biomarkers are potential candidates for studies requiring estimations of morning/pre-work blood pressure.     

Determination of the parental pronuclear origin in bovine zygotes: H3K9me3 versus H3K27me2-3

To study the dynamics of 5-methylcytosine and 5-hydroxymethylcytosine in zygotes, the parental origin of the pronuclei needs to be determined. To this end the use of the asymmetric distribution of histone modifications in pronuclei is becoming more popular. Here, we demonstrated that histone 3 lysine 27 di-tri-methylation shows a stable pattern being present in the maternal but not in the paternal pronucleus of bovine zygotes, even in late stages of pronuclear development. In contrast, the pattern of histone 3 lysine 9 tri-methylation is very variable, and therefore cannot be used to reliably determine the parental origin of bovine pronuclei.     

Desiccation and supra‐zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long‐term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes’ germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming.     

EZH2在人脂肪肉瘤的表达情况及EZH2抑制剂抗人脂肪肉瘤效果及机制研究

摘要 目的：探索组蛋白H3K27me3甲基转移酶EZH2在人脂肪瘤、高分化脂肪肉瘤和去分化脂肪肉瘤的表达情况及EZH2酶活性小分子抑制剂GSK126对人脂肪肉瘤细胞系SW872的影响,并初步探索其可能机制。方法：筛选脂肪瘤、高分化脂肪肉瘤和去分化脂肪肉瘤患者术后活检标本共计23例,其中脂肪瘤7例,高分化脂肪肉瘤9例,去分化脂肪肉瘤7例,制成组织芯片,免疫组化染色检测EZH2的蛋白表达情况。体外培养SW872 脂肪肉瘤细胞系,采用CCK-8 法检测不同浓度GSK126对细胞生存的抑制作用,流式细胞技术检测细胞凋亡情况,Realtime PCR法检测细胞的凋亡与抗凋亡（Caspase-1、Caspase-3、Caspase-7、Caspase-9、Bcl-2、Bag-3）、血管生成（VEGF-α）、干性（CD133、CD44、CD24）相关基因的表达,Western blot 检测内质网应激相关蛋白Bip和ATF4蛋白的表达量。结果：EZH2在去分化脂肪肉瘤的表达高于高分化脂肪肉瘤,在良性脂肪瘤中的阳性表达少见（均P＜0.05）。EZH2酶活性抑制剂GSK126对SW872 细胞的存活有明显的抑制作用,给药后细胞凋亡率增加（P＜0.05）,凋亡相关基因Caspase-1、Caspase-3、Caspase-7、Caspase-9均表达增强（均P＜0.05）,血管生成基因VEGF-?琢表达降低（P＜0.01）,干性基因CD133表达降低（P＜0.01）,其余基因表达无明显差别。GSK126组的内质网应激相关蛋白Bip和ATF4蛋白的表达增加。结论：EZH2蛋白表达量与脂肪肉瘤细胞分化程度呈负相关,EZH2有望成为脂肪肉瘤的生物学标志物及恶性程度标志物。EZH2抑制剂可能成为脂肪肉瘤潜在的化疗药物,可能通过增强内质网应激发挥作用。     

Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks

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