一文读懂H3K27me3

名词解释​

组蛋白甲基化是一种重要的表观遗传修饰,参与基因的表达调控、基因印记和X染色体失活,调节胚胎发育、细胞增生和分化,与细胞衰老以及肿瘤发生发展有着密切关系[1-3]

表观遗传是在基因的DNA序列不发生变化的条件下,基因功能发生可遗传的变化,其发生机制主要包括DNA甲基化、组蛋白修饰、非编码RNA调控、染色质结构重构[4]

H3K27me3是指组蛋白H3第27位赖氨酸三甲基化,H3K27me3由多梳抑制复合物2(PRC2)产生,PRC2由EZH2、胚胎外胚层发育蛋白和胚胎肝细胞抑制蛋白三部分核心亚基组成[5]

                  

 

谁能控制它的“身体”


      
H3K27me3由Zeste基因增强子同源物2(Zeste gene enhancer homolog 2,EZH2)催化产生,后者参与组成多梳蛋白抑制复合物2(polycomb repressive complex 2,PRC2)聚集至启动子区域,导致靶基因转录抑制[6]。赖氨酸特异性脱甲基酶6B(lysine-specific demethylase 6B,Kdm6B/JMJD3)和赖氨酸特异性脱甲基酶6A(lysine-specific demethylase 6A,Kdm6A/UTX)为含有Jumonji C(JmjC)结构域的双加氧酶,和EZH2作用相反,能特异性去除H3K27me3的甲基,解除转录抑制,启动靶基因表达[6]
 
  • EZH2突变与表达失衡:有研究报道EZH2在多种癌症中过表达[7],如前列腺癌[8]、乳腺癌[9]、卵巢癌[10]和结肠癌[11],并显示出H3K27me3表达水平差异。值得注意的是,EZH2过表达引起的H3K27me3变化趋势并不一致,且与具体的作用机制密切相关。比如在转移性前列腺癌中,EZH2过表达能够高度特异性催化H3K27me3,并引起H3K27me3表达水平的升高[13],导致抑制分化基因、促进细胞增值,使致癌活性增高。然而,在去势抵抗性前列腺癌(CRPC)中,EZH2过表达时H3K27me3表达水平却是降低的[14]。主要原因是在CRPC中,EZH2的作用不是通过转录抑制实现的,而是与雄激素受体结合充当关键转录因子,同时EZH2催化H3K27三甲基化的作用降低,导致H3K27me3的表达水平下调[14]

 

 
  • EED的突变:EED是识别H3K27甲基化位点的关键[13],并能够促进PRC2复合物激活,保证H3K27me3修饰,维持H3K27me3表达水平的平衡。在患有骨骼增生异常综合症(MDS)的患者中发现存在EED第363位异亮氨酸突变为蛋氨酸(EED-I363M)[13,15],该突变可削弱H3K27me3与EED的结合,从而降低PRC2催化活性的变构激活,抑制H3K27me3的识别和转录导致H3K27me3表达下调[15],从而增加髓系癌症的易感性。目前已证实I363位于EED甲基赖氨酸结合位点附近,但是EED-I363M详细作用机制尚不清楚[15]

 
  • 组蛋白H3突变:H3K27me3表达水平也与组蛋白H3编码基因突变相关。以胶质母细胞瘤(GBM)为例,当组蛋白H3.3的编码基因H3F3A上第27位赖氨酸突变为甲硫氨酸(H3F3AK27M)时,会干扰以赖氨酸为靶点的H3K27me3修饰,导致H3K27me3下调[16,17]。H3K27me3的整体功能丧失可能通过影响分化途径促使H3F3AK27M突变型儿童GBM的发病[18]。H3F3AK27M突变型GBM中H3K27me3表达水平特异性降低,可能具有生物学和诊断意义[19]

 
  • 其他因素:H3K27me3的表达水平不仅受上述因素的影响,还有其他多种因素,例如不同表观遗传组蛋白H3K36和H3K27甲基化之间的平衡[20],DNA甲基化和H3K27me3之间的拮抗作用[21],组蛋白去甲基化JMJD3[22]和UTX[23]也会影响H3K27me3的表达水平,以及其他多种直接或间接因素[24]

 
综上所述,由于蛋白修饰的交叉作用,影响H3K27me3表达水平的因素较多,不同疾病中H3K27me3水平改变也各不相同,只有结合具体作用机制才能更好地为疾病提供有效信息。

                                                      它都用在哪?
 
小鼠基因敲除实验证实EZH2在胚胎早期植入前,通过表观遗传修饰和调节细胞凋亡为胚胎发育提供必要条件[25];JMJD3是成人骨髓祖细胞向肝表型转换的表观遗传重新编程所必需的[26],JMJD3可通过调节H3K27me3的水平来调节成纤维细胞活化[27]
 
H3K27me3作为生物标志物的应用:H3K27me3与肿瘤的发生相关。在乙型肝炎病毒(HBV)诱导的肝癌发过程中,HBV蛋白(HBx)的表达可降低H3K27me3水平,而增加HBV-诱导肝癌相关宿主基因EpCAM的启动子中H3K4mel的水平,激活组蛋白修饰,从而诱导肝癌发生[28]
在一项小细胞肺癌(SCLC)化疗机制的研究中发现,H3K27me3可通过调节HOX转录反义RNA(HOTAIR)促使HOXAI DNA 甲基化进而诱导SCLC的多药耐药性,该研究还表明H3K27me3可能是SCLC化疗耐药性的潜在治疗靶点[29]
 
H3K27me3也可作为相关肿瘤预后标记。在一项鼻咽喉癌(NPC)放化疗后的研究中发现,H3K27me3的高表达与NPC患者的生存期缩短密切相关,提示H3K27me3可作为预测NPC放化疗反应和患者预后的免疫标志物[30]。然而,在结肠癌化疗中发现,H3K27me3的低水平预示着治疗效果差[31]。对于不同肿瘤而言,H3K27me3在治疗中显示出的表达上调或者下调是所代表的的意义并不一致,需要结合具体作用机制综合分析。同样,使用H3K27me3进行预后分析时,也有相似情况。例如H3K27me3的表达缺失与乳腺癌、卵巢癌和胰腺癌患者的预后不良有关[32]。与之相反,有研究者发现H3K27me3在肝细胞癌的高表达与血管浸润密切相关,并预示患者的预后差[33]
 
此外,H3K27me3作为生物标志物在相关表现遗传新药的开发和评价中也具有重要意义。在EZH2抑制剂药物研究中,不仅用于研发阶段候选药物的筛选,还可为II期临床实验推荐剂量提供依据[34]。例如通过细胞的高通量筛选法,以H3K27me3为靶点从329049个化合物中鉴定出EZH2介导基因沉默的新药NPD13668[35]。在EZH2选择性抑制剂EPZ005687对多巴胺能神经元发育的研究中,发现该药物可抑制H3K27me3并增强神经元的分化等[36]
  

                                                       如何找到它?
H3K27me3检测方法:目前H3K27me3检测方法主要有Western blot、免疫组织化学(IHC)、染色质免疫沉淀-测序(ChIP-seq)、流式细胞术(FCM)等。通常根据使用目的和样本特征选择相应方法,比如采用Western blot分析外周血细胞中H3K27me3的表达[30],IHC检测实体肿瘤组织中H3K27me3水平[37]。这两种方法能获得定性和半定量的数据,但缺乏对多细胞群的分选能力,无法同时检测到H3K27me3水平在多个细胞群中表达量的差异[38],不利于同时进行多细胞群、多参数的分析。
  • ChIP-seq技术能够显示H3K27me3在整个基因组内结合特定基因和序列的信息,为证明H3K27me3调控功能和作用机制提供重要的数据支持[39]。该技术常应用于表观遗传研究领域,但存在高度依赖抗体的缺点[40]

 
  • Cut&Tag(cleavage under targets and tagmentation)技术是蛋白质-DNA相互作用关系研究的新方法,具有抗体依赖性低、重复性好、背景值低等优点,以及从抗体结合单衔接子整合的整个反应都发生在完整细胞内的优势,有希望在单细胞水平上研究H3K27me3[41]

 
  • FCM具有分析能力强和多参数的特点。目前,FCM主要同其他检测方法联合运用于H3K27me3的定量分析,如Western blot 和FCM联合分析EZH2抑制剂药效学,评估该药对细胞群的组蛋白甲基化状态的影响[40]。随着细胞通路研究的深入,FCM有望在单细胞水平直接检测胞内H3K27me3的表达水平。此外,定量PCR、下一代测序技术(NGS)等均可用于H3K27me3定量检测。

                                                           小结
H3K27me3作为组蛋白甲基化修饰的表现形式,是表观遗传的抑制标志,在参与调节细胞分化和增值基因的表达平衡中起着关键作用。H3K27me3与多种癌症和肿瘤的发生、转移、浸润密切相关,还可作为表观遗传重要的抑制生物标志物,应用于某些疾病的预后标记,助力新药开发和评价。随着检测技术的发展和H3K27me3研究的深入,有望能够从某些基因水平掌握相关疾病形成原因,为疾病的临床诊断、靶向治疗和预后明确新方向。

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