急性白血病(AL)为克隆性恶性疾病,其主要特征表现为异常原始细胞大量增殖积聚,并不断对其他组织和器官进行浸润。研究证实,组蛋白对基因调控具有重要作用,现已发现的端粒沉默样阻断蛋白(DOT1L)是一种组蛋白,其缺乏Su(var)3-9,Enhancer-o...急性白血病(AL)为克隆性恶性疾病,其主要特征表现为异常原始细胞大量增殖积聚,并不断对其他组织和器官进行浸润。研究证实,组蛋白对基因调控具有重要作用,现已发现的端粒沉默样阻断蛋白(DOT1L)是一种组蛋白,其缺乏Su(var)3-9,Enhancer-of-zeste and Trithorax(SET)结构域,具有赖氨酸甲基转移酶(H-Lys-M)活性,可定向性促进组蛋白H3第79位赖氨酸(H3K79)的快速甲基化,这一过程与AL的发生与发展密切相关。本文根据DOT1L结构特点与功能特点,综合论述DOT1L促进H3K79的过度甲基化在AL的发生、发展及预后中的作用,借以彰显DOT1L在AL靶位研究和靶向治疗中的意义与应用前景。展开更多
目的明确甲基转移酶Dot1L是否参与成肌分化调控过程,并初步探索其调控成肌分化过程的分子机制。方法应用小鼠体外成肌分化模型C2C12细胞系,诱导C2C12细胞在0、1、3、5 d发生成肌分化,使用Dot1L特异性抑制剂抑制其甲基转移酶活性,并应用...目的明确甲基转移酶Dot1L是否参与成肌分化调控过程,并初步探索其调控成肌分化过程的分子机制。方法应用小鼠体外成肌分化模型C2C12细胞系,诱导C2C12细胞在0、1、3、5 d发生成肌分化,使用Dot1L特异性抑制剂抑制其甲基转移酶活性,并应用免疫荧光实验观察Dot1L对成肌分化过程的影响;检测成肌分化标志分子肌球蛋白重链(myosin heavy chain,MHC)和肌细胞调节因子(Myogenin)的蛋白质水平和转录水平及启动子区域的组蛋白甲基化水平;使用RNA-Seq基因组范围内检测抑制剂处理与否发生变化的基因阵列;并用CHIP-qPCR实验验证H3K79me2结合位点。结果Dot1L组蛋白底物H3K79的甲基化水平随成肌分化进程逐渐升高(P<0.01);抑制Dot1L酶活性后,成肌分化过程明显受阻;免疫荧光实验显示C2C12细胞多核肌管形成显著下降(P<0.01),MHC和Myogenin的蛋白质水平及转录水平均显著降低(P<0.01);启动子区域组蛋白H3K4甲基化水平降低(P<0.01);RNA-Seq显示受Dot1L调控的基因阵列主要集中在骨骼肌的成肌分化和肌肉的发育;经CHIP-qPCR验证参与成肌分化的部分基因存在H3K79me2结合位点。结论甲基转移酶Dot1L通过直接调控一群成肌分化相关基因的转录水平参与调控骨骼肌的成肌分化过程。展开更多
Objective· To investigate the histone methyltransferase capability of DOT1L-long form and its role in breast cancer metastasis. Methods· The existence of DOT1L-long form was confirmed by PCR, and the mRNA le...Objective· To investigate the histone methyltransferase capability of DOT1L-long form and its role in breast cancer metastasis. Methods· The existence of DOT1L-long form was confirmed by PCR, and the mRNA level of DOT1L was tested by real-time PCR. In HEK293T cells in which DOT1L canonical and DOT1L-long were overexpressed respectively, Western blotting was used to test the expression level of DOT1L and the histone methyltransferase capability. In the MCF10A cell line with inducible expression of DOT1L-long, real-time PCR was used to detect the mRNA level of epithelial-mesenchymal transition(EMT) marker, and transwell assay was used to detect the migration of breast cancer cells in which the expression level of DOT1L is low or high. Results· PCR demonstrated the existence of DOT1L-long form, and real-time PCR showed it widely exists in HCT116, T98G, MCF10A cells, etc. Western blotting showed the expression of DOT1L-long form and its H3K79 methyltransferase activity. In MCF10A cells in which overexpressed canonical DOT1L and DOT1L-long, mRNA levels of N-cadherin and fibronectine increased. Transwell showed canonical DOT1L and DOT1L-long both substantially increased the migration of breast cancer cells. Conclusion· The existence of DOT1L-long was confirmed and investigated, which is 202 amino acids longer than the canonical DOT1L, and is coded by a new exon, located between exon 27 and 28. Further, the DOT1L-long has H3K79 methyltransferase activity, and is able to promote breast cancer metastasis.展开更多
Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leuke...Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, colorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.展开更多
类端粒沉默干扰体1(disruptor of telomeric silencing 1-like,DOT1L)是一种组蛋白H3第79位赖氨酸(H3K79)甲基转移酶,参与基因转录的表观遗传调控。近来研究发现,类端粒沉默干扰体1基因在生长板(growth plate)和关节软骨中广泛表达,并...类端粒沉默干扰体1(disruptor of telomeric silencing 1-like,DOT1L)是一种组蛋白H3第79位赖氨酸(H3K79)甲基转移酶,参与基因转录的表观遗传调控。近来研究发现,类端粒沉默干扰体1基因在生长板(growth plate)和关节软骨中广泛表达,并参与维持关节软骨稳态。全基因组关联研究(genome-wide association studies,GWAS)结果显示,类端粒沉默干扰体1基因单核苷酸多态性(single nucleotide polymorphisms,SNPs)可增加欧洲人和中国汉族髋、膝关节骨关节炎(osteoarthritis,OA)患病风险。功能学实验证实,类端粒沉默干扰体1可下调去乙酰化酶沉默信息调节蛋白1(sirtuin-1,SIRT1)在关节软骨的表达,进而控制关节软骨内Wnt信号通路过度活化从而对关节产生保护作用;类端粒沉默干扰体1缺失可诱发小鼠和体外培养软骨细胞发生膝关节骨关节炎样改变。由此推断,调控类端粒沉默干扰体1-沉默信息调节蛋白1-Wnt信号通路网络可能是保护关节软骨、阻滞膝关节骨关节炎发生发展的有效措施。本文将在简述类端粒沉默干扰体1结构特点与活性的基础上,重点综述类端粒沉默干扰体1与膝关节骨关节炎发生发展之间的关系,以期为膝关节骨关节炎的诊治提供新靶点。展开更多
文摘急性白血病(AL)为克隆性恶性疾病,其主要特征表现为异常原始细胞大量增殖积聚,并不断对其他组织和器官进行浸润。研究证实,组蛋白对基因调控具有重要作用,现已发现的端粒沉默样阻断蛋白(DOT1L)是一种组蛋白,其缺乏Su(var)3-9,Enhancer-of-zeste and Trithorax(SET)结构域,具有赖氨酸甲基转移酶(H-Lys-M)活性,可定向性促进组蛋白H3第79位赖氨酸(H3K79)的快速甲基化,这一过程与AL的发生与发展密切相关。本文根据DOT1L结构特点与功能特点,综合论述DOT1L促进H3K79的过度甲基化在AL的发生、发展及预后中的作用,借以彰显DOT1L在AL靶位研究和靶向治疗中的意义与应用前景。
文摘目的明确甲基转移酶Dot1L是否参与成肌分化调控过程,并初步探索其调控成肌分化过程的分子机制。方法应用小鼠体外成肌分化模型C2C12细胞系,诱导C2C12细胞在0、1、3、5 d发生成肌分化,使用Dot1L特异性抑制剂抑制其甲基转移酶活性,并应用免疫荧光实验观察Dot1L对成肌分化过程的影响;检测成肌分化标志分子肌球蛋白重链(myosin heavy chain,MHC)和肌细胞调节因子(Myogenin)的蛋白质水平和转录水平及启动子区域的组蛋白甲基化水平;使用RNA-Seq基因组范围内检测抑制剂处理与否发生变化的基因阵列;并用CHIP-qPCR实验验证H3K79me2结合位点。结果Dot1L组蛋白底物H3K79的甲基化水平随成肌分化进程逐渐升高(P<0.01);抑制Dot1L酶活性后,成肌分化过程明显受阻;免疫荧光实验显示C2C12细胞多核肌管形成显著下降(P<0.01),MHC和Myogenin的蛋白质水平及转录水平均显著降低(P<0.01);启动子区域组蛋白H3K4甲基化水平降低(P<0.01);RNA-Seq显示受Dot1L调控的基因阵列主要集中在骨骼肌的成肌分化和肌肉的发育;经CHIP-qPCR验证参与成肌分化的部分基因存在H3K79me2结合位点。结论甲基转移酶Dot1L通过直接调控一群成肌分化相关基因的转录水平参与调控骨骼肌的成肌分化过程。
基金National Natural Science Foundation of China,31471206Basic Science Foundation of Science and Technology Commission of Shanghai Municipality,14JC1404000~~
文摘Objective· To investigate the histone methyltransferase capability of DOT1L-long form and its role in breast cancer metastasis. Methods· The existence of DOT1L-long form was confirmed by PCR, and the mRNA level of DOT1L was tested by real-time PCR. In HEK293T cells in which DOT1L canonical and DOT1L-long were overexpressed respectively, Western blotting was used to test the expression level of DOT1L and the histone methyltransferase capability. In the MCF10A cell line with inducible expression of DOT1L-long, real-time PCR was used to detect the mRNA level of epithelial-mesenchymal transition(EMT) marker, and transwell assay was used to detect the migration of breast cancer cells in which the expression level of DOT1L is low or high. Results· PCR demonstrated the existence of DOT1L-long form, and real-time PCR showed it widely exists in HCT116, T98G, MCF10A cells, etc. Western blotting showed the expression of DOT1L-long form and its H3K79 methyltransferase activity. In MCF10A cells in which overexpressed canonical DOT1L and DOT1L-long, mRNA levels of N-cadherin and fibronectine increased. Transwell showed canonical DOT1L and DOT1L-long both substantially increased the migration of breast cancer cells. Conclusion· The existence of DOT1L-long was confirmed and investigated, which is 202 amino acids longer than the canonical DOT1L, and is coded by a new exon, located between exon 27 and 28. Further, the DOT1L-long has H3K79 methyltransferase activity, and is able to promote breast cancer metastasis.
文摘Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, colorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.
文摘类端粒沉默干扰体1(disruptor of telomeric silencing 1-like,DOT1L)是一种组蛋白H3第79位赖氨酸(H3K79)甲基转移酶,参与基因转录的表观遗传调控。近来研究发现,类端粒沉默干扰体1基因在生长板(growth plate)和关节软骨中广泛表达,并参与维持关节软骨稳态。全基因组关联研究(genome-wide association studies,GWAS)结果显示,类端粒沉默干扰体1基因单核苷酸多态性(single nucleotide polymorphisms,SNPs)可增加欧洲人和中国汉族髋、膝关节骨关节炎(osteoarthritis,OA)患病风险。功能学实验证实,类端粒沉默干扰体1可下调去乙酰化酶沉默信息调节蛋白1(sirtuin-1,SIRT1)在关节软骨的表达,进而控制关节软骨内Wnt信号通路过度活化从而对关节产生保护作用;类端粒沉默干扰体1缺失可诱发小鼠和体外培养软骨细胞发生膝关节骨关节炎样改变。由此推断,调控类端粒沉默干扰体1-沉默信息调节蛋白1-Wnt信号通路网络可能是保护关节软骨、阻滞膝关节骨关节炎发生发展的有效措施。本文将在简述类端粒沉默干扰体1结构特点与活性的基础上,重点综述类端粒沉默干扰体1与膝关节骨关节炎发生发展之间的关系,以期为膝关节骨关节炎的诊治提供新靶点。
