三维基因组染色质构象捕获及其衍生技术

线性染色质经过多重折叠凝缩到真核生物的细胞核中,染色质的三维构象直接决定了真核生物的基因表达,因此染色质可以在局部或远程空间上发生互作调控基因转录。折叠成环状构象的染色质可以借助染色质构象捕获 (Chromosome conformation capture,3C) 技术来研究,基于3C技术扩展的4C/5C/Hi-C从单个位点延伸到全基因组捕捉三维构象,在此基础上,染色质构象核心技术可以与免疫共沉淀、核酸分子杂交、单细胞、基因组测序等技术偶联而产生新的衍生技术和应用,这极大地推动了染色质构象技术在基因时空特异性表达调控上的研究。文中将以3C和Hi-C等三维基因组核心技术为基础,重点介绍染色质构象捕获及其衍生技术的原理和前沿应用。     

利用基因捕获技术建立稳定表达HBV的细胞模型

pGEM-HBV1.3质粒经HindIII限制性内切酶消化,将HBV1.3全长DNA切下,与同样经HindIII限制性内切酶降解过的PU21连接,得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中,G418筛选阳性克隆并以X-gal染色,RT-PCR、Southern blot等方法验证HBV DNA的插入和表达。 PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接,该重组质粒转染HepG2细胞后经G418筛选,得到一系列阳性克隆, Southern blot证实HepG2细胞基因组中含HBV DNA,RT-PCR结果表明HBV DNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立,为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。     

下一代测序技术在表观遗传学研究中的重要应用及进展

下一代测序技术(Next generation sequencing,NGS)的出现,极大地促进了表观遗传学的研究。将NGS技术引入表观遗传学,便形成了以NGS为基础的各种表观遗传学测序及研究方法,如:全基因组亚硫酸氢盐测序法(Whole genome bisulfite sequencing,WGBS)、简化代表性亚硫酸氢盐测序法(Reduced representation bisulfite sequencing,RRBS)、甲基化DNA免疫共沉淀测序(Methylated DNA immunoprecipitationsequencing,MeDIP-seq)、染色质免疫共沉淀测序(Chromatin immunoprecipitation-sequencing,ChIP-seq)、Tet辅助重亚硫酸盐测序法(Tet-assisted bisulfite sequencing,TAB-seq)、各种染色体构象捕获测序(Chromosome conformation capture sequencing,3C-seq)技术、DnaseⅠ-seq/MNase-seq/FAIRE-seq以及RNA测序(RNA sequencing,RNA-seq)。这些方法的应用和普及改变了人们对多种表观遗传现象的传统认识,使研究人员能够更加全面地深入了解各种表观遗传标志在机体内的广泛分布,以及如何在外界因素的影响下发生相应的动态变化。文章概述了当今主要商业NGS平台的原理和特点,系统介绍了以NGS方法为基础衍生出来的各种表观遗传学测序及研究方法,并在此基础上对近年来应用NGS技术在表观遗传学研究领域中取得的最新研究成果进行了综述。     

利用水稻启动子捕获系筛选水稻胚发育相关基因的初步研究

通过对2227个启动子捕获系中报道基因β-葡萄糖苷酸酶(β-glucuronidase,gus)表达活性检测,筛选到7个水稻胚中表达GUS活性的启动子捕获系,对其中编号为W9154的捕获系作了进一步分析。Southern杂交分析表明W9154是一个单拷贝T-DNA插入系,其T-DNA插入在水稻基因组第3号染色体上一个未知蛋白基因的第2个内含子中。对该未知蛋白基因上游调控序列生物信息学分析显示,其调控序列除含有TATA-box和CAAT-box等常见启动子元件外,还含有RY基序、E盒、G盒及AACA等种子特异性启动子的特征性元件。表达特征分析发现,编码上述未知蛋白的基因在野生型水稻的胚和茎中表达,这与捕获系W9154中GUS表达活性完全一致。这些表明启动子捕获系w9154捕获的候选基因可能是一个水稻胚发育相关基因。     

利用PCR-SSCP对人MINK基因的研究

人类MINK基因的两种突变导致长QT综合征（LQTS）。我们设计了两对引物利用PCR－SSCP结合直接测序法在病人及健康者中对MINK基因进行分析，结果发现第149位密码子产生A→G转换。在所研究的对象中，此变化在病人及正常对照者中均有存在。提示MINK基因的此突变性质为非病理性的多态突变。     

利用染色质免疫共沉淀技术确定转录因子RIN调控的靶基因

以粉红期番茄果实为材料,用含不同浓度甲醛的缓冲液交联DNA和蛋白质,利用超声波将其染色质随机断裂成大小为200-1 000 bp的片段,用RIN蛋白的特异性抗体免疫沉淀与RIN蛋白结合的DNA片段,然后解交联和纯化DNA片段,最终用普通PCR试验和测序验证与转录因子RIN结合的DNA序列.结果表明,适用于番茄果实的最佳ChIP试验条件为:用1％甲醛溶液交联DNA和蛋白质的复合物;用20％功率,工作6s,间隔10s,脉冲3次超声破碎该复合物,可以得到适当大小的片段,用于后续的试验.普通PCR和测序验证结果证明转录因子RIN与LeACS2和LeACS4启动子区域的CArG box序列结合.     

蝗虫染色体分带技术的研究

本文对采自长沙岳麓山的中华稻蝗〔Oxya chinensis (Thunberg)〕和采自杭州的小稻蝗〔Oxya hyla intricata (Stal) 〕进行了染色体C带、R带、N带和A带的处理研究。结果表明,C带显示结构异染色质,其位置在不同分裂相中较为稳定;R带即G带的反带,带纹较C带丰富,在粗线期最为明显,本文观察到不同的温育条件可影响R带产生的效果;N带和Ag带可相互参照比较,从而为NOR的准确定位提供依据;A带即荧光带,本文对其显带程序作了一定的改进。 Abstract:In this paperOxya chinensis (Thunberg) collected from the Yuelu Mountains in Changsha, Hunan Province andOxya hyla intricata (Stal) collected from Hangzhou, Zhejiang province were analysed by chromosomal C-banding、R-banding、N-banding、Ag-banding and A-banding. The results showed that the constitutive heterochromatin was stained in C-banding, and it relatively stable; R-banding was the reverse of G-banding, its bands were relatively obvious at the pachytene and abundent, it may be because of the selective extracts that cause the bands evident, the different effects were surveyed under the various incubation conditions; Both N-banding and Ag-banding can stain the nucleolar organizer and the two were compared in this paper; The A-banding was a little improved in the light of the banding process.     

蝗虫染色体分带技术的研究

本文对采自长沙岳麓山的中华稻蝗〔Oxya chinensis (Thunberg)〕和采自杭州的小稻蝗〔Oxya hyla intricata (Stal) 〕进行了染色体C带、R带、N带和A带的处理研究。结果表明,C带显示结构异染色质,其位置在不同分裂相中较为稳定;R带即G带的反带,带纹较C带丰富,在粗线期最为明显,本文观察到不同的温育条件可影响R带产生的效果;N带和Ag带可相互参照比较,从而为NOR的准确定位提供依据;A带即荧光带,本文对其显带程序作了一定的改进。 Abstract:In this paperOxya chinensis (Thunberg) collected from the Yuelu Mountains in Changsha, Hunan Province andOxya hyla intricata (Stal) collected from Hangzhou, Zhejiang province were analysed by chromosomal C-banding、R-banding、N-banding、Ag-banding and A-banding. The results showed that the constitutive heterochromatin was stained in C-banding, and it relatively stable; R-banding was the reverse of G-banding, its bands were relatively obvious at the pachytene and abundent, it may be because of the selective extracts that cause the bands evident, the different effects were surveyed under the various incubation conditions; Both N-banding and Ag-banding can stain the nucleolar organizer and the two were compared in this paper; The A-banding was a little improved in the light of the banding process.     

基于高通量测序技术对染色体外环状DNA研究的最新进展

染色体外环状DNA (extrachromosomal circular DNA, eccDNA)是一种真核生物染色体外的闭合环状DNA结构,长度和染色体起源具有较高异质性。ecc DNA这一名称目前主要指大小在数百kb以内的小分子染色体外环状DNA,包括micro DNA、小多分散环状DNA (small polydispersed circular DNA, spcDNA)以及其他未分类的小分子eccDNA等。高通量测序技术(high-throughput sequencing, HTS)是一种可以同时对百万条DNA分子进行序列测定的技术,又名新一代测序技术(next generation sequencing, NGS),具有高通量、高灵敏度、高准确度等优势。近年来高通量测序结合生物信息学分析技术不仅在揭示eccDNA染色体起源、分子结构、发生机制和潜在功能以及循环系统中的eccDNA分子特征研究等方面发挥了重要作用,而且推动了eccDNA在甲基化等表观遗传学方面的研究。生物信息学软件的发展和eccDNA分析算法的开发也对其研究提供了重要帮助。血浆以及尿液等液体活检常用体液样本中...     

利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。     

染色质构象捕获及其衍生技术

染色质的构象在基因表达调节方面起重要作用．介绍了染色质构象捕获、环状染色质构象捕获、3C碳拷贝、ChIP-loop、ChIA-PET和Hi-C等技术的基本原理及发展历程,对影响实验结果准确性的主要因素进行了分析．目的是为在三维层面研究基因的表达调控介绍新的研究手段,为功能研究提供新思路,也为相关技术的应用提供理论参考．     

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.     

Heat Shock Factor 1 Drives Intergenic Association of Its Target Gene Loci upon Heat Shock

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The Zonal Organization of Odorant Receptor Gene Choice in the Main Olfactory Epithelium of the Mouse

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A Susceptibility Locus on Chromosome 13 Profoundly Impacts the Stability of Genomic Imprinting in Mouse Pluripotent Stem Cells

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Interaction in vivo between the Two Matrix Attachment Regions Flanking a Single Chromatin Loop

In interphase nuclei as in metaphase chromosomes, the genome is organized into topologically closed loop domains. Here, we have mapped the ends of the loop domain that contains the Ifng (interferon-γ) gene in primary and cultured murine T-lymphocytes. To determine whether the ends of the loop are located in close proximity to each other in the nuclear space, the 3C (chromosome conformation capture) technique, which detects protein-mediated DNA-DNA interactions, was utilized. A strong interaction was demonstrated between the two ends of the loop, which were close enough to become cross-linked in vivo in the presence of paraformaldehyde. Chromatin immunoprecipitation combined with the 3C technique demonstrated that topoisomerase IIα and MeCP2, but not topoisomerase IIβ, heterochromatin-associated protein HP1 or CTCF, were involved in this interaction. The present findings have important implications in terms of mechanisms of illegitimate recombination that can result in chromosomal translocations and deletions.