染色质免疫沉淀技术分析肺腺癌A549细胞中p53蛋白与p21^CIPI和Bim基因转录启动子的结合

为了检测肺腺癌A549细胞内抑癌蛋白p53与CDK抑制蛋白p21^CIPI和Bim基因转录调控区的结合情况,采用染色质免疫沉淀技术,用p53特异性抗体沉淀DNA,PCR检测p21^CIPI和Bim基因5’端特异性序列。结果表明,在抗体免疫沉淀的DNA片段中扩增出p21^CIPI和Bim基因5’端的特异性序列。因此证实在A549细胞内,p53蛋白可与p21^CIPI和Bim基因转录启动子的特异区域结合,进而参与两基因的表达调控。     

染色质免疫沉淀技术分析肺腺癌A549细胞中p53蛋白与p21CIP1和Bim基因转录启动子的结合

为了检测肺腺癌A549细胞内抑癌蛋白p53与CDK抑制蛋白p21CIP1和Bim基因转录调控区的结合情况,采用染色质免疫沉淀技术,用p53特异性抗体沉淀DNA,PCR检测p21CIP1和Bim基因5′端特异性序列.结果表明,在抗体免疫沉淀的DNA片段中扩增出p21CIP1和Bim基因5′端的特异性序列.因此证实在A549细胞内,p53蛋白可与p21CIP1和Bim基因转录启动子的特异区域结合,进而参与两基因的表达调控.     

体内甲醛交联及染色质免疫沉淀--研究DNA和蛋白质作用的新方法

甲醛交联及染色质免疫沉淀作用研究体内DNA和蛋白质相互作用的一种新方法,在染色质结构研究中获得了广泛的应用。该方法利用甲醛固定活细胞中的DNA与蛋白质,通过免疫沉淀分离复合物,从而分析蛋白质及其体内的DNA结合序列。     

同源盒基因A10编码蛋白的靶调节基因的筛选与鉴定

目的：筛选和鉴定同源盒基因A10编码蛋白（HOXA10）的靶调节基因。方法：以人子宫内膜细胞系AN3CA为实验对象,采用染色质免疫沉淀方法筛选HOXA10靶基因;采用平端克隆方法构建HOXA10靶基因库;采用DNA序列分析结合生物信息学方法鉴定HOXA10靶基因。结果：共获得含有HOXA10结合片段的克隆197个,选取插入片段大于100bp的质粒67个进行DNA序列分析,其中含有HOXA10结合序列TTAT的基因16个。结论：初步筛选出16个HOXA10候选靶基因,为进一步研究HOXA10的基因调节机理提供了新的思路。     

染色质免疫沉淀技术分析HePG2细胞TCF7L2蛋白与IDE基因转录启动子的结合

TCF7L2是一种重要的转录因子,通过Wnt信号途径,调节葡萄糖代谢.胰岛素降解酶(IDE)是细胞水平催化胰岛素降解的最关键的酶,与2型糖尿病(T2DM)高血糖、胰岛素抵抗、高胰岛素血症密切相关.为了检测HePG2细胞内转录因子TCF7L2与IDE基因启动子区的结合情况,采用染色质免疫沉淀技术结合PCR技术检测IDE基因启动子序列.结果表明,在特异性TCF7L2抗体免疫沉淀的DNA片段中扩增出IDE基因启动子序列,因此证实在HePG2细胞内,TCF7L2蛋白可与IDE基因转录启动子的特异区域结合,进而可能参与IDE基因的表达调控.     

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.     

染色质免疫沉淀技术——一种研究基因表达调控的工具

染色质结构在基因表达调节中起着重要的调节作用。利用甲醛固定活细胞中的DNA与蛋白质,通过免疫沉淀分离复合物的染色质免疫沉淀法是研究体内DNA和蛋白质相互作用的一种新方法,它不仅可用来研究体内反式因子与DNA的相互作用,也可以用来研究组蛋白修饰与基因表达的关系,从而分析蛋白质及其体内的DNA结合序列。目前,该方法在染色质结构研究中获得了广泛的应用。     

哺乳动物转录因子DNA结合谱

基因差异表达是生物发育和对刺激作出应答的分子基础,转录因子在这种基因差异表达中发挥着重要的调控作用。因此,要弄清楚转录因子调控基因差异表达的机理,就必须鉴定出它们全部的靶基因并构建其操纵的转录调控网络。对基因组DNA的序列特异性结合是转录因子调控基因转录的关键环节,因此,要鉴定转录因子的靶基因,就必须从它们与DNA相互作用的分子水平,鉴定它们能够识别并结合的全部DNA序列,即转录因子DNA结合谱。近年来随着DNA微阵列芯片和高通量DNA测序技术的产生和快速发展,出现了建立转录因子体内及体外DNA结合谱的一系列革命性的新技术,对该领域的研究带来重大影响。这些新技术主要包括建立转录因子体内DNA结合谱的染色质免疫沉淀-芯片技术(ChIP-chip)和染色质免疫沉淀-测序技术(ChIP-Seq),以及建立转录因子体外DNA结合谱的双链DNA微阵列芯片技术(dsDNA microarray)、指数富集配体系统进化-系列分析基因表达技术(SELEX-SAGE)、结合-n-测序技术(Bind-n-Seq)、多重大规模并行SELEX技术(MMP-SELEX)、凝胶迁移实验-测序技术(EMSA-Seq)和高通量测序-荧光配体互作图谱分析技术(HiTS-FLIP)。文章将对这些新技术做一综述。     

AP-1与STAT5相互作用协同调节血管紧张素原基因表达

为研究AP-1和STAT5协同调节血管紧张素原基因表达的作用机制,用凝胶电泳迁移率改变分析（EMSA）和染色质免疫沉淀分析（ChIP assay）检测血管紧张素Ⅱ（AngⅡ）诱导的AP-1和STAT5的DNA结合活性;用凝胶超迁移分析和免疫共沉淀方法观察AP-1和STAT5的相互作用. 结果显示,AngⅡ可分别促进AP-1和STAT5与血管紧张素原基因调控区顺式元件的结合以及二者之间的相互作用;核蛋白与含AP-1结合位点的寡核苷酸探针结合形成的复合物可与抗c-Jun抗体和抗STAT5抗体形成超迁移电泳区带;而用抗c-Jun抗体也可从STAT5 染色质免疫沉淀复合物洗脱液中检测到AP-1的存在. 而且,AP-1和STAT5的相互作用程度及其二者与DNA的结合活性与血管紧张素原表达活性具有一致关系,该效应可被JAK2特异抑制剂AG490所抑制. 上述结果提示,与顺式元件结合的AP-1和STAT5通过相互作用而形成四元复合物协同调节血管紧张素原基因的表达,JAK2对该过程具有诱导活化作用.     

血管生成素基因组结合序列的筛选与鉴定

血管生成素（angiogenin,ANG）调控细胞增殖、迁移、分化等生物学过程,但其作用的分子机制尚未完全明了. 目前认为,ANG可结合到rDNA区域促进rRNA转录,也可能与mRNA有结合.为全面鉴定细胞内可结合ANG的基因组序列,我们利用染色质免疫共沉淀结合DNA芯片技术（ChIP-chip）对HeLa细胞的基因组DNA进行了筛选,共获得了1 248个结合片段. 我们进一步分析了这些结合片段附近分布的基因,发现有699个可能受ANG结合调控的基因. 基因注释和聚类分析显示,这些可能受ANG调控的基因主要与肿瘤发生发展有关（特别是结直肠癌和前列腺癌）,并且与TGF-β和Wnt信号通路相关. 最后,我们验证了ANG不仅与WNT6、CCNE1、APC2、FZD8和EGFR基因的启动子区域有直接结合,而且调控其表达.以上研究结果为深入研究ANG的功能机制提供了线索.     

Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.     

慢病毒介导Nanog基因在小鼠ES细胞的表达

试验尝试构建小鼠Nanog基因慢病毒表达载体,培养表达外源Nanog基因的小鼠ES细胞。结果显示通过RT-PCR扩增出918bp的小鼠Nanog基因,测序正确的小鼠Nanog基因通过慢病毒介导在小鼠ES细胞表达后,表达外源Nanog基因的小鼠ES细胞生长状态同普通ES细胞无明显差异,在无LIF的ES细胞培养液培养条件下,表达外源Nanog基因的小鼠ES细胞保持正常的ES细胞集落,碱性磷酸酶、Oct4和SSEA-1免疫细胞化学检测为阳性,相同情况下未表达外源Nanog基因的小鼠ES细胞集落退化消失。试验证实了通过慢病毒载体介导培养了表达外源Nanog基因的小鼠ES细胞。试验尝试构建小鼠Nanog基因慢病毒表达载体,培养表达外源Nanog基因的小鼠ES细胞。根据小鼠Nanog基因m RNA序列设计Nanog基因引物,引物两端带有Nhe I和Xho I酶切位点。Trizol试剂处理小鼠ES细胞,通过RT-PCR扩增出小鼠Nanog基因,小鼠Nanog基因用Nhe I和Xho I酶切后连入pcDNA3.1载体中,PCR检测阳性的细菌克隆进行测序,测序正确的Nanog基因片段连接入PLL-IRES-Neo慢病毒表达载体中,包装含有Nanog基因的慢病毒感染小鼠ES细胞,在SNL细胞饲养层上G418筛选2周后,添加普通ES细胞培养液在普通小鼠胎儿成纤维细胞饲养层上培养。结果显示通过RT-PCR扩增出918 bp的小鼠Nanog基因,测序正确的小鼠Nanog基因通过慢病毒介导在小鼠ES细胞表达后,表达外源Nanog基因的小鼠ES细胞生长状态同普通ES细胞无明显差异,在无LIF的ES细胞培养液培养条件下,表达外源Nanog基因的小鼠ES细胞保持正常的ES细胞集落,碱性磷酸酶、Oct4和SSEA-1免疫细胞化学检测为阳性,相同情况下未表达外源Nanog基因的小鼠ES细胞集落退化消失。试验证实了通过慢病毒载体介导培养了表达外源Nanog基因的小鼠ES细胞。     

Quantitation of DNA fragmentation using fiberglass filters

Many types of physiological and toxicological cell killing are mediated by extensive DNA fragmentation. To date, most assays used to detect DNA fragmentation have relied on cumbersome techniques to separate intact chromatin from cleaved DNA. Here we describe a filtration assay for quantitation of DNA fragmentation. Fiberglass filtermats were used to separate intact chromatin from DNA fragments. Analysis of the separation showed that intact chromatin consistently remained on filters, while DNA fragments of all (random) sizes were consistently found in the filtrates. The assay was adaptable to different DNA detection procedures and generated results comparable to those obtained using established methods in standard model systems.     

Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

Histochemical properties of spermatozoa and somatic cells. III. Depolymerization and extraction of DNA during Feulgen acid.

The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.