全反式维甲酸诱导小鼠碱性磷酸酶基因启动子区染色体重构*★

背景：碱性磷酸酶基因是成骨细胞分化和骨形成的重要标志。在C3H10T1/2细胞中，全反式维甲酸可通过核受体上调小鼠碱性磷酸酶的表达，与MAPK通路无关。 目的：从染色体结构调控方面揭示全反式维甲酸上调碱性磷酸酶表达的分子机制。 方法：10-6 mol/L全反式维甲酸处理C3H10T1/2细胞0，1，6，12 h，DNA酶Ⅰ超敏感实验确定全反式维甲酸调控区域的位置，染色质免疫共沉淀实验检验全反式维甲酸处理细胞后一系列转录相关因子与全反式维甲酸调控区域结合的量效关系以及时相分布。 结果与结论：DNA聚合酶Ⅰ超敏感实验表明，小鼠碱性磷酸酶启动子转录起始位点上游约520 bp附近为潜在的全反式维甲酸调控区域；染色质免疫共沉淀实验表明，全反式维甲酸对小鼠碱性磷酸酶的上调作用是通过一系列转录相关因子的时序性共同作用来实现。表明全反式维甲酸诱导小鼠碱性磷酸酶基因转录的过程中伴随着染色质重构和组蛋白的修饰作用。     

FHL1基因表达与先天性马蹄内翻足：15例患者及3名正常人的对比分析

背景：前期研究表明,在马蹄内翻足患者肌肉组织中存在FHL1基因表达下调,并利用凝胶阻滞实验在体外初步验证了HOXD13和FHL1基因启动子区转录因子预测位点的结合作用,但凝胶阻滞实验结果不一定能真实地反映体内转录调控蛋白和DNA结合的状况。 目的：应用Western-blot技术进一步验证先天性马蹄内翻足患者足部肌肉组织中FHL1,HOXD13基因蛋白质水平的表达,应用染色质免疫沉淀技术验证在胚胎足部发育时在体内HOXD13和预测结合位点的结合作用。 方法：15份先天性马蹄内翻足患儿肌肉组织标本由中国医科大学附属第二临床医院小儿外科提供,3份同年龄组正常儿童足部肌肉组织由中国医科大学法医学院提供,1例孕13周流产胚胎由中国医科大学附属第二临床医院妇产科提供。所有标本使用均经患者及其家属知情并同意。应用Western-blot方法检测15例先天性马蹄内翻足患儿及3例同年龄组正常儿童足部肌肉组织HOXD13和FHL1表达情况;并用软件预测FHL1基因上游HOXD13的结合位点,染色质免疫沉淀实验验证胚胎发育时HOXD13和FHL1的相互作用,以不表达HOXD13蛋白的脑组织作为对照。 结果与结论：与同期正常儿童足部肌肉组织相比,15例人先天性马蹄内翻足患儿肌肉组织中有7例存在FHL1基因蛋白质水平表达下调,而这7例患儿中有5例同时存在HOXD13基因蛋白质表达下调。沉淀的人胚胎足部组织染色质中有预测HOXD13结合位点的扩增,无对照位点的扩增,对照脑组织中无预测位点的扩增。结果进一步验证显示了先天性马蹄内翻足患儿足部肌肉组织中FHL1、HOXD13基因表达下调;人类胚胎肢体发育过程中HOXD13蛋白可以和FHL1启动子区的预测位点结合发挥其转录调节作用。提示在人胚胎足部发育时,HOXD13表达下调导致了FHL1表达水平的下调,进而影响了足部肌肉生长发育和分化导致马蹄内翻足畸形的发生。     

REST和CoREST在神经细胞分化发育过程中的调节作用

正神经元限制性沉默因子(neuron-restrictive silencer factor,NRSF),是一种重要的锌指蛋白转录负调控因子,它与某些基因中相应的神经元限制性沉默元件(neuron restrictive silencer element,NRSE),又称RE-1(repressor element 1,RE-1)相结合,从而对许多与神经元发育及功能相关的基因的表达发挥阻遏作用。REST辅助抑制因子(REST corepressor,CoREST)是一种神经元表型调控因子,CoREST与REST的C端结构域     

siRNA沉默CCN5抑制人胶质母细胞瘤细胞的生长

目的研究小干扰RNA(siRNA)沉默高半胱氨酸蛋白61/结缔组织生长因子/肾母细胞瘤过度表达基因5(CCN5)对人胶质母细胞瘤细胞系生长的影响。方法 Western blot方法检测胶质母细胞瘤患者脑组织CNN5蛋白水平的表达;合成CCN5 siRNA小片段,并,筛选最佳干扰片段;siRNA沉默CCN5在胶质母细胞瘤细胞系中的表达,噻唑兰(MTT)方法检测细胞的生长;CCN5沉默表达后,检测半胱氨酸的天冬氨酸蛋白水解酶3(caspase-3)的酶活性。结果胶质母细胞瘤患者脑组织中CCN5蛋白表达水平较正常人脑组织中高;siRNA沉默CCN5表达抑制细胞的生长并促进凋亡效应基因caspase-3活性升高。结论 CCN5基因沉默抑制人胶质母细胞瘤细胞系的生长,为探讨CCN5在胶质母细胞瘤细胞中的作用奠定基础,并为胶质母细胞瘤的基因治疗提供潜在的靶标基因。     

RLM-RACE法确定胰岛素降解酶基因的转录起始位点

目的精确定位胰岛素降解酶基因的转录起始位点,为今后研究该基因启动子区的转录调控及其参与疾病的发病机制奠定基础。方法采用5'cDNA末端快速扩增技术,得到多个胰岛素降解酶基因cDNA 5'末端序列,连接至PUC19 T载体中,鉴定阳性克隆并测序。结果根据两次实验结果,确认胰岛素降解酶基因的5'UTR序列长度为32bp。结论胰岛素降解酶基因存在单一的转录起始位点,位于翻译起始点上游32bp处,碱基为G。     

Dravet综合征患者电压依赖性钠通道α1亚基基因5'-非翻译区外显子的遗传变异

目的 筛查Dravet综合征患者的电压依赖性钠通道α1亚基(voltage-gated sodium channel α1-subunit,SCN1A)基因5'-非翻译区外显子突变位点,分析并预测其致病易患性.方法 收集24例Dravet综合征患者的外周血,抽提基因组DNA,采用直接测序法进行SCN1A基因5'-非翻译区外显子突变位点的筛查;用生物信息学方法分析SCN1A基因5'-非翻译区外显子变异位点邻近序列的保守性及潜在的转录因子结合元件,推测其致病易患性.结果 发现位于外显子h2u上的突变位点166.642.520G>A,先证者1为新生突变,而先证者2的突变来自临床表型正常的母亲,该突变位点在100名健康对照者中均未发现.突变位点在哺乳动物中呈中度保守(62.5%),人与其他哺乳动物之间在突变位点邻近序列的平均同源率高达88.5%;166.642.520野生型位点的序列上预测得到一种转录因子结合元件,而突变型位点的序列上预测得到两种转录因子结合元件.结论 突变位点166.642.520G>A与Dravet综合征存在一定程度的相关性,其致病机制有待于进一步实验证实.     

Pumilio2在中枢神经系统的作用

Pumilio2（Pum2）是近年新发现的一种转录后调控因子,与微小RNA功能相似,通过其特定的结构域与m RNA结合以阻断翻译起始复合物的形成、抑制靶基因的表达。近年研究表明,Pum2与中枢神经系统的形态发生和功能执行密切相关,其表达变化参与中枢神经系统疾病的生物学进程。本文拟就Pum2在中枢神经系统作用的研究进展简要概述。     

C3H10T 1/2小鼠胚胎成纤维细胞Dlxin 1基因表达及骨形态发生蛋白2的调控机制

背景：Dlxin 1可与成骨相关转录因子Dlx、Msx家族成员相互作用调节其转录活性,骨形态发生蛋白2可诱导小鼠胚胎成纤维细胞(C3H10T 1/2)向成骨细胞分化。 目的：对C3H10T 1/2小鼠胚胎成纤维细胞中Dlxin 1基因的表达及骨形态发生蛋白2调控机制进行初探。 设计、时间及地点：细胞分子水平实验,于2007-03/10在暨南大学生物工程研究所实验室完成。 材料：C3H10T 1/2细胞为ATCC产品,骨形态发生蛋白2为自产,DH5α为自备。 方法：用荧光定量PCR(qRT-PCR)检测骨形态发生蛋白2对基因表达的影响,用双荧光报告载体系统搜寻Dlxin 1的上游启动子元件,用凝胶迁移实验(EMSA)对其精确定位。 主要观察指标：①细胞中Dlxin 1基因mRNA的表达情况。②pGL3-Dlxin 1系列缺失突变报告载体荧光活性及启动子片段与C3H10T 1/2细胞核蛋白的结合能力检测。 结果：在转录水平上骨形态发生蛋白2可以诱导Dlxin 1基因表达上调;Dlxin 1启动子基础活性和骨形态发生蛋白2诱导活性调控区域均位于转录起始位点上游-943 ~-728 bp之间;EMSA证实,-758 ~ -748 bp这一片段是调控核心区域。 结论：转录因子通过与Dlxin 1启动子上游TATA box结合来调控Dlxin 1基因的转录。     

小分子干扰RNA介导RhoA沉默优化骨髓间充质干细胞的培养

以往骨髓间充质干细胞的培养方法存在衰老和分化率低等问题。 目的：检测是否可以通过沉默RhoA基因的方法优化骨髓间充质干细胞培养。 方法：体外培养SD大鼠骨髓间充质干细胞，经小分子干扰RNA转染以沉默RhoA基因表达，分为3组培养：干细胞组(未转染小分子干扰RNA)、经随机打乱顺序的小分子干扰RNA 转染干细胞组、经小分子干扰RNA 转染的干细胞组。用RT-PCR，Western blot检测骨髓间充质干细胞在转染前后RhoA基因和蛋白的表达。应用细胞生长曲线、MTT比色法观察细胞生长的优化作用，采用流式细胞术测定细胞周期分布的变化。 结果与结论：与干细胞组、经随机打乱顺序的小分子干扰RNA 转染干细胞组比较，经小分子干扰RNA 转染的干细胞组RhoA基因和蛋白表达量明显降低(P < 0.05)，细胞的生长速度明显增快，细胞周期G0/G1期减少，S期细胞数增多(P < 0.05)。说明通过沉默RhoA基因的方法可以促进骨髓间充质干细胞增殖，优化培养方法。     

Deregulated Chromatin Remodeling in the Pathobiology of Brain Tumors

Brain tumors encompass a heterogeneous group of malignant tumors with variable histopathology, aggressiveness, clinical outcome and prognosis. Current gene expression profiling studies indicate interplay of genetic and epigenetic alterations in their pathobiology. A central molecular event underlying epigenetics is the alteration of chromatin structure by post-translational modifications of DNA and histones as well as nucleosome repositioning. Dynamic remodeling of the fundamental nucleosomal structure of chromatin or covalent histone marks located in core histones regulate main cellular processes including DNA methylation, replication, DNA-damage repair as well as gene expression. Deregulation of these processes has been linked to tumor suppressor gene silencing, cancer initiation and progression. The reversible nature of deregulated chromatin structure by DNA methylation and histone deacetylation inhibitors, leading to re-expression of tumor suppressor genes, makes chromatin-remodeling pathways as promising therapeutic targets. In fact, a considerable number of these inhibitors are being tested today either alone or in combination with other agents or conventional treatments in the management of brain tumors with considerable success. In this review, we focus on the mechanisms underpinning deregulated chromatin remodeling in brain tumors, discuss their potential clinical implications and highlight the advances toward new therapeutic strategies.     

SATB2 interacts with chromatin-remodeling molecules in differentiating cortical neurons

During our search for developmental regulators of neuronal differentiation, we identified special AT-rich sequence-binding protein (SATB)2 that is specifically expressed in the developing rat neocortex and binds to AT-rich DNA elements. Here we investigated whether the regulatory function of SATB2 involves chromatin remodeling at the AT-rich DNA site. In-vitro and in-vivo assays using a DNA affinity pre-incubation specificity test of recognition and chromatin immunoprecipitation showed that SATB2 specifically binds to histone deacetylase 1 and metastasis-associated protein 2, members of the nucleosome-remodeling and histone deacetylase complex. Double immunohistochemistry showed that, in the developing rat neocortex, SATB2 is coexpressed with both proteins. Using a cell culture model, we showed that trichostatin A treatment, which blocks the activities of histone deacetylases, reverses the AT-rich dsDNA-dependent repressor effect of SATB2. These findings suggested that the molecular regulatory function of SATB2 involves modification of the chromatin structure. Semi-quantitative chromatin immunoprecipitation analysis of cortices from SATB2 mutant and wild-type animals indicated that, in the knock-out brains, SATB2 is replaced in the chromatin-remodeling complex by AU-rich element RNA binding protein 1, another AT-rich DNA binding protein also expressed in differentiating cortical neurons. These results suggested that an altered chromatin structure, due to the presence of different AT-rich DNA binding proteins in the chromatin-remodeling complex, may contribute to the developmental abnormalities observed in the SATB2 mutant animals. These findings also raised the interesting possibility that SATB2, along with other AT-rich DNA binding proteins, is involved in mediating epigenetic influences during cortical development.     

Chromatin, DNA methylation and neuron gene regulation--the purpose of the package

The accessibility of cognate binding sites within a gene promoter can be modified by the condensation or relaxation of local chromatin structure. Local chromatin structure is in turn programmed by covalent modifications of cytosine bases in DNA and amino acid residues in histone protein tails. These chemical and physical adaptations around gene promoters can significantly change levels of mRNA expression. Furthermore, linear patterns of covalent modification of histone protein tails are emerging as a distinct regulatory code--another form of cellular memory. Because chromatin structure can be modified by conventional pharmacologic therapy, a novel approach to the regulation of neuronal gene expression in clinical populations is possible.     

Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene

Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction.