SATB1在基因表达调控中作用的研究进展

SATB1是一种组织特异性的核基质结合蛋白,参与了染色质高级结构的形成和组织特异性基因的表达调控,对于胸腺细胞的发育和T细胞的成熟起到了尤为重要的作用。虽然已经知道SATB1可以通过与MAR序列结合,以促进染色质重塑,调节组蛋白乙酰化和甲基化水平等多种途径对基因的表达进行调控,但是对于该过程所涉及到的分子机制仍然不是很清楚。本文对SATB1在基因表达调控方面的研究进展作一综述。     

核基质结合区结合蛋白质1--SATB1

SATB1是一种核基质结合区(MAR)结合蛋白质,以独特的模式识别并结合于MAR。近年来发现,SATB1参与了染色体的高级包装和组织特异性基因表达的负调控,敲除了SATB1基因的小鼠胸腺细胞无法正常发育,在凋亡过程中SATB1先于基因组DNA发生降解。对SATB1参与髓系细胞的分化和基因调控等方面的研究仍在进行,一般认为SATB1是通过改变染色体的高级包装行使功能。     

EZH2在心脏与血管发育中的作用

Zeste基因增强子人类同源物2(enhancer of zeste homolog 2, EZH2)是多梳蛋白抑制复合物2(polycomb repressive complex 2, PRC2)的主要元件之一，利用组蛋白甲基化酶活性发挥经典作用，抑制靶基因的表达。此外，EZH2通过甲基化其他蛋白，作为蛋白支架分子募集转录相关分子介导转录激活，与lncRNA及miRNA相互作用等非经典途径调控各项生命活动，与干细胞分化和组织器官发育关系密切。EZH2及其功能相关分子在心脏发育、血管发生等过程中发挥着至关重要的作用。靶向敲除小鼠心脏Ezh2基因会影响心肌组织及内皮源性组织的正常发育，造成广泛性的心脏发育缺陷。EZH2参与调控正常组织和肿瘤组织的血管生成，维持新生血管完整性，并参与调控内皮间质化和内皮造血转化。本文探讨了EZH2在心脏和血管发生领域的影响效应、调控机制，及其与相关疾病的关系。     

抗逆相关AP2/EREBP转录因子研究进展

干旱、低温、土地盐碱化等非生物胁迫是影响植物生长发育以及作物产量的重要因素。近年来大量研究表明,多种转录因子参与调控植物对各种生物及非生物胁迫的应答与防御反应,与此同时人们对其作用机理的探索也日渐深入。AP2/ERF转录因子家族是植物所特有的一类转录因子,在拟南芥中该家族至少有146个成员;而在水稻中该基因家族多达181个,是已知水稻转录因子基因中最大的家族。这些编码含有一个保守APETALA(AP2)结构域的蛋白质可能在植物多个发育过程及应答外界环境信号过程中发挥重要功能。综述了AP2/EREBP类转录因子的结构特征及其功能特性,并重点讨论了它们在植物抗逆中的调控作用及其在植物抗逆性分子遗传改良上的意义。     

转录因子OCT-4对靶基因AP-2γ表达调控的研究

OCT-4和AP-2γ是近年来睾丸生殖细胞瘤诊断过程中研究的热点基因.作为一个在胚胎发育中起着重要作用的转录因子,OCT-4基因在发育的不同时期、不同分化过程中发挥着多种生物学功能,其作用是通过对靶基因的调控来实现的.本研究运用多种软件分析,发现AP-2γ启动子区域的序列部分有OCT-4的结合位点.利用CHIP-PCR方法鉴定AP-2γ是OCT-4调控的靶基因.应用Luciferase assay、免疫荧光实验、小鼠隐睾模型等实验进行验证,OCT-4基因抑制AP-2γ转录活性和影响其蛋白质水平的表达.这一新发现,有利于从分子水平上研究睾丸生殖细胞瘤的发生机制.     

从转录水平研究SATB1表达调控的分子机制

目的:研究SATB1 5'转录调控区及3'UTR的调控作用,以阐明SATB1在各肿瘤细胞系中的表达和调控机制.方法:采用半定量RT-PCR分析人乳腺癌细胞系BT549和MCF7、人肺癌细胞系NCI-H-446、QG56和SPC-A1中SATB1的转录水平.并利用Western Blot方法检测各肿瘤细胞系中蛋白表达水平.分别构建SATB1两个转录本5'上游序列驱动的报告基因载体.将栽体瞬时转染QG56及SPC-A1.构建含SATB1基因3'非翻译区(3'UTR)的报告载体,瞬时转染NCI-H446、QG56及SIC-A1.运用双通道荧光素酶报告系统检测荧光素酶活性.结果:在BT549、NCI-H-446、QG56和SPC-A1中检测到SATB1的转录本2,仅在BT549及QG56有转录本1表达;但在NCI-H-446、QG56及SIC-A1中,未检测到蛋白水平的表达.在QG56中,转录本1上游-638～+404序列段荧光素酶活性最高,而在SPC-A1中转录本2上游-1218～+48序列段的荧光素酶活性最高.运用生物信息学分析-638～+404和-1218～+48两个序列段的转录因子结合位点.在NCI-H-446中,含有SATB1 3'非翻译区(3'UTR)报告载体的荧光素酶活性显著低于PGL3 control的活性(P＜0.05).结论:在肺癌细胞中,SATB1的表达与细胞转移能力的高低无关.RT-PCR、荧光素酶活性及生物信息学分析结果的一致表明SATB1的两个转录本分别受其5'上游序列调控.在NCI-H-446中,SATB1的表达受其3'UTR的调控.     

MicroRNA调控机制及在脂肪形成中的作用

MicroRNA(miRNA)是近几年发现的一类通过转录后调控机制对基因进行调控的非编码的短链RNA,广泛存在于真核生物。miRNAs在个体时序性发育、细胞增殖分化和凋亡、器官发育、脂肪代谢等许多生物发育过程中起着重要作用,并与肿瘤等疾病发生发展密切相关。近年来对miRNA的研究证实,大量miRNA参与脂肪组织发育相关的许多生物学过程调控。主要涉及miRNA的生物合成、调控靶基因转录后表达的机制(如降解mRNA序列、阻断翻译起始、处理小体转位及翻译激活),及其在脂肪形成中的作用,以期为更好地理解miRNA在脂肪形成中的作用,深入研究脂肪形成的分子机制提供参考。     

Nodal/Smad2靶基因的系统鉴定及其在早期胚胎发育中的功能机制

Nodal/Smad2信号通路在脊椎动物胚胎中内胚层诱导及其背腹分化中发挥着主导作用,但是在胚胎早期发育中,Nodal/Smad2信号调控哪些靶基因表达,这些靶基因如何在Nodal/Smad2信号下游发挥作用,人们仍然所知甚少。以国家自然科学基金委员会"细胞编程与重编程的表观遗传机制"重大研究计划为依托,王强实验室在全基因组水平上对斑马鱼胚胎原肠早期Nodal/Smad2信号通路的靶基因进行了系统鉴定,并通过分析Smad2结合区域的其他转录因子保守的结合序列的出现频率,鉴定了一批潜在的Smad2的协同转录因子。研究发现,Nodal/Smad2的靶基因主要由转录因子、发育相关基因及重要信号通路的调控因子组成,其中F-actin捆绑蛋白Fascin1a和鸟核苷酸交换因子Net1分别通过调控受体内吞与Smad2转录活性反馈调控Nodal信号转导和中内胚层形成,而BPTF做为Smad2协同转录因子,通过调节核小体滑动来调控wnt8a表达,在中枢神经系统后部化过程中发挥重要作用。相关研究工作构建了Nodal/Smad2信号在斑马鱼中内胚层诱导及体轴建立中的分子网络,为理解脊椎动物早期胚胎发育过程中的基因表达调控机制提供了有意义的线索。     

Twist2的生物学功能及其分子机制

碱性螺旋-环-螺旋(Basic helix-loop-helix protein,bHLH)家族成员Twist2对间质细胞系的发生和发育起转录调节作用,经直接或间接机制发挥分子开关功能,从而激活或抑制靶基因。Twist2能直接结合DNA上 E-box保守序列,招募共激活物或抑制剂;能与E蛋白调节因子发生蛋白-蛋白相互作用,干扰激活或抑制功能。Twist2无义突变导致Setleis综合征。对Twist2的早期研究多集中在骨骼发育,随后在多种肿瘤中发现其有表达差异,研究表明Twist2在肿瘤的上皮-间质转化(Epithelial-mesenchymal transition,EMT)中发挥着重要作用。Twist2参与了多条通路的调控,其调控作用的发挥受到时空表达、磷酸化、二聚化和细胞定位的调节,在机体的正常发育、体内平衡和疾病发生机制中研究Twist2的作用显得尤为重要。文章对Twist2在成骨分化、肿瘤形成和EMT中的作用及其分子机制进行综述,以便帮助了解Twist2的生物学功能,为进一步在疾病的诊断、发展、以及治疗等方面的转化应用研究提供依据。     

SATB1 regulates β-like globin genes through matrix related nuclear relocation of the cluster

The nuclear location and relocation of genes play crucial regulatory roles in gene expression. SATB1, a MAR-binding protein, has been found to regulate β-like globin genes through chromatin remodeling. In this study, we generated K562 cells over-expressing wild-type or nuclear matrix targeting sequences (NMTS)-deficient SATB1 and found that like wild-type SATB1, NMTS-deficient SATB1 induces out loop of β-globin cluster from its chromosome territory (CT), while it is unable to associate the cluster with the nuclear matrix as wild-type SATB1 does and had no regulatory functions to the β-globin cluster. Besides, our data showed that the transacting factor occupancies and chromatin modifications at β-globin cluster were differentially affected by wild-type and NMTS-deficient SATB1. These results indicate that SATB1 regulates β-like globin genes at the nuclear level interlaced with chromatin and DNA level, and emphasize the nuclear matrix binding activity of SATB1 to its regulatory function.     

一种宏观基因调控分子SATB1

自身多聚化的SATB1(special AT-rich sequences binding protein 1)围绕异染色质形成笼状结构分布在细胞核中,SATB1不仅结合染色质DNA的核基质结合区(matrix attachment regions,MARs),也结合核基质,能够使DNA锚定在核基质并形成袢环状结构(loop)。SATB1的磷酸化、乙酰化和小泛素化样修饰可调节其DNA结合能力和细胞核内亚结构的定位;SATB1与多种蛋白质相互作用,能够募集染色质重塑复合物和组蛋白修饰酶,实现对其靶基因表达的时空特异性调控。SATB1在调节细胞分化、细胞凋亡、肿瘤生长与转移和X染色体失活等方面起到重要作用,并有可能成为肿瘤转移的治疗靶点。     

A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition.

A human cDNA was cloned that encodes a DNA-binding protein (SATB1) that is expressed predominantly in thymus and binds selectively to the nuclear matrix/scaffold-associating DNAs (MARs/SARs). Missing nucleoside experiments showed that SATB1 selectively binds in a special AT-rich sequence context where one strand consists of mixed A's, T's, and C's, excluding G's (ATC sequences). When this feature is destroyed by mutation, SATB1 binding is greatly reduced even if the direct contact sequence remains intact. Conjunctional SATB1-binding sequences become stably unpaired in supercoiled DNA. Specific mutations that diminish the unwinding potential greatly reduce SATB1 binding. However, SATB1 does not bind single-stranded DNA. Chemical interference assays show that SATB1 binds along the minor groove with very little contact with the bases. This suggests that SATB1 recognizes the ATC sequence indirectly through the altered sugar-phosphate backbone structure present in the double-stranded DNA.     

The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation.     

SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17bp DNA sequence in the third intron.     

特异AT序列结合蛋白-1促进胰岛素样生长因子 结合蛋白-2在K562细胞中的表达

探讨过表达特异AT序列结合蛋白-1 ( special AT-rich sequence binding protein ,SATB1)核基质结合区（MAR）结合蛋白对胰岛素样生长因子结合蛋白-2（IGFBP2）基因表 达的影响,并对其影响机制进行初步探索．首先用脂质体将SATB1的真核表达载体pcDNA3.1-SATB1转染至K562细胞,通过6周G418的筛选获得阳性克隆,RT-PCR、实时PCR及Western 印迹验证过表达情况,对阳性克隆细胞中IGFBP2的表达用RT-PCR、实时PCR及Western 印迹方法进行检测;然后用RNAi的方法干扰阳性细胞中SATB1 的表达后,同样用上述3种方法再次检测IGFBP2的表达状况;用生物信息学方法对IGFBP2基因进行MAR序列与SATB1结合位点搜索分析,寻找SATB1影响IGFBP2基因表达的机制．结果显示,在稳定转染的情况下,实验组K562-SATB1细胞与转染空载体pcDNA3.1的K562-3.1细胞和未转染细胞K562相比,IGFBP2 mRNA水平上调了近7倍,而蛋白水平变化不明显．RNA干扰后,IGFBP2的表达在mRNA水平也相应下调,蛋白水平的变化同样不明显．通过生物信息学分析发现,IGFBP2第1个内含子中可能存在2. 5 kb MAR样序列,且MAR样序列上存在多个SATB1的潜在结合位点．综上所述,过表达SATB1可以使K562细胞中IGFBP2 mRNA表达水平提高,而且其调控机制可能与SATB1直接和IGFBP2基因中的MAR样序列结合有关．