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。     

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。     

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．     

CTCF和cohesin参与人成纤维细胞中HOXA基因簇染色质高级构象的组织

位于人体不同部位的成纤维细胞具有细胞特异性的HOX基因表达模式,可以作为区分不同成纤维细胞的依据之一.在个体发育的过程中,建立或维持不同HOX基因表达模式的机制始终是引人关注的问题.本实验室前期工作在NT2/D1人畸胎瘤细胞中证明了CTCF/cohesin介导的染色质高级构象在维甲酸诱导的HOXA基因共线性开启过程中发挥了重要作用.为了进一步研究原代细胞中CTCF/cohesin对HOXA基因的调控作用,本研究选取了来自体轴不同部位并且HOXA基因表达模式互补的人胚肺和包皮成纤维细胞,对HOXA基因簇中CTCF和cohesin的结合水平以及相关的染色质高级构象进行了检测.与人胚肺成纤维细胞相比,包皮成纤维细胞中的cohesin结合水平较低,相关的染色质高级构象比较"开放",并且主要表达5′端的HOXA基因.本研究还发现CTCF结合位点CBSA56处于HOXA基因簇染色质高级构象中的核心位置,并且该位点参与的染色质相互作用在两种成纤维细胞中呈现出明显的差异,说明CBSA56是一个关键的CTCF结合位点.以上结果表明,CTCF和cohesin参与了人原代成纤维细胞中HOXA基因簇染色质高级构象的组织和HOXA基因的表达调控,并且提示细胞类型特异性的染色质高级构象与HOXA基因的空间共线性表达模式之间存在协同关系.     

特异AT序列结合蛋白2(SATB2)的研究进展

特异AT序列结合蛋白2(SATB2)是与核基质结合区结合的转录因子,在转录调控和染色质重组等过程中发挥重要作用。SATB2是成骨细胞分化和骨基质形成过程中的关键调控分子,其突变可导致先天性颌面部畸形的发生;SATB2是中枢神经系统发育过程中的核心分子,在胼胝体、脑桥等正常发育中起重要作用;SATB2也参与肿瘤的发生,在乳腺癌等恶性肿瘤中的表达升高。文章就上述各领域中SATB2的研究进展进行了综述。     

染色质构象与基因功能

在真核细胞中,DNA序列以染色质为载体,高度凝缩并存储于细胞核内,其复制、修复和转录表达等过程受到染色质构象的精准调控。越来越多的研究表明,特定的染色质构象可选择性激活或沉默基因,从而控制细胞自我维持或定向分化,决定细胞的组织特异性和细胞命运。因此,对染色质构象的深入研究已成为准确解析基因功能的一个关键切入点,也是当前基因组学研究所面临的一个巨大挑战。本文对染色质构象的研究历史、结构特征、动态调控机制进行了综述,并重点论述了不同维度构象特征对基因转录调控的影响,对该领域的研究难点进行了讨论,展望了其未来的发展方向,期望通过有效梳理染色质构象与基因调控之间的脉络关系,为未来该领域的研究提供参考。     

DNA的核骨架结合序列（MARs）研究进展

核骨架结合序列(Matrix-associated Reg-ions,MARs)是存在于真核细胞染色质中的一段与核骨架特异性结合的DNA序列。它常常位于基因的侧翼区内,与一些调控因子相邻;序列具有A-T丰富的特点,而且常常含有拓扑异构酶Ⅱ的酶切位点。近年来的研究表明MARs参与了细胞的许多重要的生命活动,包括染色质的组装,基因的复制和表达。因此,MARs被认为是一种决定染色质结构性和功能性区域的新的顺式调控因子。本文对MARs的特点,生物学功能以及研究方法等方面的最新进展进行了阐述。     

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.     

用生物信息学方法寻找肝癌特异性表达基因转录调控模式

为了对肝癌(hepatocellularcarcinoma,HCC)的分子发病机理进行研究,首先对肝癌基因表达谱数据用t-检验算法进行了分析,找到了肝癌中特异性表达基因(characteristicgenes).然后把这些基因结合已知的肝HNF家族转录因子染色质免疫共沉淀结合DNA启动子芯片(ChIP-chip)实验数据用SAEM算法进行分析,得到了肝癌特异性表达基因的转录调控关系,并寻找到了多个HNF家族转录因子调控单基因的转录调控模式.结果表明HNF家族转录因子对大量具有重要功能的肝癌特异性表达基因进行了转录调控,并且多个HNF家族转录因子调控单基因可以形成前馈环和多输入调控等模式.     

染色质重塑在植物顶端分生组织中的调控作用

高等植物的生长发育依赖于顶端分生组织的维持和定向分化。顶端分生组织一方面需要维持自身的结构,同时又需要通过特定细胞的分化来起始各种器官的发育,这就需要通过精准的调控来决定各个细胞的命运。其中,染色质重塑就是一类广泛参与这些命运决定过程的调控方式。染色质重塑通过形成一系列重塑复合体对染色质修饰进行精确控制,影响各类转录调节因子与染色质结合的难易程度,进而使细胞感受各种信号和环境剌激,并在调控基因时空特异性表达与沉默方面发挥重要作用,形成一系列分子开关。现概述染色质重塑参与顶端分生组织调控的最新研究进展,梳理参与该调控的各类途径及重要调控因子,并展望该领域未来的研究方向。     

SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes

Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3’-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.     

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.     

The structural basis for the oligomerization of the N-terminal domain of SATB1

Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.     

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.     

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.