【作者】 李大力；

【导师】 吴秀山； 刘明耀；

【作者基本信息】 湖南师范大学 ， 遗传学， 2007， 博士

【摘要】 转录因子识别结合目的基因的调控序列是转录过程的第一步，也是基因表达调控的关键环节，受到顺式、反式作用元件严谨而有序的调节与控制。虽然基因转录产物都是由RNA聚合酶复合体直接合成的，但是每个基因的表达时间和空间模式都不尽相同。这种表达的特异性就是由于众多转录因子协调控制的结果。因此对于基因转录调控的研究一直是分子生物学的一个热点，同时也是研究的难点之一。本人在读博期间，研究了心脏肌肉发育相关基因Smyd1和性成熟相关基因KiSS1的转录调控机制，从分子水平揭示了它们的表达是如何被上游基因或细胞外信号所控制的，为体内研究提供了直接的分子证据；同时研究了与细胞周期蛋白结合的螺旋—环—螺旋转录因子GCIP在肌肉细胞分化中的功能，并参与了其它课题的研究工作，具体体现在以下几个方面：1．Smyd1基因促进肌肉发育并受SRF和Myogenin的直接调控Smyd1是一个包含有SET和MYND结构域并在心脏和肌肉特异性表达的基因，同时它作为一个修饰H3-K4残基甲基化的甲基转移酶激括下游基因转录。人类Smyd1基因在成体的心脏和骨骼肌表达。在C2C12细胞中过表达Smyd1基因促进肌肉分化和肌管形成，这是由于肌肉特异性转录因子的上调所致。在肌母细胞中过表达血清应答因子（SRF）和肌细胞生成素（Myogenin）会促使Smyd1的表达量升高。我们克隆了Smyd1基因的启动子并分析了在肌生成中可能控制Smyd1表达的DNA调控元件。Smyd1基因近端的启动子包含有一些参与肌母细胞分化的转录因子结合位点，这些因子包括Myogenin、SRF和myocyte enhancer factor 2（MEF2）等。EMSA和ChIP分析表明SRF和Myogenin分别结合Smyd1启动子区域的CArG和E-box元件，共转染实验表明SRF和Myogenin的结合位点对于激活启动子是必须的。综合以上研究表明Smyd1基因被肌肉分化的核心调节因子直接调控，并且具有促进肌肉分化的功能。2．GCIP／CCNDBP1与E47和MyoD形成功能性复合物调节肌肉分化骨骼肌的分化是一个高度有序的多步骤的过程，也被称为肌肉发生。这一过程包括肌肉特异性基因的表达、肌细胞脱离分裂周期进入分化程序以及最后融合形成多核的肌管直至形成成熟的肌纤维。肌肉发生由一类肌肉特异性的碱性螺旋—环—螺旋转录因子家族基因（如MyoD）以及广泛表达的E—box基因共同协同调控。我们的研究表明，最近发现的螺旋—环—螺旋亮氨酸富急蛋白GCIP／CCNDBP1调节肌肉特异性基因表达以及E47／MyoD异源二聚物的形成。在C2C12成肌肉细胞分化过程中，GCIP的mRNA和蛋白表达水平都有所提高。在C2C12中过表达GCIP能促进E47／MyoD蛋白复合物的形成，激活肌肉特异性下游基因表达并能促进肌管形成，而过表达GCIP的突变体则减弱E47／MyoD复合物形成。这些结果表明GCIP蛋白能够通过与E47／MyoD形成异源多聚物而促进肌肉分化。3．雌激素同受体α和Sp蛋白复合物调节KiSS1基因表达Kisspeptins是KiSS1基因编码的分泌型神经肽，后来被证明是G蛋白偶联受体-54（GPR54）的天然配体。刺激KiSS1／GPR54信号途径会激活下丘脑-垂体-性腺（HPG）轴并启动青春期发育。最近的证据表明在雌鼠中，雌激素（E₂）在前腹室旁核（AVPV）（一个重要的生殖内分泌下丘脑核区）激活KiSS1的表达，但是在弓状核（Arc）的作用却相反。虽然目前对KiSS1基因有了一些初步研究，但是对于E₂调控KiSS1表达的分子机制还知之甚少。我们证明在雌激素受体（ERα）阳性下丘脑GT1-7细胞中，加雌激素处理，KiSS1基因的表达水平增强。将KiSS1基因启动子偶联荧光素酶瞬时转染，在ERα存在时E₂增强启动子的活性。KiSS1启动子的缺失分析证明E₂调节启动子活性的作用依赖于启动子近端的Sp1位点。我们用凝胶阻滞（EMSA）实验证明Sp1和Sp3蛋白与启动子中多个保守的Sp1识别位点结合。有趣的是，Sp1转录激活KiSS1启动子活性，而Sp3则起转录抑制作用。Sp蛋白和ERα形成一个复合物，而E₂诱导的对KiSS1启动子的激活作用并不依赖于ERα的DNA结合结构域。染色质免疫沉淀（ChIP）分析证明Sp1和Sp3两者都结合KiSS1启动子上的GC富集序列，而ERα与KiSS1启动子的结合是依赖于E₂的激活，并随时间推移而结合增强。总之，这些结果证明了E₂通过ERα激活KiSS1基因的表达，而ERα则通过结合Sp1／Sp3蛋白并利用Sp1／Sp3识别结合KiSS1基因启动子上的GC富集区来刺激KiSS1基因转录。这有助于我们在分子水平理解类固醇激素对KiSS1的反馈调节作用。4．其它基因的研究证明在高转移性黑色素瘤细胞中，转移抑制基因KiSS1的表达受Sp1／DRIP130蛋白复合物的调控；证明GCIP基因在结肠癌中具有抑癌基因的特性，抑制结肠癌细胞系的生长；参与人类ZNF394、ZNF480、ZNF411、WDR26以及SNX-L等基因的克隆、表达和功能研究，这些基因多数在心脏中表达，将是心脏发育相关候选基因；通过对基因剔除小鼠的研究，证明GPR48／LGR4对小鼠骨骼以及眼睛发育有非常关键的作用。该基因的缺失将导致胚胎软骨终末发育迟缓、成骨细胞分化和矿化下降，成体骨密度，骨体积，骨形成率和类骨质都下降；导致眼前段发育不全，包括小眼、虹膜发育不全、角膜发育异常和白内障，在老年小鼠中还能检测到神经节细胞丧失、内核层变薄和早发型青光眼。更多还原

【Abstract】 The recognition and association of transcriptional factors tothe target regulatory element of downstream gene are the firstand pivotal step of gene transcription which is highlyregulated by both cis-and trans-acting factors. Although genetranscripts are synthesized by RNA polymerase, the special andtemporal expression pattern of each gene is distinct, which isdue to the combinatorial regulation of multipletranscriptional factors. During my doctoral thesis research,the projects including the studies of transcriptionalregulations of a cardiac and muscle specific gene Smyd1 and apuberty related gene KiSS1. These studies revealed how theexpressions of these two genes are regulated by upstream genesor extracellular signals on the molecular level, which are thedirect molecular evidences to support in vivo studies. At thesame time, functional studies of a HLH transcriptional factor,named GCIP, during myogenesis has been done as wellas some ofcollaborative researches.1. Smyd1 gene, the direct target of SRF and Myogenin, promotes myogenesisSmyd1 is a heart and muscle specific SET-MYND domaincontaining protein functioning as a methyltransferase whichmodifies H3-K4 residues and activates downstream genetranscription. In adult human tissues tested, Smyd1 isrestricted in heart and skeletal muscle. Over-expression ofSmyd1 promotes myoblasts differentiation and myotube formationin C2C12 cells due to up-regulation of muscle specifictranscriptional factors. The expression of Smyd1 is elevatedwhen serum response factor （SRF） and Myogenin is over-expressedin myoblasts respectively. We isolated the Smyd1 gene promoterand identified DNAregulatory elements that might control Smyd1expression during Myogenesis. The proximal promoter of theSmyd1 gene contained binding sites for several factors involvedin myoblasts differentiation including Myogenin, SRF, andmyocyte enhancer factor 2 （MEF2）. EMSA. and ChIP assaysdemonstrated that SRF and Myogenin bind to CArG and E-boxelements on Smyd1 promoter region respectively.Co-transfection experiments suggested that binding sites ofboth Myogenin and SRF are necessary for activation of thepromoter. Taken together, these studies indicate that Smyd1 isa key regulator of myogenic differentiation which acts as a downstream target of muscle regulatory factors.2. GCIP/CCNDBP1 regulates myogenic differentiation by forminga functional protein complex with E47 and MyoDDifferentiation of skeletal muscle is a highly orderedmulti-step process called myogenesis, which involves theexpression of muscle-specific genes, withdrawal of cell cycleand formation of multinucleated myotube. It is controlled bya family of muscle-specific basic helix-loop-helix （bHLH）transcription factors, such as MyoD, by heterodimerizationwith ubiquitous bHLH proteins, called E proteins, to activatemuscle genes by binding E-boxes （CANNTG）. In this study, weshow that GCIP/CCNDBP1, a recently identified HLH leucine-richprotein without a predicted basic DNAbinding region, regulatesmuscle specific gene expression and E47/MyoDheterodimerization. Both the mRNA and protein expressionlevels of GCIP were up-regulated during myogenicdifferentiation of C2C12 cells. Over-expression of GCIP inC2C12 cells promotes E47/MyoD complex association, activationof muscle specific transcription,and myotube formation duringskeletal muscle cell differentiation while the mutant form ofGCIP reduced the E47/MyoD heterodimerization and inefficient muscle differentiation. These findings identify a novelpro-myogenic role for the recently identified GCIP/CCNDBP1protein in forming a functional protein complex with MyoD/E47heterodimers that are essential for myogenesis.3. Estrogen regulates KiSS1 gene expression through estrogenreceptorαand SP protein complexesKisspeptins, secreting neuropeptides encoded by the KiSS1（KiSS-1） gene, are natural ligands of a newly identified Gprotein-coupled receptor-54, GPR54. Activation ofKiSS1/GPR54 signaling results in potent activation ofhypothalamus-pituitary-gonadal axis and initiates puberty.Recent data has shown that in female mice KiSS1 is positivelyregulated by estradiol （E₂） in the anteroventralperiventricular nucleus （AVPV）, an important reproductiveneuroendocrine brain region, but negatively regulated in thearcuate nucleus （Arc）. However, little is known about themolecular mechanisms governing E₂ modulated KiSS1 expression.Here, we demonstrate that the expression level of KiSS1 genewas upregulated with the administration of E₂ in estrogenreceptor alpha （ERα） positive hypothalamus GT1-7 cells. Usingtransient transfection of the human KiSS1 gene promoter coupled to luciferase, E₂ increases promoter activity in the presenceof ERα. Deletion analysis of the KiSS1 promoter indicatesthat the E₂-regulated increase in promoter activity dependedon Spl sites located on the proximal promoter region. Usingelectrophoretic mobility shift assays （EMSA）, we determinedthat both Spl and Sp3 proteins associate with the four putativeSp1 sites in vitro. Interestingly, Spl transactivates KISS1promoter activity whereas Sp3 functions as a transcriptionalrepressor. Sp1 and ERαform a complex in vivo and the DNAbinding domain of ERαis not necessary to mediate the E₂ inducedactivation of KiSS1 promoter. Chromatin immunoprecipitation（ChIP） analyses demonstrated that both Sp1 and Sp3constitutively bound to GC-rich motif on the KiSS/ promoterwhile the association of ERαwith the KiSS1 promoter isdependent on E₂ exposure. Together, these results demonstratethat E₂ dependent transcriptional activation of the KiSS1 geneis mediated by ERαthrough the interaction of Sp1/Sp3 proteinswith GC-rich motifs of KiSS1 promoter, providinga molecularnechanism in our understanding how steroid hormones feedbackregulate KISS1 expression in the HPG axis.4. Research on other genesThe expression of metastasis suppressor gene KiSS1 is regulated by Sp1/DRIP130 complex in highly metastatic melanoma cells.GCIP/CCNDBP1, a helix-loop-helix protein, suppressestumorigenesis. Identification and characterization of humannovel genes including ZNF394, ZNF480, ZNF411, WDR26 and SNX-L,some of which are expressed in the heart tissue. Through geneknockout method, GPR48/LGR4 has been identified as an orphanGPCR involved in bone and eye development.更多还原