【作者】 李江夏；

【导师】 龚瑶琴；

【作者基本信息】 山东大学 ， 遗传学， 2009， 博士

【摘要】 第一部分人类LRR5基因的转录调控机制研究低密度脂蛋白受体相关蛋白5基因（low-density lipoprotein receptor related protein 5, LRP5）定位于人类染色体11q13,有23个外显子,编码一个属于低密度脂蛋白受体超家族的含有1615个氨基酸的单个跨膜受体蛋白。LRP5在成人和胚胎组织中广泛表达,其中肝脏表达最高。在骨骼组织中,LRP5主要在骨内膜和小梁骨表面的成骨细胞中表达,而不在破骨细胞中表达。LRP5在胚胎发育过程中调节多个发育过程,在个体成熟过程中也起着维持生理平衡作用。LRP5基因缺失功能突变导致骨质疏松,而获得功能突变导致骨密度升高,表明LRP5基因在骨形成中发挥着重要的作用,这种作用主要是通过Wnt信号传导通路来完成的。此外,LRP5在血管形成或再生以及正常的脂质代谢和糖代谢中也发挥重要作用。尽管对LRP5的功能进行了大量研究,但LRP5基因的表达调控机制目前仍不清楚。为此,我们对LRP5基因的表达调控机制进行了分析,取得了以下结果：1.确定了LRP5基因转录起始点：采用引物延伸方法,我们分析了LRP5基因转录起始点,发现人类LRP5基因的转录起始点位于翻译起始点上游114bp处,距离LRP5基因cDNA序列NM₀02335的5′末端40bp处。2.为寻找LRP5基因转录起始点上游顺式调控元件,我们采用MatInspector和AliBaba 2.1分析软件对LRP5基因翻译起始点上游2495bp片段进行了分析,发现该DNA片段缺乏经典的TATA盒和CAAT盒,但是含有多个可以与Sp1结合的GC盒,以及KLF15,CDE,MAZ,ZBPF等转录因子结合位点。利用荧光素酶报告基因系统,我们分析了该基因组片段在U20S细胞和HEK293细胞中的转录活性,发现该片段具有启动子活性。3.为了定位启动子核心区域,我们构建了系列截短载体pWT-2015, pWT-1594, pWT-1442, pWT-1309, pWT-1167, pWT-1085, pWT-953, pWT-359, pWT-219, pWT-167, pWT-72, pWT-53和pWT-40,瞬时转染U20S细胞和HEK293细胞并测定荧光素酶活性,结果表明载体在两种细胞中的转录活性趋势一致,提示这些区域内不含有组织特异性调控元件。对比分析不同截短载体表达活性发现人类LRP5基因的基本启动子位于-72至-53之间。4.软件分析发现在-72至-53之间存在一个KLF15识别结合位点和一个Spl识别结合位点,且两个位点部分重叠。为确定二者在LRP5基因转录调控中的作用,我们构建了两个位点单突变和双突变表达载体,荧光素酶活性分析表明KLF15结合位点和Sp1结合位点是决定LRP5基因基本启动子活性所必需的。5. EMSA分析发现,包含-72至-53区域的野生型探针能特异性地结合核蛋白,非标记的野生型探针能有效竞争标记探针与核蛋白形成复合物,而非标记的突变型探针则无明显竞争作用,说明该区域的KLF15和Sp1结合位点能特异性结合核蛋白。利用抗Sp1抗体和抗KLF15抗体进行ChIP分析,发现在两个抗体沉淀的染色质复合物中能检测到-72至-53区域DNA片段,进一步证实该区域能特异性与KLF15和Sp1结合。6.在缺乏KLF15和Sp1蛋白的Drosophila SL2细胞株中共转染pPac-KLF15或者pPac-Sp1表达载体,发现Sp1或KLF15增加LRP5基因启动子活性依赖于LRP5基因-72至-53bp区相应结合位点的存在。以上研究从Sp1和KLF15结合序列到转录因子以及转录因子与结合位点结合等方面证实LRP5基因-72bp-53bp区域内的Sp1和KLF15结合位点是人LRP5基因启动子功能的所需要的。第二部分KLF15调节内源性LRR5基因表达KLF15属于锌指蛋白转录因子家族,既具有转录激活作用,又具有转录抑制作用。本文第一部分利用报告基因分析方法发现,KLF15和Spl共同激活LRP5基因转录。为进一步证实KLF15在生理状态下也可调控LRP5基因转录,本部分以C2C12细胞株为研究对象,该细胞株属于小鼠骨骼肌成肌细胞系,具有多种分化潜能。可以在不同条件诱导下分化为肌管、脂肪细胞和成骨细胞。采用适时定量PCR和Western blotting分析不同诱导分化条件下KLF15表达情况及其对LRP5的转录调控作用,取得了以下结果：1.用含有2%马血清的DMEM培养基能诱导C2C12细胞分化为肌管,可以观察到细胞形态由典型的梭形和多角形的成纤维样细胞分化为呈平行排列走向,相互融合形成中央核的多核性长管状初生肌管；实时定量PCR检测到肌管的标志基因myogenin表达明显上调。在C2C12诱导分化为肌管过程中,KLF15的mRNA和蛋白质表达水平明显增高,LRP5 mRNA表达水平也明显增加。2.用5μM的罗格列酮生长培养基能诱导C2C12细胞分化为脂肪细胞,油红0染色可以观察到明显的脂肪细胞产生的脂滴；实时定量PCR检测到脂肪细胞标志基因aP2表达明显上调。在C2C12向脂肪细胞分化过程中,KLF15的mRNA和蛋白质表达水平明显增高,LRP5 mRNA表达水平也明显增加。3.用成骨培养基能诱导C2C12细胞分化为成骨细胞,茜素红染色可以观察到骨结节形成；实时定量PCR检测到成骨细胞标志基因ALP表达明显上调。在C2C12向成骨细胞分化过程中,KLF15的mRNA和蛋白质表达水平均明显增高,LRP5mRNA表达水平也明显增加。通过分析C2C12细胞诱导分化为肌管、脂肪细胞和成骨细胞过程中KLF15和LRP5的表达变化,进一步证实LRP5是KLF15转录因子的靶基因,KLF15可以激活LRP5基因转录。第三部分人类KLF15基因的转录调控机制研究如前所述,KLF15可以激活基因转录,也可以抑制某些基因转录。前两部分结果均显示KLF15可以在多种细胞分化过程中上调并激活LRP5基因转录。但是到目前为止,对KLF15的转录调控机制尚不明确。为此,本部分对KLF15基因的转录调控机制进行了分析。1.采用MatInspector和AliBaba 2.1网上软件对KLF15基因启动子区域序列进行分析,发现KLF15启动子缺乏经典的TATA盒,但是富含可以结合Sp1的GC盒子,同时也含有USF1、Smad 4、AP2、CREB、Smad3和NFY等识别结合位点。2.采用荧光素酶报告基因分析系统,我们构建了序列截短荧光素酶报告基因表达载体pGL3-1804, pGL3-1505, pGL3-863, pGL3-792, pGL3-514, pGL3-217,pGL3-189, pGL3-80, pGL3-45和pGL3-5,瞬时转染HEK293细胞后测定荧光素酶活性。分析这些表达载体荧光素酶表达活性发现人类KLF15基因的转录调控关键元件位于转录起始点上游-189至-80和-80至-45之间的两个区域。3.软件分析发现-189至-80区域内含有四个GC盒,分别命名为GC1、GC2、GC3和GC4。突变分析发现,GC2和GC3起主要作用,GCl位点作用较小,但在GC2或GC3突变后GC1可能发挥替代作用。4.在-80至-45之间存在一个E-box保守序列和一个CREB识别结合位点,且两个位点部分重叠。缺失和突变分析发现,E-box是维持人类KLF15基因启动子活性所必需的。利用RNA干扰CREB基因实验排除了该区域CREB识别结合位点的作用。5. EMSA分析证明-189至-155区域内的GC盒序列可以与核蛋白形成特异性DNA蛋白质复合物,未标记的野生型探针能有效竞争复合物形成,而未标记的突变型探针不影响复合物形成。ChIP分析也证实该区域序列能特异性与Spl蛋白结合。6. EMSA分析证明-80至-45区域内的E-BOX能与核蛋白结合形成特异性DNA蛋白质复合物,未标记的野生型探针能有效竞争复合物形成,而未标记突变探针不影响复合物形成。ChIP实验进一步证实该区域可以特异性与转录因子USF1结合。综上所述,本研究采用缺失和突变分析、EMSA、ChIP分析等方法证实USF1和SP1通过分别与KLF15基因启动子近侧-80至-45区域内的E-box和-189至-155区域的GC-box结合调节KLF15基因转录。更多还原

【Abstract】 Part 1. Regulation of human LRP5 gene transcriptionLRP5 (low-density lipoprotein receptor related protein 5), located on human chromosome 11ql3, contains 23 exons encoding a 1615 amino acid single-pass transmembrane receptor that belongs to the low density lipoprotein (LDL) receptor superfamily. LRP5 is expressed in multiple adult and embryonic tissues with strongest expression occurring in the live. In bone, it is expressed by osteoblasts of the endosteal and trabecular bone surfaces but not osteoclasts.LRP5 regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in maturity. Loss-of-function mutation in LRP5 can result in osteoporosis, and gain-of-function mutation leads to high bone mass (HBM), indicating that LRP5 plays an important role in bone formation. Such role is through the Wnt signaling pathway. In addition to these roles, the LRP5 is also required for normal cholesterol and glucose metabolism.Despite lots of effort has been done in study of function of LRP5, the molecular regulation of LRP5 expression remains unclear. Therefore, we studied the transcriptional regulation of LRP5 and got the following results:1. Determination of the transcriptional start site of LRP5 gene. Using primer extension, we identified the transcriptional start site of LRP5 gene, and found that the transcriptional start site is located at the 114bp upstream of the translational start site,40 nucleotides upstream of the 5’end of the published cDNA sequence (GenBank accession no. NM002335).2. In order to identify putative transcriptional factor binding sites, we analyzed a 2495bp region upstream of translational start site of LRP5 using the programs Matlnspector (www.genomatix.de/products/MatInspector/) and AliBaba 2.1 (www.gene-regulation.com/pub/programs.html), and failed to find any canonical TATA box or CAAT-like sequence. However, several potential transcription factor binding motifs were identified in the promoter region close to transcription start site, including Sp1, KLF15, CDE, MAZ, and ZBPF. Using luciferase reporter gene, we tested the transcriptional activity of this fragment in U2OS cells and HEK293 cells, and found this region has promoter activity.3. To determine the minimal region required for basal activity of the promoter, a series of deletion constructs were generated and designated as pWT-2015, pWT-1594, pWT-1442, pWT-1309, pWT-1167, pWT-1085, pWT-953, pWT-359, pWT-219, pWT-72, pWT-53 and pWT-40 based on their variable 5’end. These constructs were tested for their ability to drive the luciferase reporter gene in transiently transfected U2OS and HEK293 cells. Similar trends were observed in these two cell lines, suggesting that cell-specific element may not be present in those sequences. Comparing the expression activity of different deletion constructs, the proximal promoter has been located at-72 and-53.4. With software analysis, two overlapped transcriptional factor binding sites, one for KLF15 and the other for Sp1, were found in-72 to-53. To determine the contribution of the Spl and/or KLF15 sites to the promoter activity, constructs with either single or double mutant reporters were generated. The luciferase activity assay showed both KLF15 and Spl binding sites between-72 and-53 of human LRP5 promoter contributes to the basal activity of human LRP5 transcription.5. It is found in EMSA that wild type probe containing the region-72 to-53 can specificly binds to nuclear proteins. Unlabeled wild type probe can effectively abolish the complex of labeled probe and nuclear protein, while unlabeled mutant probe can’t, indicating that the KLF15 and Spl sites can specificly bind to nuclear proteins. In ChIP analysis using anti-Sp1 and anti-KLF15 antibodies, DNA fragment from-72 to-53 can be detected from co-immunoprecipitated chromatin complex. This result further confirmed this region can specificly bind to KLF15 and Sp1.6. By co-transfecting Drosophila SL2 cells lacking KLF15 and Spl proteins with pPac-KLF15 and pPac-Spl, it is found that the increase of LRP5 promoter activity by Spl or KLF15 relies on the existence of corresponding binding sites in-72 to-53.The foregoing study from different aspects, including the binding sites of Spl and KLF15, transcription factor and their binding, proved the Spl and KLF15 binding sites in-72 to-53 of LRP5 gene are essential for its promoter activity.Part 2. KLF15 regulates the transcription of endogenous LRP5Kruppel-like factors (KLFs), a subclass of the zinc-finger family of transcriptional regulators, can function as transcriptional activators and/or repressors depending on promoter context. In the first part, we demonstrated that both KLF15 and Sp1 functions as a positive regulatory element and activates the human LRP5 promoter. C2C12 cell line, a muscle satellite cell line, can differentiate into myotubes, adipocytes and osteoblasts under different condition. Using quantitative PCR and Western blotting, the expression of KLF15 and its regulation of transcription of LRP5 under different differentiating condition were detected, and the results are as following.1. C2C12 cell matained in DMEM medium with 2% horse serum was induced to the typical morphology of multinucleated myotube. Myotube-specific myogenin gene was dramaticlly upregulated during myogenic differentiation as determined by real-time PCR. During myogenic differentiation, both mRNA and protein of KLF15 were significantly increased. Consequently, LRP5 transcription was significantly upregulated.2. C2C12 cells matained in the adipogenic medium containg 5μM rosiglitazone became adipose-like morphology and contain lipid droplet which can be stained with Oil red O. The successful induction was confirmed by the significant upregulation of aP2 mRNA, an adipose-sepcific marker, during the adipogenic differentiation. The KLF15 expression was significantly upregulated at both mRNA and protein levels. LRP5 mRNA level was also increased during the adipogenic differentiation.3. C2C12 matained in osteogenic differentiation medium was induced to differentiate toward osteoblast. On day 24 after treatment, the mineralized nodules were detected by Alizarin red staining. The ALP mRNA, osteoblast-specific gene, was upregulated during the differentiation process. Both mRNA and protein levels of KLF15 gene were significantly induced during differentiation. Consequently, LRP5 mRNA was significantly upregulated.Taken together, inducing C2C12 cells to differentiate toward myogenis, adipogenis and osteogenis can alter the expression of KLF15 and LRP5. These further confirmed that LRP5 is the target gene of KLF15 and KLF15 can activate the transcription of LRP5 gene.Part 3. Regulation of human KLF15 gene transcriptionKLF15 can activate, as well as repress, gene transcription. The results of last two parts all showed KLF15 can be upregulated during cell differentiation and activate the transcription of LRP5 gene. However, the regulation of transcription of KLF15 is still unknown. Therefore, in this part we studied the regulation mechanism of KLF15 transcription.1. Using the programs MatInspector (www.genomatix.de/products/MatInspector/) and AliBaba 2.1 (www.gene-regulation.com/pub/programs.html) to analyze the promoter sequence of KLF15 gene, we failed to find any canonical TATA box. However, multiple potential transcription factor binding motifs were identified, including five GC-boxes that can bind to Spl, as well as USF1, CREB, Smad3, Smad4, AP2, and NYF sites.2. Serial deletion constructs with luciferase reporter gene, pGL3-1804, pGL3-1505, pGL3-863, pGL3-792, pGL3-514, pGL3-217, pGL3-189, pGL3-80, pGL3-45 and pGL3-5, were generated, and luciferase activity were measured after transient transfecting these constructs into HEK293 cells. According to the luciferase activity of these deletion constructs, it is found that the proximal transcription regulator is located in-189 to-80 and-80 to-45.3. Four GC boxes, designated as GC1, GC2, GC3 and GC4, were found within-189 to-80 by software analysis. Through site-direct mutagenesis, we found GC2 and GC3 play an important role in regulation of KLF15 transcription. The effect of GC1 is not so strong, but might become important after GC2 or GC3 mutated.4. There are two partially overlapping sites, one E-box consensus sequence and one CREB binding sits within-80 to-45. After deletion and mutation, E-box was found as an essential element that maintains the promoter activity of human KLF15 gene. The possibility of effect of CREB on promoter activity was excluded using RNA interference of CREB.5. EMSA results showed that GC boxs located in-189 to-155 can form specific DNA-protein complex with nuclear protein. Unlabeled wild type probe can effectively abolish this complex, whereas the unlabeled mutant probe can’t. ChIP results also confirmed that Spl protein can specificly bind to this region.6. Also with EMSA was E-Box found to be able to form specific DNA-protein complex. Unlabeled wild type probe can effectively abolish this complex, whereas the unlabeled mutant probe can’t. ChIP results also confirmed that transcription factor USF1 can specificly bind to this region.In summary, this study adopting different approaches, such as deletion and mutation analysis, EMSA, ChIP, to prove that USF1 and Spl can bind to E-box in-80 to-45 and GC-box in-189 to-155 in KLF15 promoter, respectively, thus regulate the transcription of KLF15 gene.更多还原