人类SCAN-nf蛋白家族基因的发现及其功能研究

【作者】 罗坤甜；

【导师】 余龙；

【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士

【摘要】 锌指蛋白是真核生物基因组中最丰富的蛋白之一，其功能多样，在DNA识别、RNA包装、转录激活、蛋白折叠和装配、细胞凋亡调控以及脂类结合中均发挥着重要的作用。C₂H₂型锌指蛋白则是其中最常见的一种类型，是一类重要的调控基因表达的转录因子。它含有数目不等的串联锌指基序，其氨基端常有各种极其保守的结构域，如POZ、KRAB和SCAN结构域。 其中，SCAN结构域是一个高度保守的区域，并且特异的存在于脊椎动物中。众多含有SCAN结构域的C₂H₂型锌指蛋白组成了一个C₂H₂型锌指蛋白的亚家族。虽然已有一些研究表明某些该家族的蛋白能够调控一些重要的蛋白转录、表达，例如：生长因子、脂类代谢过程中的关键基因以及其他一些与细胞存活和分化相关的基因，但是大部分该家族成员的功能尚不明了。 在本项研究中，我们使用生物信息学与实验相结合的方法鉴定了人类基因组中属于SCAN家族的54个成员，分析了它们在染色体上的定位和分布情况以及各个基因的结构。这些SCAN家族的基因在染色体上成簇分布，如3p21，6p21.3，7q22，15q25，16p13.3，17p11.2，18q12，19q13.4，以及Xq26等位点。在研究过程中我们通过生物信息学方法发现SCAN家族的基因具有多种形式的可变剪接本，其中一类剪接方式广泛存在于该家族的20个成员之中，在这一类剪接方式中这些基因产生的可变剪接本缺失了原有的锌指结构，我们称之为nf（no zinc finger）剪接本，而对于原来含有锌指结构的剪接本我们称之为fu（full length with zinc finger）剪接本，并且我们通过实验验证了其中14对可变剪接本的存在。 为了分析这种可变剪接本存在的意义，我们克隆了该家族部分成员的fu与nf剪接本，使用免疫共沉淀、GST-pull down等实验分析了它们各个基因自身可变剪接本之间的相互作用情况，以及不同基因的可变剪接本之间的相互作用情况。实验结果表明fu剪接本与nf剪接本之间能够发生相互作用，并且我们用片段缺失的实验证实了它们之间相互作用是通过SCAN结构域来实现的。对于作为转录因子的锌指蛋白而言，它们需要通过羧基端的锌指结构与特异识别的DNA相结合，才能发挥其转录调控作用，但同时它们的氨基端所含有的SCAN结构域又能介导蛋白与蛋白之间的相互作用。在这样的情况下，缺失锌指区但仍含有SCAN结构域的可变剪接本如果能通过SCAN结构域与含有锌指结构的剪接本相作用，那么这些多样的可变剪接本的产生很可能大大增加了转录调控的多样性与复杂性。而转录因子发挥作用需要进入到细胞核中，因此，我们进一步分析了这些基因的不同剪接本在细胞内定位的情况，结果显示我们所检测的5个成员的nf剪接本都能被相应的fu剪接本带入细胞核内，并且这种作用是通过SCAN结构域所介导的。 随后，我们用双荧光素酶报告系统进一步分析了这些基因的不同剪接本在转录调控方面的作用。实验结果显示ZNF191fu、ZNF434fu具有显著的转录抑制活性，ZNF396fu则具有较弱的转录抑制作用，而ZNF397fu、ZNF447fu的转录调控作用则不显著。而这些成员的nf剪接本与它们对应的fu剪接本的转录调控功能有所不同，其中ZNF191nf与ZNF434nf剪接本的转录调控活性相对它们的fu剪接本显著降低，呈现较弱的转录抑制作用，但ZNF447nf剪接本却呈现出显著的转录激活作用，同时ZNF397nf剪接本则表现出显著的转录抑制作用，ZNF396nf剪接本的转录调控作用与其fu剪接本相当。nf剪接本这种与fu剪接本不尽相同的转录调控活性以及能与fu剪接本互作的能力，大大增加了转录调控作用的复杂性，这使得通过少量的转录因子作用来实现众多基因时空表达的不同成为可能。 为了进一步了解这种可变剪接本之间相互作用的生物学意义，我们还以ZNF191与ZNF434为例，分析其对信号通路的影响，并根据结果分别以HSE与AP1（PMA）信号通路的激活程度为检测指标，进一步分析了nf剪接本对fu剪接本功能的可能影响，结果显示ZNF191与ZNF434的nf剪接本均呈现出对fu剪接本功能的抑制作用。 在研究SCAN家族的过程中，为了能够深入了解其成员功能，我们还对该家族的一个成员ZNF445进行了深入的研究。该基因全长9105bp，编码1031个氨基酸，含有8个外显子，定位于染色体3p21.32。其编码的蛋白分子量约为119KDa，在氨基端含有一个SCAN结构域与一个KRAB结构域，在羧基端含有14个C₂H₂型锌指结构。人体16种组织表达谱分析显示ZNF445在睾丸、骨骼肌、子宫、胸腺、小肠、结肠、胰腺以及外周血白细胞中有着不同程度的表达。我们对ZNF445进行转录调控活性检测发现该蛋白是一个转录抑制因子。细胞内定位分析显示ZNF445定位于细胞核内，这种定位支持了其转录功能的发挥。在分析ZNF445对信号通路的影响时我们发现ZNF445能够显著激活AP1与SRE信号通路，由于AP1与SRE是MAPK信号通路的重要下游效应分子，因此我们进一步分析了ZNF445对AP1、SRE的激活作用是否是通过MAPK通路作用来实现的。我们首先使用了MEK的抑制剂PD98059进行检测，结果发现ZNF445对AP1与SRE的激活作用能够被该抑制剂所抑制，这表明ZNF445通过MAPK信号通路作用于AP1与SRE。随后我们进一步分析了ZNF445对MAPKN路的可能影响，我们发现转染ZNF445的细胞中Raf-1／MEK／ERK／Elk-1四种蛋白的磷酸化水平与阴性对照组相比有明显的增强，随后我们通过浓度梯度实验进一步分析了这种增强磷酸化水平的作用。在此基础上，我们构建了ZNF445片段缺失的载体，寻找对MAPK通路产生影响的主要结构域。结果显示在ZNF445三个功能区——SCAN、KRAB与锌指结构中，SCAN结构域对AP1以及SRE通路的激活作用最为显著。因此我们进一步分析了SCAN结构域对MAPK通路的影响，结果显示ZNF445的SCAN结构域能够增强Raf-1／MEK／ERK／Elk-1这四种蛋白的磷酸化水平。此外我们通过克隆形成率实验初步分析了ZNF445在细胞生长、增殖方面的作用，结果显示ZNF445能够促进L02细胞的克隆形成。 综上，我们在研究人类SCAN家族的过程中，发现了一类该家族成员的可变剪接本nf。这类剪接本不具有锌指结构，但能够通过氨基端的SCAN结构域与含有锌指结构的fu剪接本相互作用，并且能够通过这种相互作用影响fu剪接本的生物学功能，这一发现对于进一步了解锌指蛋白转录因子调控作用的发挥有着重要意义，很可能是有限的转录因子能够调控众多的基因时空表达这一现象的重要解释之一。在对该家族成员ZNF445的深入研究中，我们证实了该蛋白对MAPK通路的重要影响，以及这种影响的生物学意义，并且我们发现这种对MAPK通路的作用主要与其SCAN结构域相关。这是对于SCAN家族成员功能研究的一个重要补充，对于细胞生长、增殖这一生物现象的研究具有重要的学术价值。更多还原

【Abstract】 Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse including DNA recognition, RNA packaging, transcriptional activation, protein folding and assembly, regulation of apoptosis, and lipid binding. The C₂H₂ type, which is a kind of important transcriptional factors, is the most common one in this family. There are some tandem zinc-finger motifs with varied numbers in C₂H₂ zinc-finger proteins. The N-terminus usually has some highly conserved domains, such as POZ, KRAB and SCAN domains.Among them, the SCAN domain is a highly conserver motif that is vertebrate-specific. The SCAN-ZFPs constitute a subfamily of C2H2 zinc finger proteins. Although some genes of the SCAN family have been implicated in the transcriptional regulation of growth factors, genes involved in lipid metabolism, as well as other genes involved in cell survival and differentiation, the function of most members of the SCAN family is unknown.In this study, we utilize a bioinformatics approach with experiments to define the structures and gene locations of the 54 members of the human SCAN family. The genes encoding SCAN domains are clustered in the human genomes, such as 3p21, 6p21.3, 7q22, 15q25, 16p13.3, 17p11.2, 18q12, 19q13.4, and Xq26. We also found that many genes of the SCAN family were capable of generating isoforms. And one type of the isoforms doesn’t have the zinc fingers, we named them nf （no zinc finger）, and fu （full length with zinc finger） to the isoforms with zinc fingers.To study the function of the nf isoforms, we cloned several members of the SCAN family, including fu and nf isoforms. Then we confirm their interactions through co-IP and GST-pull down. Our results showed that the fu isoforms can interact with the nf isoforms, and this interaction was mediated by the SCAN domain. It is known that the C₂H₂ zinc finger proteins need binding to specific DNA segments via their zinc fingers and interact with other cellular factors to control the transcription of target genes. Thus, if the isoforms without zinc fingers can interact with the ones with zinc fingers, the multi types of transcripts will make the mechanism of transcription regulation more complicated and variegated. So our results suggested that the nf isofrms may be very important to the complexity of the transcription regulation. And we know that the transcriptional factors need to enter the nuleusbefore regulating the transcriptional activity of their target genes. So we detected the subcellular localization of the isoforms. And we found that the nf isofroms we studied can be transferred to the nucleus by the fu isforms through the interaction meditated by the SCAN domain.Since the C₂H₂ zinc finger proteins are important transcription factors, we used the dual-luciferase reporter system to study the function on transcription regulation of the SCAN family members. The results showed that the ZNF191fu and ZNF434fu functioned as transcription repressors while the ZNF396fu can only weakly suppress the transcription activity of the reporter, and the ZNF397fu and ZNF447fu showed no signanificant capability of transcription regulation. Besides that, we also studied the function on transcription regulation of the nf isoforms, and we found that they showed different capability of transcription regulation to the fu isoforms. The ZNF191nf and ZNF434nf showed weaker regulation ability than their fu isoforms, while the ZNF447nf can significantly activate the transcription activity of the reporter gene. And ZNF397nf function as transcription repressors. The different transcriptional regulation activity of the nf isoforms and their interaction with the fu isoforms would contribute to the complexity of the transcription regulation.To further examine the biological function of interaction between different isoforms we also analyze the potential role of ZNF191 and ZNF434 on signaling pathway. We found that ZNF191fu can significantly enhance the HSE-luciferase activity, while ZNF434fu can enhance the AP1 （PMA）-luciferase activity, then we analyze whether the nf isoforms have impact on the fu isoforms. And we found the nf isoforms of ZNF191 and ZNF434 can inhibit the activity of their fu isoforms respectively.In this study, we also identified a novel gene encoding a SCAN family member named ZNF445. The ZNF445 mRNA consists of 9105 nucleotides and has a 1031-amino acid open reading frame. It is mapped to chromosome 3p21.32. The predicted 119-kDa protein contains a SCAN domain at the N-terminus, followed by a well-conserved Kriippel-associated box （KRAB） domain. At the C-terminus of the protein, there are 14 C₂H₂ （Cys2-His2） zinc finger motifs. Northern blot analysis indicates that a 9.1 kb transcript specific for ZNF445 is expressed in uterus, thymus, small intestine, colon, pancreas, peripheral blood leukocyte, and especially at a higher level in the testis and skeletal muscle in human adult tissues. Reporter gene assays showed that ZNF445 is a transcriptional repressor, and ZNF445 protein was located inthe nucleus when overexpressed in cultured cells. Overexpression of ZNF445 in the HEK 293T cells activates the transcriptional activities of AP1 and SRE. Since AP1 and SRE are important nuclear effectors of the MAPK pathway, we examine whether this activation was related to the MAPK pathway. We found that the activation of AP1 and SRE by ZNF445 can be supressed by PD98059, an inhibitor of MEK. This suggested that the ZNF445 affected AP1 and SRE through the MAPK pathyway. Then we studied the potential impact on MAPK pathway of ZNF445. The results showed that expression of ZNF445 can increase Elk-1, ERK1/2, MEK and Raf-1 phosphorylation and this impact is dose-dependent. In addition, deletion studies showed that the SCAN domain of ZNF445 may be involved in this activation. These results clearly indicate that ZNF445 is a member of the SCAN family of zinc finger transcription factor and may function in MAPK pathway through Raf-1/MEK/ERK /Elk-1 signals. And we also study the function of ZNF445 on cell growth and proliferation by the experiment of cloning efficiency in L02. The result showed that ZNF445 can significantly increase the cloning efficiency when compared with the control.In summary, we found a kind of isoforms when we study the human SCAN family. This kind isoforms don’t contain zinc fingers so called nf isoforms. And they can interact with the fu isoforms which contain the zinc fingers through the SCAN domain. This interaction between them can affect the biological function of the fu isoforms. This finding is important to futher study the transcriptional regulation function of the zinc finger proteins, and may be one important answer to the problem - how can such a small group of transcription factors regulate so many genes expression. And in the study of ZNF445, one member of the SCAN family, we confirmed that the protein can affect the MAPK pathway, and found its biological function. We also found the SCAN domain of ZNF445 may be involved in this function. This is an important supplementary to the study of the SCAN family, and threw some light on the question about the cell growth and proliferation.更多还原