【作者】 张洁；

【导师】 施扬； 宋后燕； 汤其群； 马端； 于敏；

【作者基本信息】 复旦大学 ， 分子医学， 2009， 博士

【摘要】 赖氨酸特异的脱甲基酶1(也称为LSD1,BHC110或者AOF2)是第一个发现的组蛋白脱甲基酶。在CoREST(corepressor for REST)、BHC80和HADC等辅助因子帮助下,LSD1能够使组蛋白H3-K4位脱去一个或二个甲基。组蛋白脱甲基后引起染色质重塑或改变靶基因的转录活性。通常H3-K4脱甲基化抑制靶基因转录活性,而H3-K9脱甲基化则导致靶基因转录被激活。LSD1与很多疾病有密切的关系。例如,在人肿瘤细胞中LSD1表现为沉默,而一些LSD1作用的靶基因却大量表达。这些基因在癌症的发展中有重要的作用。又如,lsd1敲除的小鼠在围产早期死亡,这也导致难以深入研究lsd1在发育过程中的作用及其机制。模式生物斑马鱼胚胎透明、体外发育、个体小、繁殖率高以及其心血管、血液、消化道、肝脏、肾脏以及视觉系统与人类相应系统有许多共同点,基因组序列与人的同源性更是达到75%以上。此外,斑马鱼所需部分的氧可以通过体表渗透,因此特别适用于心血管和血液系统早期发育的研究。本课题就是利用斑马鱼作为模型来研究LSD1对于发育的影响并对其机制进行了初步的探索。第一部分LSD1在脊椎动物中的同源性及其在斑马鱼胚胎发育早期的表达LSD1的cDNA共编码689个氨基酸残基,蛋白质分子量约为110KD。生物信息学的方法聚类比较显示LSD1在脊椎动物的进化过程中,蛋白质结构非常保守(同源性＞65%)。不同物种的LSD1蛋白质分子上有两个很保守的结构域:SWIRM和FAD结构域;在斑马鱼发育中,lsd1在胚胎上表达从受精后一直持续到整个发育早期,而且lsd1的表达量始终处于高水平,特别是在神经系统中。第二部分斑马鱼中lsd1基因表达下调方法的建立本研究通过吗啡啉修饰的反义寡核苷酸技术(morpholino)干扰目标mRNA翻译的方法下调lsd1表达,并观察斑马鱼发育受到的影响。我们设计了两个具有剪接功能的吗啡啉修饰反义寡核苷酸,下调了lsd1基因在斑马鱼中的表达。外显子11和17是LSD1功能蛋白必需的编码序列,失去其中任何一段,LSD1即丧失脱甲基酶活性。RT—PCR结果显示外显子11和17在两种MO注射后分别被剪切掉,这也验证了吗啡啉修饰的反义寡核苷酸的有效性,说明LSD1 MO能够有效下调内源性的lsd1。此外,当斑马鱼暴露在一定浓度的PCPA(＞20μM,反环苯丙胺)中,LSD1具有的组蛋白脱甲基酶活性H3-K4(2m)也被强烈抑制。第三部分lsd1基因表达下调对斑马鱼血液系统发育的影响斑马鱼是研究血管和造血的良好模型,常被应用于血管异常和造血障碍的研究。体外实验提示LSD1对于血细胞的分化起着重要作用。因为在小鼠模型中致死性,所以原始造血系统功能与LSD1的关系了解不多。我们利用斑马鱼第一次发现PCPA可以诱导斑马鱼的贫血现象,进一步研究显示贫血是由于原始造血系统被抑制造成的。这种贫血现象是由于与LSD1相关的表观遗传学变化引起的;吗啡啉下调lsd1后,斑马鱼中出现了相似的贫血表型。表明原始造血系统的发育需要LSD1的H3K4脱甲基酶参与。制备sc1、lmo2、gata2、gata1、globin、pu.1、mpx、L-plastin和flk1探针进行了胚胎整体原位杂交。结果显示,血管发育基本不受影响,但是原始红系细胞数量减少。红系发育受阻的同时髓系发育则基本无影响。此外,血细胞流动速度也受到影响。第四部分lsd1基因表达下调对斑马鱼神经系统发育的影响lsd1在斑马鱼的CNS部位持续高表达表明LSD1在神经系统发育上有一定的作用。PCPA曾作为临床上使用的抑郁药,因对患者有副作用导致使用范围减小。可是副作用分子机制不详。据报道,PCPA可以共价结合LSD1蛋白的FAD结构域从而对其酶活性产生不可逆的抑制,从而调控与神经发育相关的基因。我们观察到浓度大于或等于75μM的PCPA处理的斑马鱼较对照组活跃程度下降。原位杂交显示soxla和huc的表达水平下调,TUNEL检测结果说明上述现象是由于神经元细胞过度凋亡。注射LSD1吗啡啉后胚胎神经细胞也表现为过渡凋亡。RT-PCR结果显示,P53依赖的信号通路参与了LSD1介导的凋亡过程。更多还原

【Abstract】 Lysine-specific demethylase 1(LSD1 also known as BHC110 or AOF2) is the first discovered histone lysine demethylase(110×10~3 Daltons).With the help of its cofactors CoREST,BHC80 and HADC,it specifically demethylates mono-and dimethylated histone H3 lysine 4(H3-K4) or H3-H9,thus exhibiting diverse transcriptional activities by chromatin reconfiguration.Methylation of H3-K4 is predominantly associated with transcriptionally active genes,whereas methylation of H3-K9 marks silent genes or heterochromatin.A loss-of-function LSD1 in the human colon carcinoma cells causes a phenotype with a reexpression of multiple and aberrantly silenced genes which are important in the development of colon cancer.Using standard gene targeting technology in mouse embryonic stem cells,lsd1 knockout mice die in prenatal-perinatal.Despite of the sudden cardiac death phenotype,lsd1 knockout strategy resulted in big impact to development of pituitary and neural system at very early stage.More detailed phenotypic analysis of animals with lsd1 mutations will be required to uncover its biological function.The zebrafish,Danio rerio,offers several distinct advantages as a genetic and embryological model system,including the external fertilization,rapid development and optical clarity of its embryos.Being a vertebrate,the zebrafish has a notochord, blood,heart and vasculature,kidney and optical systems that share many features with corresponding human systems.The zebrafish system bridges the gap between fruit fly/worm and mouse/human genetics(genomic sequences similar identity＞75%), making it feasible to address issues of early development,organ formation,integrative physiology,pharmacology and complex disease.Even in the total absence of blood circulation,they receive part of oxygen by passive diffusion to survive and continue to develop in a relatively normal fashion for several days,thereby allowing a detailed analysis of animals with severe cardiovascular defects.So far,we used zebrafish as a modle to study the functions of LSD1 and related mechanisms.Section 1 Evolutionarily Conserved Structure and Spatiotemporal Expression of lsd1 in ZebrafishThe complete open reading frame of the LSD1 cDNA encodes a 689 amino acid protein with a calculated molecular weight of 110 kDa.The zebrafish LSD1 protein sequence shares high similar identity with other species’sequences.Further analysis of the zebrafish LSD1 sequence predicts the presence of several motifs(SWIRM and FAD domain) important to function of histone demethylation,lsd1 is expressed throughout the developing stages from one cell embryo.As embryonic development progresses,the expression pattern becomes restricted to the anterior of the embryo and in particular of the neural structures.Section 2 Loss-of-function LSD1 Zebrafish Can Be Obtained by LSD1 MO and PCPA TreatmentTo study the effects of LSD1 down regulation on the development of the embryonic zebrafish,two types of well designed morpholino oligonucleotide antisenses were injected into zebrafish embryos which can splice the exon11 and exon17 of lsd1 at one or two cells stage.Exon11 and exon17 play a crucial role in function of histone demethylation.After injection,the validity of morpholinos were tested by RT-PCR and sequencing.Furthermore,LSD1 histone demethylase is be inhibited when embryos were exposure to Trans-2-phenylcyclopropylamine(referred to as PCPA hereafter;also known as tranylcypromine and Parnate,＞20μM).Western blot showed that the H3K4 methylation level of PCPA treatment group was upregulted compared to the control.Section 3 Requirements of LSD1 in Zebrafish Hematopoietic SystemDevelopmentIn recent years,zebrafish has emerged as an exciting animal model system for studying vertebrate organ development,in particular,the development of hematopoietic system.In vitro studies suggested that lsd1 expression was of particular importance during establishment or regeneration of the mammalian definitive hematopoiesis.But little is known about its in vivo functions during primitive hematopoiesis because of embryonic lethality of mammalian knockout models.In the present study,we turn to the zebrafish embryos to pursue the role of LSD1 during hematopoiesis.In the present study,we noticed for the first time that antidepressant PCPA treatment induces short time anemia in zebrafish.The phynotype was caused by inhibition of pirimitive hematopoiesis in zebrafish embryos.We predict that it is an epigenetic events associated with LSD1.Furthermore,we also found that lsd1 knockdown by morpholino-modified antisense oligonucleotides resulted in similar phenotype(anemia).It is suggested that LSD1 H3-K4 histone demethylase is required for primitive erythropoiesis in zebrafish.Whole-mount in situ hybridization with scl, lmo2,gata2,gata1,globin,pu.1,mpx,L-plastin and flk1 RNA probes showed that the number primitive erythropoiesis cell was decrease,but it did not nearly affect vascular development.Based on these findings we put forward an assumption that down regulation of lsd1 expression leads to inhibition of primitive erythropoiesis but not myelopoiesis.Besides,we found that gatal+of blood cells flow slowly in LSD1 MO embryos which drops a hint that LSD1 are involved in appropriate circulation of blood cells.Section 4 LSD1 Is Essential for Zebrafish Neural System DevelopmentWe previously showed that lsd1 is highly expressed in zebrafish CNS during zebrafish embryogenesis.PCPA is used clinically as an antidepressant.The study of its adverse reaction has shown its side effect to patients.However,little is known about the molecule mechanism of the adverse reaction.Recent study showed that PCPA is an irreversible inhibitor of LSD1 which regulates neuronal specific genes by covalently modifying the FAD cofactor.We provide the first evidence that zebrafish embryos showed sluggish action at PCPA treatment(75μM) compared to the controls. Down-regulation of some neuron marker genes expression(soxla and huc) is evaluated by whole mount in situ hybridization,which indicates the decrease in the number of nerve cells.Then,nerve cells apoptosis is determined by TUNEL assay in PCPA-treated embryos as well as LSD1 MO embryos.We demonstrate that the PCPA induces apoptosis mediated by LSD1 demethylase activity,and our data also suggest that p53-depended signaling pathway may be required for the maintenance nerve cell apoptosis in LSD1-deficient zebrafish embryo.更多还原