Effects Of LSD1 On Zebrafish Development And Related Mechanisms

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.