| Lsdl (Lysine specific demethylase 1) is the first discovered histone lysine demethylase (HKDM), which demethylates H3K4me1/2 and H3K9mel/2 and modifies chromatin structure and transcriptional activity. With the rapid development of epigenetic research, it has been recognized that epigenetic regulation plays key role in the process of embryonic development, cell differentiation, and disease. Of course, dynamic regulation of histone methylation is important for the establishment of different "Epigenome" for the diverse tissues/cells during development. However, the detailed biological role of Lsdl in embryonic development is still unclear. Therefore, we used mouse ES (embryonic stem) cells as in vitro and zebrafish as in vivo models to investigate the biological function of Lsdl. Several approaches including lentivirus-mediated RNAi, morpholino-modified antisense oligonucleotide, and specific demethylase inhibitor, were used to disrupt the expression or function of Lsd1. RT-qPCR, chromatin imunoprecipitation and microarray were used to explore the molecular mechanism.Firstly, we successfully decreased the expression of Lsd1 in mouse ES cells by lentivirus-mediated shRNA. We found that knockdown of Lsd1 does not impair the morphology and self-renewal of mouse ES cells, as identified by the expression of pluripotent markers. TCP (tranylcypromine, an inhibitor for Lsd1 H3K4mel/2 demethylse activity) treatment reduced the expression of erythroid markers during EB (embryonic body) differentiation, but did not later the expression of enotherlial or early differentiation markers. Lsd1 knockdown not only inhibited erythropoiesis but also delayed the upregulation of mesodermal maker Brachyury during EB differentiation. ChIP (chromatin immunoprecipitation) assay showed that Lsd1 bind to the promoter region of cKit gene, and that H3K4me2 levies were altered in several hematopoietic factors when EBs were treated with TCP. Our result suggested that Lsd1 regulates early mesodermal differentiation and primitive erythropoiesis. H3K4me1/2 demethylase activity is required for primitive erythropoiesis but mesodermal differentiation regulation depends on other activity of Lsdl. Secondly, we used zebrafish as in vivo model to study the role of Lsd1 in development. We found that TCP exposure caused "bloodless" phenotype, but Pargyline and Nialamide had no effect. Further study revealed that TCP did not disrupt the vascular formation but inhibits the primitive erythropoiesis. TCP-treated Tg(gatal:EGFP) zebrafish embryos displayed less or no circulating EGFP+ blood cells at 30 hpf, acomponied with reduced expression of embryonic globin genes. Recombinant GSt-zLsdl displayed the demethylase activity to H3K4me2 in vitro and TCP could inhibit such activity. Consistently, interfering the normal splicing of zLsdl mRNA by morpholino-modified anti-sense oligonucleotide also caused "bloodless" phenotype. Microarray analysis also showed that knockdown zLsdl decreased the erythrocyte-associated genes. RT-qPCR analysis revealed that TCP or lsdl-MO did not inhibit the early hematopoietic specification.Taken together, our results revealed that Lsdl plays key role in early embryonic development and primitive erythropoiesis. Lsdl regulates erythroid differentiation via its H3K4m1/2 demethylase activity, but its role in mesodermal development might be dependent on other activity. The detailed mechanism of Lsdl regulating development or differentiation of distinct tissues and its role in blood disease need further research.
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