Epigenetic Mechanism Studies Of Myogenesis And Epc1/2 Mediating Hematopoiesis Defects In Copper Overload Zebrafish Embryos

There are motion defect,reduced disease resistance and hypoxia tolerance in fishunder environmental stress.However,the potential regulatory mechanisms are rarely reported.In this study,we will investigate the molecular mechanisms which copper stress leads to muscle cell development defects in zebrafish and copper stress regulates the development of hematopoietic stem cells by regulating the differential expression of zebrafish epc1/2 genes.1.Cu²⁺regulates zebrafish muscle development via epigenetic regulationCopper is an indispensable trace element in organism.However,copper overload or accumulation is harmful to the health of body.In this study,we aimed to exploring the molecular mechanism that Cu²⁺regulated muscle cell development through epigenetic regulation in zebrafish.The results are as follows:1)Cu²⁺stress causes the defects of zebrafish skeletal myofibrillogenesisImmunostaining was used to detect the myofibrils and sarcomere in the trunk of Cu²⁺-stressed embryos.The results showed that f-Actin,α-Actinin,and s My HC in Cu²⁺treated embryos were arranged irregularly.TEM analysis exhibited that the Z-disc in Cu²⁺-stressed embryos developed normally.However,the sarcoplasmic reticulum with triads was defective compared with the control.Those results indicated that Cu²⁺stress caused the defects of zebrafish skeletal muscle fiber development.2)Cu²⁺inhibits the transcriptional activity of SRF and Myog on the smyd1b promoterLuciferase activity analysis indicated Cu²⁺inhibited the expression of smyd1b by suppressing the transcription activity of SRF and Myog on the smyd1b promoter.WISH results displayed that the expression of mylpfa,smyhc1l and smyd1b genes in the embryos of cox17^-/-and atp7b^-/-mutants was also significantly down-regulated in compared with Cu²⁺-stressed wild-type embryos,revealing that the inhibition of the above genes expression by Cu²⁺stress was independent of the integral function of Cox17 or Atp7b.3)Muscle-related genes in Cu²⁺-stressed embryos are regulated via DNA methylationBioinformatics analysis of genome-wide methylation sequencing data indicated that the promoter regions of muscle-related genes,rab3ip,fgfbp2b and smyd5,in the Cu²⁺-stressed group were hypermethylated.The promoter region of arpc1a was demethylated.Promoter methylation analysis further verified that the smyd5 promoter in the Cu²⁺-stressed group was hypermethylated.2.EPC1,EPC2 regulated the development of hematopoietic stem cells viaepigenetic regulationCu²⁺stress can lead to immune system deficiency of zebrafish embryos.EPC1 and EPC2 are important components of chromatin regulatory complex,and their dysfunctions have been found to be associated with human diseases including leukemia.However,little is known of their functions in hematopoiesis especially in definitive hematopoiesis.In this study,we found the differential expression of epc1 and epc2genes in Cu²⁺overload hematopoietic stem cells.The experimental techniques,such as knockout/down,q RT-PCR,WISH and so on,were applied to explore the underlying molecular mechanism of hematopoietic stem cells development regulated by epc1/2.The main results of this study are as follows:1)Loss of epc1/2 leads to reduced hypoxia tolerance of embryos/larvaeHypoxia stress of epc1^-/-and epc2^-/-mutants at 24hpf,48hpf and 72hpf showed that the hypoxia tolerance of epc1^-/-mutants was not affected,while epc2^-/-mutants were more sensitive to hypoxia stress.2)Depletions of epc1 and epc2 result in the reduced proliferation of hematopoietic stem cellsThe results of WISH analysis and confocal microscopy displayed that knockdown/knockout of epc1/2 significantly down-regulated the expression of hematopoietic stem cell marker c-myb/runx1 and thymus marker rag1 in the AGM region.Cell-direct q PCR analysis showed that deletion of epc1/epc2 resulted in the down-regulated expression of c-myb/runx1 in hematopoietic stem cells.This manifests that the loss of epc1 and epc2 causes the defects of hematopoietic stem cells and thymus development.Proliferation test of hematopoietic stem cell showed the decreasing number of Brd U⁺runx1⁺cells in the AGM and CHT regions of epc1^-/-and epc2^-/-mutants.Apoptosis test revealed that the ratio of Annexin V-PE-labeled apoptotic runx1⁺cells to the total number of runx1⁺cells did not change in epc1^-/-and epc2^-/-embryos.This indicates that the defect of hematopoietic stem cells caused by the loss of epc1/2 may result from the reduction of hematopoietic stem cell proliferation.3)epc1 and epc2 regulate hematopoietic development via modulating the level of histone acetylationWestern Blot and Immunostaining revealed the down-regulated expression of the acetylated histones（H3K9Ac,H3K27Ac and H3K56Ac）in epc1^-/-and epc2^-/-embryos and in runx1⁺cells sorted from epc1^-/-and epc2^-/-mutants.Cell-direct q PCR analysis showed that the deletion of epc1 and epc2 affected the expression of a variety of acetylation/deacetylation-related genes in runx1⁺cells.WISH results showed that histone deacetylase inhibitors partially rescued the down-regulated expression of hematopoietic stem cell marker gene c-myb and vascular marker genes fli1/flk1 in AGM region of epc1^-/-and epc2^-/-mutants at 24hpf.These results indicate that epc1 and epc2may regulate hematopoietic development via modulating the level of histone acetylation.4)napl14a,dlst,mbd3b may be the downstream targets of epc1/2Transcriptome analysis of epc1^-/-mutant and WISH analysis revealed that dlst,napl14a and mbd3b displayed decreasing expressions in the AGM region in epc1^-/-mutant embryos.Deletion of epc1 leaded to the down-regulated expression of napl14a and dlst,and the up-regulated expression of mbd3b in runx1⁺cells.WISH results revealed that the expression of runx1/c-myb was significantly decreased in napl14a and dlst mutants/morphants,and increased in mbd3b mutants/morphants.Collectively,dlst,napl14a and mbd3b may lie downstream of epc1 and involved in zebrafish definitive hematopoiesis.5)EPC1/2 regulate DLST,MBD3 through FOXR2 or SRFDual luciferase activity analysis found that co-transfection of FOXR2 or SRF with EPC1/EPC2 resulted in a significantly increasing luciferase activity of DLST promoter,and significantly down-regulation of the MBD3 promoter luciferase activity.This indicated that FOXR2 or SRF may promote the expression of DLST and inhibit the expression of MBD3 by recruiting EPC1/2.In summary,the studies on Cu²⁺regulating zebrafish muscle fiber differentiation through histone methylation/DNA methylation and epc1 and epc2 regulating hematopoietic stem cell development,can further improve the epigenetic regulation mechanism in muscle/hematopoietic stem cell development,and provide a molecular theoretical basis for exploring the epigenetic regulation of muscle/hematopoietic stem cell development.