| Eaf1 and Eaf2 are ELL-related factors,and ELL is RNA polymerase II elongation factor that plays an important role in acute myeloid leukemia and embryogenesis.However,the regulatory functions of these two genes in the development and differentiation of erythrocytes and neurocytes remain unclear.In this paper,the regulatory functions and potential regulatory mechanisms of Eaf1/2 on erythrocyte genesis and neurocytes development in zebrafish（293T cell line）will be studied mainly around the ELL-related factors Eaf1 and Eaf2,respectively.The study is as follows:1.Zebrafish eaf1/2 regulates erythrocyte production by regulating gata1 expression and Wnt signaling and promotes the tolerance of fish hypoxia.In this study,we used CRISPR/Cas9 gene-editing technology to construct successfully a mutant of eaf1/2 gene deletion in zebrafish and to explore the regulatory function and epigenetic regulation mechanism of eaf1 and eaf2 on the development of zebrafish erythrocytes.1)The loss of function of eaf1 and eaf2 leads to reduced hypoxia tolerance and bacterial susceptibility in zebrafish.By hypoxic stress eaf1-/-and eaf2-/-homozygous mutants as well as wild-type embryos and adults,it was found that the hypoxia tolerance of eaf1-/-and eaf2-/-mutant was worse than that of wild-type mutants;Experiments with Aeromonas hydrophila（AH）infestation have found that juvenile eaf1-/-and eaf2-/-zebrafish are more sensitive to AH infection compared with wild-type.2)The lack of function of eaf1 and eaf2 leads to a decrease in the number of erythrocytes in zebrafish embryos.The number of erythrocytes in eaf1-/-and eaf2-/-mutants and wild-type embryos were detected using O-dianisidine staining.The number of hemoglobin in eaf1-/-and eaf2-/-mutants was found to be reduced significantly.The expression of red blood cell（RBC）marker genes hbbe1,hbbe2,and hbbe3 in eaf1-/-and eaf2-/-mutants was detected by WISH,and significant downregulation of hemoglobin marker genes in mutant embryos was found.In summary,it is shown that the lack of function of eaf1 and eaf2 leads to a decrease in the number of erythrocytes in zebrafish embryos.3)Zebrafish eaf1-/-and eaf2-/-mutants in other hematopoietic cells and hematopoiesis regulate the expression of transcription factors.Using WISH to detect the expression of other hematopoietic cell marker genes and vascular marker genes in eaf1-/-and eaf2-/-mutants,the results showed that gata1,an important transcription factor that regulates the development of hematopoietic erythroid line,was downregulated at 14hpf and 24hpf,lmo2 and scl were upregulated at 14hpf but downregulated at 24hpf;while there was no difference in the expression of gata2,fli1,flk1;and the expression of hematopoietic stem cell marker gene c-myb/runx1 is upregulated at 33hpf;myeloid marker genes mpx and L-plastin are downregulated in33hpf embryos.4)eaf1 and eaf2 regulate classical Wnt/β-catenin signaling in a developmental-specific manner to regulate scl and lmo2 transcriptionLoss of eaf1 or eaf2 function leads to disorders of primordial erythrocyte production in zebrafish embryos.The canonical Wnt/β-catenin activity underwent significant dynamic changes,upregulated at 16 hpf and downregulated at 24 hpf,showing consistent dynamic expression characteristics with their target genes scl and lmo2 upregulated at 14hpf but downregulated at 24 hpf.Ch IP-qPCR results showed that the loss of function of eaf1 or eaf2 led to a significant decrease in the binding enrichment of TCF4 on the promoter of the gene scl and lmo2 in the 24 hpf zebrafish mutant embryos,which in turn could lead to a significant decrease in the expression of scl and lmo2.Conclusion:eaf1and eaf2 regulate the expression of scl or lmo2 by modulating Wnt/β-catenin signaling pathway in a developmental-specific manner,thereby regulating primordial erythrocyte production in zebrafish.5)Eaf1 and Eaf2 regulate gata1 transcription through epigenetic modification mechanismsThe loss of function of eaf1 and eaf2 in zebrafish leads to significant downregulation of zebrafish embryonic histone H3K27Ac,H3K4me1,H3K4me3,and significant upregulation of histone H3K27me3.The Ch IP-qPCR results showed that the binding abundance of histone H3K27me3 to the gata1 promoter in the dysfunctional mutants of zebrafish eaf1 and eaf2 was upregulated,while the binding to the scl and lmo2promoters did not change.These data suggest that eaf1 and eaf2 modulate gata1transcription by modifying gata1 promoter H3K27me3 in combination with enrichment.2.Eaf1 and Eaf2 apparently regulate the development of neural stem cellsIn this study,the epithemic regulatory mechanism of Eaf1/2 on the development and differentiation of neural stem cells was explored by Eaf1/2 functional knockdown HEK293T cell lines（h Eaf1/2 KD cell line）.The results of this project are as follows:1)Loss of eaf1 and eaf2 function leads to developmental defects in zebrafish neurons,myelin sheaths,axonsIn eaf1-/-and eaf2-/-homozygous mutants,neuron-related genes aldocb,tbr1b,bdnf expression upregulated,myelin marker genes plp1a,pou3f1,elav13,olig2,and neurodevelopment-related genes mbp,vim,amotl expression are all upregulated.2)Eaf1 and Eaf2 functional knockdown promote neural stem cells and differentiation processesIn the h Eaf1/2 KD cell line,neural stem cell differentiation-related genes SLIT3,PCDHB10,CRMP1,BAI1,CHRRNB2,GABRA5,SALL4,OLFM1,SEZ6L,SCRG1,GDPD5,LGALS1,NES,etc.were upregulated.In the h Eaf1/2 KD cell line,more cells showed immunofluorescence positive signals of neural stem cell marker proteins SOX2,PAX6,NESTIN,TUBBLIN,NEUN,MAP2,glial cell marker proteins MBP,VIMENTIN,GFAP,OLIG2,and synaptic proteins SYNPSIN,PSD95,GABA,SV2A,showing the function of h Eaf1/2 KD cell line to promotes cell transdifferentiation of neurotype cells derived from the ectoderm.3)Expression of histone proteins in Eaf1 and Eaf2 functional knockdown cellsIn the h Eaf1/2 KD cell line,the epimodeled histones H3K4me1 and H3K27Ac are downregulated in h Eaf1/2 KD cells,while the expression of H3K27me3 and H3K4me3 is upregulated.In this paper,the study of zebrafish ELL-related factor Eaf1/2 regulating erythrocyte production by regulating gata1 expression and Wnt signaling,and regulating neurocytes development through Eaf1/2,which further provides a basis for the theoretical study of erythroid development and neurocytes development of Eaf1/2 on zebrafish,and improves the Wnt/β-catenin signaling pathway in ELL-related factor Eaf1/2 The function of regulating the development of red blood cells and the role of apparent regulation in the development of erythrocyte and neurocytes provide a molecular theoretical basis for exploring the development of erythrocyte/neurocytes. |
