Stage-Specific Transcriptome Analysis Of Cell Cycle-Related Genes In Erythropoiesis

Research purposes:Erythropoiesis is the process of hematopoietic stem cells going through progenitor proliferation,terminal erythroid differentiation,enucleation and maturation to produce red blood cells.The erythroid progenitor cells undergo self-renewal and proliferation.The terminal erythropoiesis is accompanied by proliferation and differentiation and finally exits cell cycle and matures into functional erythrocytes by enucleation.Cell cycle changes according to specific stages in erythropoiesis.Previous studies have shown that some cell cycle related genes played important roles in different stages of erythropoiesis,but it's still unclear that how cell cycle related genes affect erythropoiesis at different stages.In this project,we used public databases to accumulate cell cycle-related genes,clustered their expression patterns in stagespecific transcriptome of cultured erythroblasts,compared gene functions of different expression patterns,and predicted key genes using stage-specific protein interaction networks.This work explored the temporal changes of cell cycle-related genes in erythropoiesis.Methods:RNA-seq raw data was filtered and processed by transcriptome analysis tools such as Fast QC,Tophat and Cufflinks.Gene expression was quantified and differential expressed genes between stages were identified.Cell cycle related genes was accumulated from four public databases: GO,Pathcards,Reactome,and Cyclebase.The stag-specific expression data of cell cycle-related genes during erythropoiesis was extracted for further analysis such as principal component analysis,correlation analysis and cluster analysis to investigate their temporal changing pattern.Gene ontology enrichment was used to understand function of different gene clusters with specific expression pattern.Protein-protein interaction was also constructed from known database to identify connection and core genes for each cluster.We obtained six gene clusters with specific expression patterns,including gene cluster that were highly expressed only in BFU-E-CFU-E or only in Ortho.Results:Quality control showed good quality of RNA sequencing and read mapping for further analysis.A total of 4557 cell cycle-related genes were obtained from public databases.Principal component analysis and correlation analysis illustrated modular characteristic of stages in erythropoiesis: Burst forming unit-erythroid(BFU-E)and colony forming unit-erythroid(CFU-E),proerythroblasts(Pro)and basophilic erythroblasts(Baso),polychromatic erythroblasts(Poly),orthochromatic erythroblasts(Ortho),which was consisted with known phenotypes.We identified 6 gene clusters with different expression pattern: genes with high expression in BFU-E-Baso,genes with high expression in BFU-E-CFU-E,genes with increasing expression from Pro and decreasing expression after Baso,genes with increasing expression from Pro and decreasing expression after Poly,genes that were highly expressed in Poly and Ortho,and genes that were only highly expressed only in Ortho.Gene ontology analysis showed that genes highly expressed in BFU-E-CFU-E were enriched in m RNA metabolism and translation process,which hinted these GO terms may be associated with self-renewal in early progenitor cells.Genes highly expressed in Pro-Poly were enriched in cell division process and may affect the differentiation of terminal differentiation through mitosis regulation.Genes highly expressed in Ortho were enriched in cell cycle arrest and ubiquitin-dependent protein catabolic process,which may contribute to enucleation.Protein-protein interaction network was also constructed and analyzed for each cluster,key genes in each cluster were also identified.Comparison of transcriptome clustering between erythroblasts cultured from hematopoietic stem cells derived from cord blood and fetal liver showed overall consistency.Due to different sources,the functional enrichment results of some gene sets are different.The major difference between them were the negative regulation of proliferation,apoptotic pathway in cluster with high expression in BFUE-CFUE,which may be associated with the progenitor proliferation difference between CB and FL.Conclusion:This project performed a comprehensive analysis of cell cycle-related genes in cultured erythropoiesis.We identified 6 specific expression clusters and provided GO terms and key genes that were potentially related to self-renewal and enucleation.The self-renewal of early progenitor cells is related to the process of m RNA metabolism,the process of co-translational protein targeting to membrane,and the translation process.The enucleation may be associated with cell cycle arrest and proteasomemediated ubiquitin-dependent protein catabolic process in vitro.Temporal comparison of erythroblasts cultured from hematopoietic stem cells derived from FL and CB implied overall similar regulation in in-vitro systems and potential mechanisms of progenitor proliferation.These results not only enhanced our understanding of early self-renewal and terminal cell cycle exit during erythropoiesis,but also provided new cluses for proliferation and enucleation in in-vitro erythropoiesis.