过表达GATA2对鸡前脂肪细胞转录表达的调控作用及机制

Action and mechanism of GATA2 overexpression regulating the transcriptome expression of chicken preadipocytes

目的阐明过表达GATA2对鸡前脂肪细胞转录表达的影响。方法培养鸡前脂肪细胞ICP1,转染pCMV-myc-GATA2或pCMV-myc质粒,Western blot验证细胞中GATA2过表达情况,RNA-seq研究过表达GATA2对细胞转录组表达变化,ChIP-seq研究GATA2在基因组的结合情况,Real-time PCR验证部分差异表达基因、ChIP-PCR验证GATA2对TAF3基因的集合情况。结果转染pCMV-myc-GATA2可以在ICP1细胞过表达GATA2蛋白,过表达GATA2后,ICP1细胞中942个基因表达上调,840个基因表达下调(P<0.05),富集分析显示差异表达基因与核糖体和线粒体的结构和功能相关(P<0.01);GESA分析显示,过表达GATA2的ICP1细胞中RNA聚合酶Ⅱ转录起始复合物、核糖体生物生成、丙酸代谢和氧化磷酸化相关基因集表达上调;组蛋白赖氨酸N甲基转移酶、核转录抑制复合体形成、脂肪因子和钙离子等信号通路相关基因集表达下调。ICP1细胞中ChIP-seq鉴定出2833个GATA2结合的基因组峰,涉及2018个基因。Motif分析显示GATA2结合(T/A)GATA模序;富集分析显示这些基因参与胚胎发育、信号传导、细胞代谢和细胞间相互作用。差异表达基因和GATA2结合基因取交集获得105个基因,富集分析显示这些基因与转录、转录后调控、细胞间相互作用、信号传导和细胞周期相关(P<0.001)。结论GATA2可以与鸡前脂肪细胞的基因组结合调节其转录组表达模式。

Objective To clarify the impact of GATA2 overexpression on transcriptional expression in chicken preadipocytes.Methods Chicken preadipocytes ICP1 were cultivated,pCMV-myc-GATA2 or pCMV-myc plasmid was transfected,Western blot was used to verify the overexpression of GATA2 in cells,RNA-seq was used to study the changes in cell transcriptome expression caused by overexpression of GATA2,ChIP-seq was used to study the binding of GATA2 in the genome,Real time PCR was used to verify some differentially expressed genes,ChIP-PCR was used to verify the collection of GATA2 to TAF3 genes.Results Transfection of pCMV-myc-GATA2 allowed overexpression of GATA2 protein in ICP1 cells;overexpression of GATA2 resulted in up-regulation of 942 genes and down-regulation of 840 genes in ICP1 cells(P<0.05),and enrichment analysis showed that differentially expressed genes were associated with ribosome and mitochondrial structure and function(P<0.01);GESA analysis showed that the expression of GATA2-overexpressing ICP1 cells showed up-regulation of RNA polymeraseⅡtranscription initiation complex,ribosome biogenesis,propionate metabolism,and oxidative phosphorylation-related gene sets,and down-regulation of histone lysine N-methyltransferase,nuclear transcription repression complex formation,adipokines,and calcium ions signaling pathway-related gene sets.ChIP-seq in ICP1 cells identified 2833 GATA2-binding genomic peaks involving 2018 genes.motif analysis revealed GATA2-binding(T/A)GATA motifs;enrichment analysis showed that these genes are involved in embryonic development,signaling,cellular metabolism,and cell-cell interactions.Differently expressed genes and GATA2-binding genes were taken to intersect to obtain 105 genes,and enrichment analysis showed that these genes were associated with transcription,post-transcriptional regulation,cell-cell interactions,signaling,and cell cycle(P<0.001).Conclusion GATA2 can at least bind to the genome of chicken preadipocytes to regulate their transcriptome expression patterns.