Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin accessibility and looping (RNA-Seq)

ETO2 functions as a transcription repressor and is required for the embryonic erythropoiesis and the hemoglobin switch. To gain insight into ETO2 regulatory function during human erythropoiesis, we performed RNA-seq for WT and ETO2 KO K562 cells and found that up-regulated genes upon ETO2 loss in human cells included many markers of mature erythroid cells EPB42, ALAS2, GYPA and SLC25a37. Notably, the alpha-globin genes (HBA1, HBA2 and HBZ) and embryonic and fetal beta-globin genes (HBE1, HBG1, and HBG2) were significantly increased after deletion of ETO2. By contrast, deletion of ETO2 down-regulated the transcription factor genes (ETS1, KLF8 and SOX6) which play a negative role in globin gene expression and hemoglobin synthesis. To further explore different domain function of ETO2, we have analyzed our RNA-seq data from domain deletion cell lines compared to the cell line expressing wild type ETO2. Generally, 702 genes were found to co-regulated by three domain function of ETO2. Interestingly, the regulation of fetal globin genes, erythroid regulator (SOX5) as well as epigenetic factor (HDAC7) is required by three domain function of ETO2. During mouse embryonic erythropoiesis, our FACS-sorted and normal RNA-seq data in E14.5 fetal liver cells indicated that eto2 promoted a critical developmental transition and played an important role in globin switch from embryonic to adult beta-globin transcription since its function is essential for key regulators (PU.1, BCL11A and ZBTB7A) and globin genes (Hbb-y and Hba-x) regulation. RNA-seq was performed between human erythroleukemia K562 control cells and ETO2 KO cells in order to identify the differentially expressed genes. RNA-seq was performed in WT control, KO control, rescued ETO2 full length (FL), rescued ETO2 full length without domain TAF110 (D1), rescued ETO2 full length without domain HHR (D2) and rescued ETO2 full length without domain ZF (D4) K562 cells to identify the differentially expressed genes and genes regulated by different functional domains. RNA-seq was performed using FACS-sorted and total embryonic day 14.5 (E14.5) fetal liver cells between control and Eto2 knockout mice to identify the differentially expressed genes which are essential for mouse development and embryonic erythropoiesis.ETO2 作为转录阻遏因子（transcription repressor），对胚胎红细胞生成（embryonic erythropoiesis）及血红蛋白转换（hemoglobin switch）不可或缺。为深入解析ETO2在人类红细胞生成过程中的调控功能，我们对野生型（wild type, WT）及ETO2敲除（knockout, KO）的K562细胞开展了RNA测序（RNA-seq），结果发现人类细胞中ETO2缺失后上调的基因包含众多成熟红细胞标志物，如EPB42、ALAS2、GYPA及SLC25A37。值得注意的是，α-珠蛋白基因（HBA1、HBA2及HBZ）以及胚胎型、胎儿型β-珠蛋白基因（HBE1、HBG1及HBG2）在ETO2缺失后均显著上调。与之相反，ETO2缺失会下调那些在珠蛋白基因表达及血红蛋白合成中发挥负调控作用的转录因子基因，包括ETS1、KLF8与SOX6。为进一步探究ETO2不同结构域的功能，我们分析了携带结构域缺失的细胞系与表达野生型ETO2的细胞系的RNA测序数据。整体而言，共有702个基因受到ETO2三个结构域的共同调控。有趣的是，胎儿珠蛋白基因、红细胞调控因子SOX5以及表观遗传因子HDAC7的调控，均依赖于ETO2的三个结构域功能。在小鼠胚胎红细胞生成过程中，我们对E14.5胎肝细胞的流式分选（fluorescence-activated cell sorting, FACS）细胞及总细胞开展的RNA测序数据分析显示，eto2可促进关键发育转换，并在胚胎型向成年型β-珠蛋白转录的转换过程中发挥重要作用，这是因为其功能对关键调控因子（PU.1、BCL11A及ZBTB7A）及珠蛋白基因（Hbb-y与Hba-x）的调控至关重要。我们针对人类红白血病K562对照细胞与ETO2敲除细胞开展RNA测序，以鉴定差异表达基因。我们在野生型对照、敲除对照、回补全长ETO2（full length, FL）、回补缺失TAF110结构域的全长ETO2（D1）、回补缺失HHR结构域的全长ETO2（D2）以及回补缺失ZF结构域的全长ETO2（D4）的K562细胞中开展RNA测序，以此鉴定差异表达基因及受不同功能结构域调控的基因。我们针对对照小鼠与ETO2敲除小鼠的E14.5胎肝细胞（包括流式分选细胞及总细胞）开展RNA测序，以鉴定对小鼠发育及胚胎红细胞生成至关重要的差异表达基因。