RNA-seq expression profiles during terminal erythropoiesis. Mus musculus

It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA Polymerase II (Pol II) occupancy and multiple post-translational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels-in particular, H3K79me2 and H4K16ac-were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, while gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data maps the epigenetic landscape of terminal erythropoiesis and suggests that control of transcription elongation regulates gene expression during terminal erythroid differentiation. Overall design: Mouse fetal liver cells are double-labeled for erythroid-specific TER119 and non erythroid-specific transferrin receptor (CD71) and then sorted by flow-cytometry. E14.5 fetal livers contain at least five distinct populations of cells (R1 through R5); as they progressively differentiate they gain TER119 and then gain and subsequently lose CD71. CFU-E cells and proerythroblasts make up the R1 population; R2 consists of proerythroblasts and early basophilic erythroblasts; R3 includes early and late basophilic erythroblasts; R4 is mostly polychromatophilic and orthochromatophilic erythroblasts; and R5 is comprised of late orthochromatophilic erythroblasts and reticulocytes. We have sorted for R2-R5 cells for RNA-seq experiment.

目前尚不清楚在终末红细胞生成过程中，表观遗传变化如何调控红细胞特异性基因的诱导。本研究采用全局mRNA测序（mRNA-seq）与染色质免疫沉淀结合高通量测序（CHIP-seq），探究红细胞祖细胞分化为晚期成红细胞时，mRNA水平、RNA聚合酶II（Pol II）结合水平以及多种组蛋白翻译后修饰所发生的变化。在该发育转变过程中被诱导的基因中，Pol II结合水平、激活型组蛋白修饰标记H3K4me2、H3K4me3、H3K9Ac与H4K16Ac，以及延伸相关甲基化标记H3K79me2的水平均显著升高。与之相反，在分化过程中被抑制的基因，其H3K79me2水平相对降低，但Pol II结合与活性组蛋白标记的水平与红细胞祖细胞相似。我们还发现，组蛋白修饰水平的相对变化——尤其是H3K79me2与H4K16ac——对基因表达模式的预测能力最强。本研究结果表明，在终末红细胞生成过程中，启动子相关与延伸相关的修饰标记均参与红细胞特异性基因的诱导激活，而基因抑制则以介导Pol II延伸的组蛋白修饰变化为特征。本研究的数据绘制了终末红细胞生成的表观遗传图谱，并提示转录延伸的调控在终末红细胞分化过程中参与基因表达的调节。

整体实验设计：对小鼠胎肝细胞采用红细胞特异性标记TER119与非红细胞特异性转铁蛋白受体（CD71）进行双荧光标记，随后通过流式细胞术分选出目标细胞群。E14.5胎肝细胞包含至少5种不同的细胞群（R1至R5）；随着细胞逐步分化，其表面会先获得TER119表达，随后先获得CD71表达，再逐步丢失CD71。红细胞集落形成单位（CFU-E）与早幼红细胞构成R1群；R2群由早幼红细胞与早期嗜碱性成红细胞组成；R3群包含早期与晚期嗜碱性成红细胞；R4群主要为嗜多染性与正染性成红细胞；R5群则由晚期正染性成红细胞与网织红细胞构成。本研究针对R2至R5细胞群开展了RNA-seq实验。