Global chromatin occupancy and epigenetic signature analysis reveal new insights into the function of GATA1 N-terminus in erythropoiesis

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N-terminus, are seen in patients with Diamond-Blackfan Anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic genes were altered by the loss of the N-terminus, including aberrant up-regulation of Gata2 and Runx1. Mass spectrometry studies demonstrated there was a global increase in H3K27 methylation in the erythroid progenitors. By contrast, chromatin biding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in transcriptional regulation and red blood cell maturation. Fetal liver cells from Gata1s embryos were stained with antibodies against CD71 and Ter119, CD71hiTer119neg/low population (R1/2) and CD71hiTer119hi (R3) population were sorted for analysis comparing with corresponding populations of fetal liver cells from wild type embryos.GATA1的突变可导致缺失GATA1 N端的GATA1s异构体表达，此类突变可见于戴森-布莱克凡贫血（Diamond-Blackfan Anemia, DBA）患者。为深入阐明GATA1s与DBA之间的致病关联，本研究对Gata1s小鼠的红细胞生成过程开展了系统性研究。研究发现，该模型小鼠的卵黄囊与胎肝造血功能存在显著缺陷，具体表现为红系细胞终末成熟受阻，以及红系祖细胞数量大幅减少。RNA测序（RNA-sequencing）结果显示，红系与巨核细胞相关基因的表达均因GATA1 N端缺失发生紊乱，其中包括Gata2与Runx1的异常上调。质谱分析（Mass spectrometry）证实，红系祖细胞内存在H3K27甲基化的全局性升高。与之相对，染色质结合实验显示，尽管GATA1与GATA1s的染色质结合占有率相近，但Gata2及Runx1基因调控区域的H3K27me3水平显著降低。已有研究表明GATA2过表达会损伤红细胞生成，与此观察一致，我们发现Gata2单倍剂量不足可有效挽救Gata1s胎鼠的红系发育缺陷。综上，本研究对转录组与表观遗传特征的整合基因组分析表明，尽管Gata1s小鼠无法精准模拟DBA的病理特征，但该模型为揭示GATA1 N端在转录调控及红细胞成熟过程中的关键作用提供了全新视角。本研究使用CD71与Ter119抗体对Gata1s胚胎的胎肝细胞进行染色，分选得到CD71hiTer119neg/low群体（R1/2）与CD71hiTer119hi群体（R3），并与野生型（wild type）胚胎胎肝的对应群体开展对比分析。