Uncovering a Key Metabolic Role of the GATA1 N-Terminus in Red Blood Cell Development

Mutations in GATA1 that result in skipping of exon 2, which encodes the N-terminus, are associated with the myeloid leukemia of Down syndrome and Diamond-Blackfan anemia (DBA). To elucidate the molecular functions of the N-terminus, we employed single-cell RNA sequencing (scRNA-seq) of fetal liver cells derived from Gata1 mutant embryos that express the GATA1 short (GATA1s) isoform in place of full-length GATA1 (GATA1FL). scRNA-seq highlighted defects in erythropoiesis and revealed that the absence of the N-terminus resulted in elevated expression of genes involved in glycolysis, such as the rate-limiting pyruvate kinase PKM. To elucidate the regulation of the PKM gene, we performed precision nuclear run-on sequencing and cleavage under targets and release using nuclease on erythroid cells following acute deletion of GATA1 and determined that PKM is a direct target of GATA1. Mechanistically, we found that substitution of GATA1FL with GATA1s led to increased glycolysis in erythroid progenitor cells but did not affect oxidative phosphorylation. We further discovered that the expression of PKM is significantly elevated in DBA patients with RPS19 mutations, consistent with a role for GATA1 regulation of glycolysis in erythropoiesis. Together, these findings reveal that GATA1 controls not just heme metabolism, but also glycolysis. Overall design: HUDEP2 cells expressing FKBP12F36V-GATA1 (two clones 6F8 and 7C4) were cultured under differentiation conditions for 24 hours. PRO-seq was conducted at 0.5, 3, and 6 hours in the presence of the acute degradation inducer dTAG-13 (100 nM) to monitor the nascent transcripts.

GATA1基因中导致外显子2（编码N端结构域）发生跳读的突变，与唐氏综合征髓系白血病及戴蒙德-布莱克范贫血（Diamond-Blackfan anemia, DBA）密切相关。为阐明GATA1 N端结构域的分子功能，我们针对表达GATA1短亚型（GATA1s）而非全长GATA1（GATA1FL）的Gata1突变胚胎来源的胎肝细胞，开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。scRNA-seq分析揭示了红细胞生成过程中的缺陷，并发现缺失GATA1 N端结构域会使糖酵解相关基因的表达上调，其中包括限速型丙酮酸激酶PKM。

为阐明PKM基因的调控机制，我们对急性敲除GATA1后的红细胞系细胞开展了精准核运行测序（precision nuclear run-on sequencing, PRO-seq）与靶标切割及核酸酶释放测序（cleavage under targets and release using nuclease, CUT&RUN），并证实PKM是GATA1的直接靶基因。

机制层面的研究显示，将全长GATA1（GATA1FL）替换为GATA1s，可使红细胞系祖细胞的糖酵解水平升高，但不会影响氧化磷酸化过程。

我们进一步发现，携带RPS19突变的戴蒙德-布莱克范贫血患者体内PKM的表达显著上调，这与GATA1通过调控糖酵解参与红细胞生成的功能相符。

综上，本研究结果证实GATA1不仅调控血红素代谢，还参与糖酵解过程的调控。

实验设计：将表达FKBP12F36V-GATA1的HUDEP2细胞（包含6F8与7C4两个克隆株）在分化诱导条件下培养24小时。于加入急性降解诱导剂dTAG-13（100 nM）后的0.5、3及6小时分别开展PRO-seq，以监测新生转录本的动态变化。