An Essential Role for the N-Terminus of GATA1 in Metabolic Regulation during Erythropoiesis

Several lines of evidence confirm that the N-terminus of GATA1 plays a pivotal role in normal erythroid differentiation, including the phenotype of patients with N-terminal truncations, the inability of GATA1 N-terminal deleted iPSCs to generate erythroid cells, and red blood cell defects in mice expressing only the short isoform (known as GATA1s). However, the molecular mechanisms underlying the erythropoietic defects associated with GATA1 N-terminal mutations remain poorly understood.

We performed single-cell multi-omics on total fetal livers of Gata1s E13.5 embryos and, similar to prior flow cytometry-based assays, identified an accumulation of early erythroblasts and a deficiency of late-stage mature erythroblasts. Gene set enrichment analysis revealed disturbances in metabolic pathways in Gata1s erythroblasts, particularly at the basophilic and polychromatic erythroblast stages. Specifically, we observed enrichment of glycolytic pathways with a prominent increase in expression of pyruvate kinase M2 (PKM2), a rate-limiting pyruvate kinase that catalyzes the last step within glycolysis. To assess the relevance of these observations in murine cells to humans, we studied the effect of GATA1 modulation on growth and differentiation of HUDEP-1 cells, a cell line model widely used to study human erythropoiesis. We found that loss of both full length GATA1 and GATA1s suppressed growth and differentiation, while loss of full length with persistent expression of GATA1s led to similar defects. Of note, we were able to successfully derive HUDEP-1 clones that lack GATA1, but only when GATA1s was overexpressed, supporting the model that high levels of GATA1s can rescue steady state growth of erythroblasts; however, GATA1s HUDEP-1 cells failed to differentiate. RNA-sequencing revealed that GATA1s HUDEP-1 cells express significantly higher levels of PKM2 along with an enrichment in glycolytic pathways. Seahorse Glycolysis Stress Test assays confirmed that selective expression of GATA1s promotes glycolysis of the modified HUDEP-1 cells. Increased total PKM2 protein and its phosphorylation were confirmed by western blot analysis, and pyruvate kinase activity assays further corroborated the hyperactivation of glycolysis in HUDEP-1 cells expressing only GATA1s. We also generated HUDEP-2 cells with an FKBP12^F36V-tagged GATA1, which can be inducibly degraded by addition of the dTAG (degradation tag) ligand. PKM nascent transcripts were markedly elevated upon GATA1 degradation and CUT&RUN experiments confirmed that PKM is a direct target of GATA1. Importantly, consistent with a detrimental effect of elevated PKM2 activity on erythropoiesis, we found that PKM2 activation suppressed erythroid colony formation of wild-type induced pluripotent stem cells.

Next, to molecularly define the requirement for the N-terminus, we performed co-immunoprecipitation followed by mass spectrometry on differentiated HEL cells expressing epitope tagged GATA1 or GATA1s. This study revealed associations between GATA1 and various epigenetic complexes, including NuRD, SWI/SNF, and REST/CoREST, emphasizing their potential roles in GATA1-mediated chromatin regulation. Of note, follow-up co-IP assays suggested that GATA1s binds the SWI/SNF complex to a lesser extent than full length GATA1, implying that the impaired activation potential of GATA1s may in part be due to decreased recruitment of SWI/SNF to chromatin. Finally, we observed altered degrees of histone modifications, including H3K4me3, H3K4me1, H3K27me3, and H3K27ac, as well as differential chromatin accessibility in Gata1s murine fetal erythroblasts and differentiated HUDEP-1 cells expressing only GATA1s. We also observed that GATA1 recruits H3K27ac better than GATA1s on its targets, while GATA1s retains H3K4me3 and H3K27me3 during differentiation.

Together our findings suggest that the loss of the GATA1 N-terminus leads to gene expression dysregulation through the failure of epigenetic regulation during erythroid maturation. In addition, our data shed light on the complex molecular mechanisms underlying GATA1 N-terminal defects in erythropoiesis and identify a novel role for GATA1 in the regulation of glycolysis.

Crispino:Cellarity: Consultancy; Syndax: Research Funding; Alethiomics: Membership on an entity's Board of Directors or advisory committees.