O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis

Zhen Zhang,Matthew P Parker,Stefan Graw,Lesya V Novikova,Halyna Fedosyuk,Joseph D Fontes,Devin C Koestler,Kenneth R Peterson,Chad Slawson

doi:10.1074/jbc.ra118.005993

Abstract

The addition of a single β-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after β-estradiol (E2) addition. We observed that during G1E-ER4 differentiation, overall O-GlcNAc levels decrease, and physical interactions of GATA-1 with both OGT and OGA increase. RNA-Seq-based transcriptome analysis of G1E-ER4 cells differentiated in the presence of the OGA inhibitor Thiamet-G (TMG) revealed changes in expression of 433 GATA-1 target genes. ChIP results indicated that the TMG treatment decreases the occupancy of GATA-1, OGT, and OGA at the GATA-binding site of the lysosomal protein transmembrane 5 (Laptm5) gene promoter. TMG also reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. Sustained treatment of G1E-ER4 cells with TMG before differentiation reduced hemoglobin-positive cells and increased stem/progenitor cell surface markers. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for O-GlcNAcylation in regulating hematopoietic cell fate.

Highlights

The addition of a single ␤-D-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins
We demonstrate that 1) erythropoietic cell differentiation resulted in a marked reduction of total O-GlcNAc levels; 2) interaction among GATA-binding protein 1 (GATA-1)–ER, OGT, and OGA was enhanced after E2-induced differentiation; 3) perturbation of O-GlcNAc cycling affected 1,173 differentially expressed genes, including 47 erythroid-specific GATA-1 target genes; 4) OGA inhibition by Thiamet-G (TMG) led to an amplification of GATA-1–mediated activation or repression of GATA-1 target genes; 5) TMG treatment impaired myeloid cell differentiation into neutrophil-like cells; and 6) prolonged TMG treatment disrupted erythroid differentiation in G1E-ER4 cells
To determine whether O-GlcNAcylation is involved in erythroid differentiation, we treated GATA-1–ER cells with E2 to trigger erythroid differentiation

Summary

Introduction

The addition of a single ␤-D-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. TMG reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for OGlcNAcylation in regulating hematopoietic cell fate. GATA-1, a dual zinc finger DNA-binding protein that recognizes WGATAR motifs, is a key erythroid-specific transcription factor that coordinates erythropoietic gene programs during maturation [2,3,4,5].

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