GSE122104
O-GlcNAc Homeostasis Controls Cell Fate Decisions During Hematopoiesis
The addition of O-GlcNAc (a single ß-D-N-acetylglucosamine sugar at serine and threonine residues) by O-GlcNAc transferase (OGT) and removal by O-GlcNAcase (OGA) maintains homeostatic levels of O-GlcNAc. We investigated the role of OGlcNAc homeostasis in hematopoiesis utilizing G1E-ER4 cells carrying a GATA-1 transcription factor fused to the estrogen receptor (GATA-1ER) that undergo erythropoiesis following the addition of ß-estradiol (E2) and myeloid leukemia cells that differentiate into neutrophils in the presence of all-trans retinoic acid. During G1E-ER4 differentiation, a decrease in overall O-GlcNAc levels and an increase in GATA-1  interactions with OGT and OGA were observed. Transcriptome analysis on G1E-ER4 cells differentiated in the presence of Thiamet-G (TMG), an OGA inhibitor, identified expression changes in 433 GATA-1 target genes. Chromatin immunoprecipitation demonstrated that the occupancy of GATA-1, OGT, and OGA at Laptm5 gene GATA site was decreased with TMG. Myeloid leukemia cells showed a decline in O-GlcNAc levels during differentiation and TMG reduced the expression of genes involved in differentiation. Sustained treatment with TMG in G1E-ER4 cells prior to differentiation caused a reduction of hemoglobin positive cells during differentiation. Our results show that alterations in O-GlcNAc homeostasis disrupt transcriptional programs causing differentiation errors suggesting a vital role of O-GlcNAcylation in control of cell fate. Overall design: To determine whether the erythroid gene transcription network was altered by disruptions in O-GlcNAc homeostasis during G1E-ER4 differentiation. We performed Next Generation RNAsequence analysis on G1E-ER4 cells without treatment (Ctrl), with TMG or E2 treatment only, and E2+TMG treatment for 30 hrs.  Thiamet G (TMG) is a highly selective inhibitor of OGA that blocks the enzymes ability to remove the O-GlcNAc moiety. Cells treated with TMG lose the ability to remove the O-GlcNAc moiety leading to increased cellular O-GlcNAc levels, and ultimately alters O-GlcNAc cycling. Cells were harvest and total RNA was isolated by TRI Reagent (Sigma) for library preparation. Briefly, cDNA library was prepared using Illumina TruSeq Stranded mRNA sample preparation kit (Illumina) as manufacturer's instruction. 800 ng of the total RNA was used for each library preparation. The quality of total RNA was evaluated by Agilent 2100 Expert bioanalyzer and RNA with RIN = 9.80 was used. The libraries were sequenced paired-end using Illumina HiSeq2500. FastQC (0.11.2) and RSEM (1.2.22) software were used to assess the quality of the RNA sequencing results, align the reads to the mouse genome reference GRCm38/mm10, and calculate the gene expression values. R (3.2.2) and EdgeR (3.14.0) software were used to normalize the expression values using the TMM-method (weighted trimmed mean of M-values), followed by differential expression analysis. To reduce the burden of multiple testing in differential gene expression analyses, a filter was initially applied to reduce the number of genes. Genes were removed if they did not present a meaningful gene expression across all samples; only genes with cpm (counts per million) of >10 in least two samples were considered in differential expression analyses. The Benjamini and Hochberg procedure was used to control the false discovery rate (FDR).