| dc.description.abstract | O-GlcNAc is a post-translational modification on serine or threonine residues by β-N-acetylglucosamine. O-GlcNAc is added and removed from serine and threonine residues by the O-GlcNAc processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment and return to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis is critical to the regulation of many cellular functions. However, the relationship between O-GlcNAc homeostasis, OGT, OGA, and gene transcription is unknown. In this dissertation, I seek to address some of the fundamental mechanisms into the control of transcription by O-GlcNAcylation. First, I explored how changes in O-GlcNAc homeostasis affect the transcription of OGT and OGA. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT and increased OGA protein expression. OGT transcription level slightly declined, but OGA significantly increased with TMG treatment. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein in the presence of TMG; nor did TMG stabilize OGT and OGA transcription when cells were treated with RNA transcription inhibitor actinomycin D. RNA Pol II chromatin immunoprecipitation at the OGA promoter showed that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc. Next, we investigated how O-GlcNAcylation regulates Aγ-globin transcription. Erythropoiesis is the process of generating erythrocytes from erythroid progenitor cells. During this process, numerous genes are up-regulated or down-regulated. Transcription factor GATA-1 is the master regulator for erythropoiesis. However, the mechanism underling how GATA-1 regulate gene activation or repression is not well understood. Here, we utilized different cell and animal models with OGA inhibitor TMG to address this question. We first studied the Aγ-globin gene repression at adult stage mediated by GATA-1/FOG-1/Mi2β repressor complex at the -566 GATA site at the Aγ-globin gene promoter. We demonstrated that OGT and OGA interact with the Aγ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduced OGT and OGA promoter interactions in β-YAC bone marrow cells (BMCs). When WT β-YAC BMCs are treated with an OGA inhibitor Thiamet-G (TMG), the occupancy of OGT, OGA, and Mi2β at the Aγ-globin promoter was increased. In addition, OGT and Mi2β recruitment was increased at the Aγ-globin promoter when γ-globin becomes repressed in post-conception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we showed that Mi2β is modified with O-GlcNAc and both OGT and OGA interacts with Mi2β, GATA-1, and FOG-1. Taken together, our data suggested that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation, mediated by modulating the assembly of the GATA-1/FOG-1/Mi2β repressor complex at the -566 GATA motif within the promoter. Then, we asked if O-GlcNAc regulates GATA-1 target gene transcription during erythropoiesis. In this study, we utilized a well-established cell model of erythropoiesis, G1E-ER4 cells, a murine GATA-1 null erythroblast line that undergoes erythroid differentiation when GATA-1 activity is restored by the addition of β-estradiol (E2). Interestingly, overall O-GlcNAc levels dramatically decreased after GATA-1 activation in G1E-ER4 cells, with a slight increase in OGA and a decrease in OGT protein levels. GATA-1 interacts with OGT/OGA, and this interaction increased during erythropoiesis. Transcriptome analysis of G1E-ER4 cells treated with E2 and Thiamet-G (TMG, an OGA inhibitor) revealed that 1,173 genes changed expression patterns compared to E2 treatment only, including 433 GATA-1 target genes. These data suggest a subset of GATA-1 target genes are regulated through O-GlcNAcylation. Next, we demonstrated that the occupancy of GATA-1 and OGT/OGA, and the overall O-GlcNAc level at Laptm5 GATA binding site decreased when OGA was inhibited by TMG during erythropoiesis. Our data suggests that O-GlcNAcylation plays a role in regulating a subset of GATA-1 targeted erythroid genes and suggest O-GlcNAcylation as a mechanism regulating GATA-1 function at specific GATA-1 targeted genes. | |
