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The Functional Role of O-GlcNAcylation in Gene Transcription: A New Approach to Study O-GlcNAc Regulated Gene Transcription
Parker, Matthew P
Parker, Matthew P
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Abstract
Sickle cell disease (SCD) and β-thalassemias are the most common monogenetic diseases diagnosed in the world. Increasing fetal hemoglobin (HbF) via activation of ?-globin chain synthesis is widely accepted as the most effective treatment for SCD. Repression of ?-globin expression in adult erythropoiesis is mediated by several multi-protein complexes, recruited to the promoters by DNA-binding moieties including BCL11A, TR-2/TR-4 (DRED), LRF, and GATA-1, all of which ultimately recruit the NuRD chromatin remodeling complex to establish complete gene silencing. The identification of druggable targets to reactivate HbF with specificity and without side effects remains a challenge. The role of post-translational modification (PTM) of proteins in regulating developmental and differentiation processes is understudied, but recently we established that O-GlcNAcylation regulates erythropoiesis. O-GlcNAc is a single N-acetylglucosamine sugar attached to serine or threonine residues in nuclear, cytoplasmic, or mitochondrial proteins. Two enzymes control O-GlcNAcylation, O-GlcNAc transferase (OGT), which adds the modification, and O-GlcNAcase (OGA), which removes it. O-GlcNAcylation responds to inputs from multiple metabolic and stress pathways including, glucose, amino acid, fatty acid, and nucleotide metabolism. Thus, O-GlcNAc acts as a general sensor of cellular homeostasis to regulate a wide variety of cellular processes including RNA polymerase function, epigenetic dynamics, and transcription factor activity.Our previous work demonstrated that O-GlcNAc plays a role in ?-globin gene transcription. Our published data showed that OGT and OGA interact with the GATA-1-FOG-1-NuRD repressor complex. Specifically, O-GlcNAcylation modulates the formation of the multi-protein NuRD repressor complex. OGT adds O-GlcNAc to CHD4, a subunit of NuRD, to stimulate the formation of this repressor complex, whereas removing this PTM by OGA results in the activation of ?-globin gene expression. In addition, the GATA-1 binding protein, GATAD2A, interacts with CHD4 and organizes it within the NuRD complex. The domain of GATAD2A that interacts with CHD4 is O-GlcNAcylated. Finally, the NuRD complex proteins MBD2, RBBP4, HDAC2, and MTA2 are also modified by O-GlcNAc; however, the function for these modifications remains unknown. Based on this data, we hypothesize that O-GlcNAcylation controls NuRD repressor assembly and is a novel ?-globin regulatory mechanism. More recently, we found that GATA-1, a master transcriptional regulator of erythropoiesis and hemoglobin switching, interacts with OGA and OGT at the onset of erythropoiesis. We investigated the role of O-GlcNAc in erythropoiesis using G1E-ER4 cells, in which the only copy of GATA-1 is fused to the estrogen receptor (GATA-1ER). These cells undergo erythropoiesis when β-estradiol is added to the culture. Transcriptome analysis of G1E-ER4 cells differentiated in the presence of the highly selective OGA inhibitor, Thiamet-G (TMG), identified expression changes in 433 GATA-1 target genes, including 47 erythroid-specific genes. In light of this data, we hypothesize that at the onset of erythroid lineage commitment, GATA-1 functions as an adaptor protein to deliver OGT and OGA to erythroid-specific cis-regulatory DNA elements, where OGT and OGA modify the O-GlcNAc status of bound protein complexes to direct transcriptional networks necessary for normal erythroid development and terminal differentiation. Testing these hypotheses presents many challenges. Technologies to readily manipulate O-GlcNAcylation at specific DNA loci for functional analysis without pleiotropic consequence are lacking. In recent years, CRISPR-Cas9-based technologies have enabled precise perturbation of DNA sequences and chromatin epigenetic regulatory elements to elucidate the role of various transcriptional regulatory elements. Thus, we hypothesis that similar systems can be developed to study chromatin and transcriptional O-GlcNAcylation, which would circumvent pleiotropic effects afflicting conventional genetic and pharmacological approaches. Here, we describe the development of a programmable CRISPR-Cas9-based system that allows for highly specific O-GlcNAc manipulation of chromatin elements. The tools described consist of nuclease-null Cas9 (dCas9) fused to OGT and OGA. Previously, we demonstrated that O-GlcNAc plays a role in regulating human ?-globin gene transcription. To validate our new CRISPR/Cas9 tools and to set the stage to explore the function of O-GlcNAc in ?-globin gene transcription, we targeted both dCas9-OGT and OGA fusion proteins to DNA sequences flanking the -566/567 GATA site of the ?-globin gene promoters. RT-qPCR data revealed an increase in ?-globin gene expression when dCas9-OGA was targeted to this area and a decrease in ?-globin gene expression when dCas9-OGT was targeted to identical sequences, further supporting our previous data, which implicates O-GlcNAcylation in ?-globin gene regulation. RT-qPCR data coupled with Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays are beginning to demonstrate the robust and highly specific nature of our targeting system, which can be employed to investigate the large number of O-GlcNAc events known to exist within the eukaryotic genomes.
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2021-01-01
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University of Kansas
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Biochemistry, Molecular biology, CRISPR/Cas9, Hemoglobin, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), O-GlcNAcylation, Transcription