Author:
Parker Matthew P.,Dias Wagner B.,Brautman Will,Lowe Nick,Fedosyuk Halyna,Peterson Kenneth R.,Slawson Chad
Abstract
AbstractEukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, epigenetic chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia identifiedO-linked β-N-acetylglucosamine (O-GlcNAc) as an abundant chromatin post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. Thus, O-GlcNAc regulation ofcis-regulatory elements is essential for proper gene expression. O-GlcNAc is a single N-acetylglucosamine sugar attached to serine or threonine residues in nuclear, cytoplasmic, or mitochondrial proteins. Two enzymes cycle O-GlcNAc on or off protein; O-GlcNAc transferase (OGT) adds the modification, and O-GlcNAcase (OGA) removes it. O-GlcNAcylation responds to inputs from multiple metabolic and stress pathways including glucose, amino acid, fatty acid, and nucleotide metabolism. Therefore, O-GlcNAc acts as a sensor of cellular homeostasis able to link environmental conditions with gene transcription; however, decoding the precise function of millions of O-GlcNAc regulated elements remains challenging. Technologies to readily manipulate O-GlcNAcylation at specificcis-regulatory elements for functional analysis without pleiotropic consequences are lacking. We have employed novel CRISPR-based gene targeting tools to probe the function of O-GlcNAc regulatedcis-elements. First, we developed a programmable CRISPR-Cas9-based targeting system. This was accomplished by fusing a catalytically dead Cas9 (dCas9) to O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA), which allows for highly specific O-GlcNAc manipulation at chromatincis-regulatory elements. Previously, we demonstrated that O-GlcNAc plays a role in regulating humanAγ-globin gene expression by regulating CHD4 function and the formation of the NuRD (Nucleosome Remodeling and Deacetylase) complex at the -566 GATA repressor-binding site. Thus, as a proof of principle and to further explore the function of O-GlcNAc inγ-globin gene transcription, we targeted both dCas9-OGT and -OGA fusion proteins to theAγ-globin gene promoter. When dCas9-OGT or dCas9-OGA was targeted to the -566 GATA silencer site of theAγ-globin promoter, gene expression decreased or increased, respectively. This data strongly correlates with our previous findings and implicates O-GlcNAc cycling inγ-globin gene regulation. Importantly, this method can be employed to investigate O-GlcNAc events known to exist within the eukaryotic genome in a highly specific manner. Together, this tool will be fundamental in elucidating the function of O-GlcNAc in gene transcription.
Publisher
Cold Spring Harbor Laboratory