Repurposing the CRISPR-Cas9 System for Targeted Chromatin O-linked β-N- acetylglucosamine Editing

Abstract

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, epigenetic chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia identified O-linked {beta}-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 of cis-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 specific cis-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 regulated cis-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 chromatin cis-regulatory elements. Previously, we demonstrated that O-GlcNAc plays a role in regulating human A{gamma}-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 in{gamma} -globin gene transcription, we targeted both dCas9-OGT and -OGA fusion proteins to the A{gamma}-globin gene promoter. When dCas9-OGT or dCas9-OGA was targeted to the -566 GATA silencer site of the A{gamma}-globin promoter, gene expression decreased or increased, respectively. This data strongly correlates with our previous findings and implicates O-GlcNAc cycling in{gamma} -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.