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, chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia have identified O-linked β-N-acetylglucosamine (O-GlcNAc) as an abundant post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, chromatin dynamics, and cofactor activity. Decoding the precise function of all the intracellular O-GlcNAc-regulated elements identified in these studies remains challenging. Technologies to manipulate O-GlcNAcylation at specific DNA loci for functional analysis without pleiotropic consequences were lacking, but the advent of CRISPR-Cas9-based technologies has enabled the precise perturbation of DNA sequences and delivery of proteins to specific cis-acting DNA regulatory elements to elucidate their role in transcription. We reasoned that a CRISPR-Cas9 system could be developed to study the function of O-GlcNAcylation in transcription, which would circumvent the problems inherent in conventional gene manipulation and pharmacological approaches. We developed a programmable CRISPR-Cas9-based system that allows for O-GlcNAc manipulation of transcriptional complex function at specific cis-regulatory elements. The tools consist of nuclease-null Cas9 (dCas9) fused to O-GlcNAc transferase (OGT), which adds the O-GlcNAc modification, or O-GlcNAcase (OGA), which removes the modification. Previously, we demonstrated that O-GlcNAc plays a role in regulating human □-globin gene transcription. Increasing fetal hemoglobin (HbF) via activation of □-globin chain synthesis is widely accepted as the most effective treatment for SCD and certain types of β-thalassemias. O-GlcNAcylation modulates the formation of a GATA-1-FOG-1-NuRD repressor complex that binds the -566 GATA site of the A□-globin promoter when □-globin gene expression is silent. OGT and OGA interact with GATA-1 and CHD4, a component of the NuRD complex. O-GlcNAcylation of CHD4 stimulates the formation of this repressor complex, whereas removing this PTM results in activation of A□-globin gene expression. As proof of principle to demonstrate the utility of our system, we targeted dCas9-OGT or dCas9-OGA fusion proteins to DNA sequences flanking the -566 GATA site of the A□-globin gene promoter. Increased □-globin gene transcription was measured by RT-qPCR when dCas9-OGA was targeted to this area, whereas □-globin gene expression decreased when dCas9-OGT was targeted to the identical sequences. These data support our published work, confirming that O-GlcNAcylation regulates A□-globin gene transcription. RT-qPCR data coupled with Cut and Run assays demonstrate the robust and highly specific nature of our targeting system, which can be employed as a generally applicable tool to study the role of O-GlcNAcylation in transcriptional regulation.