O‐GlcNAc Transferase and O‐GlcNAcase Interact with Mi2β at the Aγ‐Globin Promoter

• DOI: 10.1096/fasebj.30.1_supplement.803.3
• Publication Date: 2023-11-26T16:48:44Z
• Authors: Zhen Zhang; Flávia C. Costa; Ee Phie Tan; Nathan Bushue; Catherine E. Cost...; Mark E. McComb; Stephen A. Whelan; Kenneth R. Peterson; Chad Slawson

Sickle Cell Disease (SCD), caused by a point mutation in the adult β‐globin gene, leads to chronic damage to multiple organs and stroke, as well as cardiovascular abnormalities and dysfunction. However, SCD patients are phenotypically normal if they carry compensatory mutations that result in continued expression of fetal γ‐globin. Thus, a logical clinical goal for treatment of SCD is to up‐regulate fetal γ‐globin synthesis. One mode of γ‐globin silencing occurs at the GATA binding sites located at −566 or −567 relative to the Aγ‐globin or Gγ‐globin transcription start site, respectively, and is mediated through the DNA binding moiety GATA‐1 (GATA Binding Protein 1) and its recruitment of co‐repressor partners, FOG‐1 (Friend of GATA‐1) and Mi2β (CHD4, Chromodomain Helicase DNA Binding Protein 4). Post‐translational modification of transcription factors can regulate their activity. One such modification is O‐GlcNAcylation, which is the attachment of a single N‐acetyl‐glucosamine moiety to serine or threonine residues on nuclear, cytoplasmic and mitochondrial proteins. O‐GlcNAc is added to proteins by O‐GlcNAc transferase (OGT) and removed by O‐GlcNAcase (OGA). Here, using ChIP (Chromatin Immunoprecipitation) assays, we demonstrate that both OGT and OGA interact with the Aγ‐globin promoter when Aγ‐globin is repressed in wild‐type murine CID (Chemical Inducer of Dimerization)‐dependent human β‐YAC (β‐globin locus yeast artificial chromosome) bone marrow cells (BMCs). In addition, Mi2β and OGT are recruited to the −566 Aγ‐globin GATA silencer site in mouse embryonic day E18 fetal liver when Aγ‐globin is repressed. Furthermore, we demonstrate that OGT and OGA interact with Mi2β and FOG‐1, and Mi2β and FOG‐1 are O‐GlcNAcylated using co‐immunoprecipitation experiments. In order to confirm Mi2β O‐GlcNAcylation, we mapped one O‐GlcNAc site at S85 by mass spectrometry. Our data suggested that OGT and OGA can regulate GATA‐1/FOG‐1/Mi2β repressor complex activity at the Aγ‐globin promoter.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes for Health under grant P20 GM12345, National Institute of Diabetes and Digestive and Kidney Diseases R01 DK100595 to C. Slawson and K. Peterson, and by National Heart, Lung, and Blood Institute contract HHSN268201000031C and NIGMS grant P41 GM104603 to C. E. Costello.