Fine Tuning of Hemoglobin Switching and Erythropoiesis

Sickle cell disease (SCD) and β‐thalassemias are the most common monogenetic diseases diagnosed in the world. Increasing fetal hemoglobin (HbF) via activation of γ‐globin chain synthesis is widely accepted as the most effective treatment for SCD. Repression of γ‐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 remains a challenge. Understanding the role of post‐translational modifications (PTMs) in erythropoiesis and hemoglobin switching may lead to the development of novel therapeutic approaches. One PTM that functions in reactivation of γ‐globin is O‐GlcNAcylation (O‐GlcNAc). 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 functions including transcription, translation, and signaling. We hypothesize that O‐GlcNAcylation modifies transcriptional complexes that affect gene expression during erythropoiesis. We investigated the role of O‐GlcNAc in erythroid differentiation using mouse models and erythroid cell lines. Our work demonstrates that; 1) CHD4, a component of NuRD, is modified by O‐GlcNAc at T1041, 2) OGT and OGA interact with CHD4, 3) alterations in O‐GlcNAc cycling change NuRD‐regulated β‐like globin gene expression, and 4) alterations in O‐GlcNAcylation alter erythroid cell differentiation. 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. Now, we will report on novel CRISPR/Cas9‐based OGA and OGT genome tools that target O‐GlcNAcylation to transcription complexes bound at specific DNA cis‐regulatory elements. This new platform will allow for the precise targeting of OGA and OGT to GATA‐1 target genes without perturbing O‐GlcNAc levels globally, which, until now, has not been possible. These new tools will provide new insight regarding the role of O‐GlcNAc in the modulation of erythropoiesis and hemoglobin switching.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.