MYB represses ζ-globin expression through upregulating ETO2

Abstract

Reactivating the embryonic ζ-globin gene represents a potential therapeutic approach to ameliorate the severe clinical phenotype of α-thalassemia and sickle cell disease. The transcription factor MYB has been extensively proven to be a master regulator of the γ-globin gene, but its role in the regulation of ζ-globin remains incompletely understood. Here, we report a mechanistic study on the derepression of ζ-globin both in vivo and in vitro. We show that MYB depletion in mouse models and human hematopoietic stem cells leads to consistent and remarkable reactivation of ζ-globin. Furthermore, multiomics analysis and functional validation of MYB-knockout and wild-type cell lines reveal that ETO2 functions as a novel repressor of ζ-globin through coordination with NuRD nucleosome remodeling and the deacetylation complex to modulate histone deacetylation of ζ-globin. Additionally, we evaluate the clinical significance of these findings by knocking out ETO2 in primary CD34+ cells from nondeletional hemoglobin H patients, which results in a significant increase in ζ-globin expression. The RNA-seq data reveal that key erythroid genes are more co-regulated by Myb and Eto2 than by Myb and Klf1, highlighting a distinctly enhanced erythroid-specific transcriptional impact within the MYB-ETO2 regulatory axis. Compared with ETO2 knockout alone, codepletion of ETO2 and BCL11A did not significantly activate ζ-globin, suggesting that the MYB-ETO2 pathway primarily silences ζ-globin. Our study reveals a linear MYB-ETO2 signaling pathway crucial for ζ-globin repression and offers new targets for treating α-thalassemia and sickle cell disease.

1. Introduction

Thalassemia is among the most prevalent monogenic disorders worldwide and poses a significant health and economic burden. Approximately 1%–5% of the world’s population is reported to carry α-thalassemia mutations, and 3% is reported to carry β-thalassemia mutations [1–3]. Over the last three decades, considerable progress has been achieved in β-thalassemia treatment, highlighted by research on γ-β hemoglobin switching and pharmacological reactivation of fetal hemoglobin [4–10]. However, gaps are pronounced in the case of α-thalassemia. Mammalian α- and β-like globin genes, which are arranged on the basis of their expression during development, transition from embryonic/fetal to adult forms, reflecting their role across different gestational stages and erythroblast maturation sites. The α-like globin locus in both humans and mice harbors three functional genes, including an embryonic ζ-globin gene and two duplicated adult α-globin genes [11]. The embryonic ζ-globin gene is specifically expressed in primitive erythrocytes derived from the yolk sac, followed by transcriptional repression as a transition to definitive erythropoiesis occurs [12]. Previous experimental evidence has confirmed that the induction of embryonic ζ-globin, by acting as a substitute for deficient adult α-globin, rescues the viability of transgenic mouse models of α-thalassemia and sickle cell disease (SCD), suggesting a potential therapeutic approach for both conditions [13,14]. However, relatively limited attention has been given to the regulatory mechanisms of ζ-globin reactivation.

Erythropoiesis is a differentiation process in which hematopoietic stem cells progressively transform into mature red blood cells through a series of cellular events and the precise regulation of globin gene expression, which is highly regulated by specific transcription factors and growth factors. Substantial efforts have been made over the past few decades to investigate the genetic mechanisms underlying globin gene expression and fetal-to-adult hemoglobin switching, leading to the identification of BCL11A, MYB, KLF1 and LRF as the major modifiers in the regulation of γ-globin gene expression [15–17]. The transcription factor MYB plays an indispensable role not only in definitive erythropoiesis but also in its capacity to repress embryonic (ε-globin) and fetal gene (γ-globin) expression within the β-globin locus. The traditional model posits that MYB action occurs through KLF1, affecting the downstream factors BCL11A and LRF to regulate γ-globin gene expression [16,18–20]. Biallelic deleterious mutations in KLF1 reportedly cause hemolytic anemia with a dramatic increase in ζ-globin expression [21]. Recent studies provided evidence that BCL11A and LRF, two master γ-globin regulators, might have a direct effect on ζ-globin repression.