Mi2β-Mediated Silencing of the Fetal γ-Globin Gene in Adult Erythroid Cells

Abstract 825

Epigenetics has emerged as a key regulator of the fetal to adult hemoglobin switch during development. Understanding the mechanisms of fetal 𝛄-globin gene silencing offers the promise of effective targeted therapy of β- thalassemia and sickle cell anemia. Previous studies carried out by our group using adult erythroid cells from mice transgenic for a yeast artificial chromosome containing the entire human β-globin locus (β-YAC) and primary human erythroid cells have shown that methyl-CpG binding protein 2 (MBD2) is critical for full silencing of the fetal 𝛄-globin gene. MBD2 binds to methylated CpG rich promoters and silences the associated target genes by recruiting the Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. Absent or ≥75% decreased expression of MBD2 has been shown to have a 10–50 fold stimulatory effect on the expression of the 𝛄-globin gene in adult β-YAC transgenic mice and a 5–6 fold effect in CD34+ progenitor derived human erythroid cells in culture. Mi2, a major component of the NuRD complex, is an ATP-dependent chromatin remodeler consisting of two isoforms, Mi2a (also known as CHD3) and Mi2β (also known as CHD4). We have observed that 80% knockdown of Mi2β leads to a significantly higher expression of the 𝛄-globin gene in βYAC containing murine adult hematopoietic CID cells (∼100 fold) when compared to equivalent knockdown of other components of the MBD2/NuRD complex (∼5-10 fold), including MBD2. Remarkably, in CD34+ progenitor derived adult human erythroid cells, as little as 40% knockdown of Mi2β resulted in a ∼30 fold increase in 𝛄/𝛄 + β mRNA levels. Moreover, simultaneous knockdown of MBD2 and Mi2β resulted in no greater 𝛄-globin gene expression than Mi2β knockdown alone. This suggests that Mi2β is acting independently of as well as through its role in the MBD2-NuRD complex to exert its silencing effect on the 𝛄-globin gene. Complete conditional knockout of Mi2β in transgenic mouse hematopoietic cells has been shown by others to result in arrest of erythroid differentiation. We observed that in human CD34+ progenitor cells ≥80% Mi2β knockdown also altered differentiation. In contrast, 40–50% knockdown of Mi2β does not affect erythroid differentiation compared to scramble shRNA controls and resulted in 25 – 40% 𝛄/𝛄 + β globin RNA expression versus 2–3% in scramble shRNA controls. To determine possible mechanisms of Mi2β-mediated silencing outside of its role in the MBD2-NuRD complex, we examined the effect of Mi2β knockdown on two key mediators of 𝛄-globin gene silencing, Bcl11A and KLF-1 (EKLF). Our results showed that RNA and protein levels of these proteins are diminished by 40% and 70% respectively in cells with 40–50% Mi2β knockdown. Knockdown of MBD2 caused no decrease in either Bcl11A or KLF-1 levels. Thus at least part of the effect of Mi2β on 𝛄-globin gene silencing is through its positive effects on KLF-1 and Bcl11A expression. In summary, we have shown that less than a 50% decrease in Mi2β expression in primary human adult erythroid cells resulted in high levels of 𝛄/𝛄 + β gene expression without altering erythroid differentiation. The silencing effects of Mi2β occur both through the MBD2-NuRD repressor complex and through positive regulation of the KLF-1 and Bcl11A genes. These results also suggest that partial inhibition of Mi2β function could be of therapeutic benefit in the treatment of β- thalassemia and sickle cell anemia.