Investigations of the gene regulatory protein, ZBTB7A, and its role in red blood cells

Abstract

Haemoglobin, the oxygen carrying protein in erythrocytes, is composed of two alpha- and two beta-like globin subunits. Its composition changes during gestation to meet varying oxygen demands of the developing embryo. A developmental switch occurs at birth, where foetal γ-globin expression is replaced by adult β-globin. Mutations in the γ-globin promoter can impair the switch and thereby cause elevated levels of foetal haemoglobin in adulthood, known as hereditary persistence of foetal haemoglobin (HPFH). This condition alleviates symptoms of adult β-globin disorders such as β-thalassemia and sickle cell disease. Reactivation of γ-globin expression currently offers a promising therapeutic approach for these diseases. A group of HPFH mutations clustered approximately 200 basepairs upstream of the γ-globin gene (-200 site) are believed to disrupt binding of transcriptional repressors of γ-globin expression. We have previously demonstrated that ZBTB7A, a transcriptional repressor highly expressed in erythroid cells and required for normal erythroid development, is able to bind this site in vitro, and binding is abolished by mutations in the -200 site. The purpose of this study was to further analyse the binding of ZBTB7A in vivo, as well as determine how it is regulated in erythroid cells. Here we use ChIP-Seq to demonstrate that ZBTB7A binds the γ-globin -200 site in cultured cells. Previous results suggested that Zbtb7a is regulated by the master regulator of erythropoiesis, KLF1, however, whether this effect was direct or mediated through other KLF1 target genes was unknown. We use a Klf1-/- mouse model, and the KLF1 inducible cell line, K1ER, to show that Zbtb7a is directly activated by KLF1. We also find that ZBTB7A has a novel transcription start site in both humans and mice that is highly expressed in erythroid cells and is induced upon erythroid cell differentiation. These findings suggest ZBTB7A plays a role in haemoglobin switching. As ZBTB7A is an important factor in erythroid maturation and survival, completely abolishing its expression may be detrimental. However, it may be possible to target the erythroid specific transcript to reduce ZBTB7A expression in erythroid cells alone, which may be sufficient to boost foetal haemoglobin as a potential therapeutic for β-haemoglobinopathies.