Ikaros drives human haemoglobin switching by facilitating active chromatin hub formation

The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5'--G-A- - β-3'. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of -globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the -globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the -globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease