ChIP analysis of epigenetic cofactor binding across the β-globin locus in SCD mice before and after RN-1 treatment. (A) Schematic diagram of the β-globin locus in SCD mice. A 9.7-kb DNA fragment containing the human ^Aγ-globin gene and human sickle β-globin (β^S) gene replaced the mouse β^major- and β^minor-globin genes.³³  Primers were designed to amplify discrete regions across the β-globin locus, including (I) the εy-globin promoter DR site, (II) the βh1-globin promoter DR site, (III) the β^major-globin upstream 5.9 kbp (presumptive negative control, (IV) the ^Aγ-globin promoter DR site, (V) a +3-kb region downstream of the ^Aγ-globin gene, and (VI) the β-globin promoter CAAT box (precise positions of the primer pairs used for these assays are given in supplemental Table 1). (B-H) ChIP analyses of LSD1, H3K4me2, H3K9me2, CoREST, TR2, TR4, and DNMT1 binding to each region of the β-globin locus (A) in BM cells of SCD mice before or after RN-1 treatment. The level of H3K4me2 and H3K9me2 is normalized to the fraction of total histone 3, whereas the level of all other proteins is normalized to control IgG. Data are presented as mean ± SD (n = 4-6 mice per group) (*P < .05; **P < .01 vs untreated SCD mice).