Role of AP1/NFE2 binding sites in endogenous α-globin gene transcription by Melanie R. Loyd, Yasuhiro Okamoto, Mindy S. Randall, and Paul A. Ney Blood Volume 102(12):4223-4228 December 1, 2003 ©2003 by American Society of Hematology

Site-directed mutagenesis of AP1/NFE2 binding sites in HS-26. Site-directed mutagenesis of AP1/NFE2 binding sites in HS-26. (A) AP1/NFE2 binding sites (stars) are replaced with a neo gene, which is flanked by LoxP sites (triangles), through homologous recombination (HR). This is the floxed mutation. The selectable marker is removed by Cre-mediated recombination (Cre). This is the ΔNFE2 mutation. Solid boxes represent exons of the gene C16orf35. The restriction enzyme sites are StuI (S), HindIII (H), EcoRI (E), XbaI (X), Pst I (P), EcoRV (V), and Bgl I (B). (B) Murine α-globin locus with the sequence of the ΔNFE2 mutation. (C) Southern blot analysis of tail DNA from F2 mice. With HindIII, an external probe (▦) hybridizes to a 4.9-kb wild-type band (WT) or a 6.7-kb band (floxed). With XbaI, an internal probe (▨) hybridizes to a 1.8-kb band (WT) or a 2.4-kb band (ΔNFE2). The probes are shown in panel A. Melanie R. Loyd et al. Blood 2003;102:4223-4228 ©2003 by American Society of Hematology

Floxed mutation causes α-thalassemia. Floxed mutation causes α-thalassemia. (A) Dissected E14.5 embryos. HS-26 genotypes are indicated at the top. (B) Blood smears from adult mice, stained with Wright-Giemsa (original magnification, × 100). Note the target cells, hypochromic cells, and prominent fragments in the blood from the floxed/floxed mice. A white blood cell (purple) is shown in each panel for size comparison. Melanie R. Loyd et al. Blood 2003;102:4223-4228 ©2003 by American Society of Hematology

Allele-specific RNA PCR Allele-specific RNA PCR. (A) The RNA PCR product from both adult α-globin genes of most non-AKR strains is bisected by an MspI restriction site. Allele-specific RNA PCR. (A) The RNA PCR product from both adult α-globin genes of most non-AKR strains is bisected by an MspI restriction site. (B) Allele-specific RNA PCR on blood from HS26+(nonAKR)/+(AKR) non-AKR × AKR F1 mice. Controls are water (W) and C57BL6 (B6). (C) Allele-specific RNA PCR on bone marrow from HS26floxed(nonAKR)/+(AKR), HS26+(nonAKR)/+(AKR), and HS26ΔNFE2(nonAKR)/+(AKR) non-AKR × AKR F1 mice. Melanie R. Loyd et al. Blood 2003;102:4223-4228 ©2003 by American Society of Hematology

HS-26 is formed in the absence of AP1/NFE2 binding sites. HS-26 is formed in the absence of AP1/NFE2 binding sites. (Top) Diagram of 4.9-kb wild-type HindIII DNA fragment. Cleavage at HS-26 yields a 3.6-kb band in Southern blots. The probe (▦) and other symbols are the same as in Figure 1. (Bottom) DNase I hypersensitivity in E14.5 fetal liver cells. Genotypes are indicated at the top. Melanie R. Loyd et al. Blood 2003;102:4223-4228 ©2003 by American Society of Hematology

Assay for activators. Assay for activators. In fetal liver cells from HS-26ΔNFE2(nonAKR)/+(AKR) embryos, the activity of the wild-type allele of HS-26 can be directly compared with the mutated allele, under different trans-acting conditions. This assay can be used to determine the ability of a transcription factor to function through the intact AP1/NFE2 binding sites. Melanie R. Loyd et al. Blood 2003;102:4223-4228 ©2003 by American Society of Hematology