Generation of Non-Deletional Hereditary Persistence of Fetal Hemoglobin (HPFH) Beta-Yac Transgenic Mouse Models: -175 Black HPFH and -195 Brazilian HPFH

Fetal hemoglobin (HbF) is a major genetic modifier of the phenotypic heterogeneity in patients with the major β-globin disorders sickle cell disease (SCD) and β-thalassemia. Although the normal level of HbF postnatally is approximately 1% of total hemoglobin, some individuals have a condition known as hereditary persistence of fetal hemoglobin (HPFH), characterized by elevated synthesis of γ-globin in adulthood. HPFH is caused by small or large deletions in the β-globin locus (deletional HPFH), or point mutations in the ^Aγ-globin or ^Gγ-globin gene promoters (non-deletional HPFH). Pharmacological agents such as butyrate, decitabine, and hydroxyurea are effective in inducing HbF in vitro and in vivo. To date, hydroxyurea is the only drug approved for clinical use in sickle cell patients, although the efficacy level is variable between patients and the long-term effects of this drug remain uncertain. Therefore, current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin gene expression in adult life. Many studies have demonstrated the role of stage-specific transcription factors in β-like globin gene switching, indicating their potential as therapeutic targets in the treatment of β-hemoglobinopathies. In order to better understand the molecular pathways involved in the regulating γ-globin gene expression, we used β-YAC transgenic mice, produced with a 213 Kb β-globin locus yeast artificial chromosome, containing a 187 Kb human chromosomal insert encompassing the entire 82 Kb β-globin locus from 5’HS5 of the LCR to 3’HS1, approximately 20 Kb downstream from the β-globin gene. Four different transgenic mouse lines were included in this study: 1) wild β-YAC mice, with the normal sequence of the human β-globin locus; 2) mutant β-YAC mice with the ^Aγ-globin -117 G>A HPFH mutation 3) mutant β-YAC mice with the ^Aγ-globin -175 T>C HPFH mutation, and 4) mutant β-YAC mice with the ^Aγ-globin -195 T>C HPFH mutation. Adult -175 and -195 mutant β-YAC mice displayed an HPFH phenotype with an increased level of HbF. As measured by HPLC, -175 HPFH mice had the highest average level of γ-globin chains [16.4% γ/(γ+β)], followed by -195 HPFH mice (8.4%).  Wild-type β-YAC control mice averaged 2.8% and -117 Greek HPFH β-YAC control mice displayed an average of 7.4%. Measurement of ^Aγ-globin mRNA by RNase protection analysis (RNAP) supported the HPLC data; γ/(γ+β) was 34%, 12.1%, 14.1% and less than 0.5% for -175 HPFH, -195 HPFH, -117 HPFH and wild-type β-YAC animals, respectively. Relative mRNA levels as determined by RT-qPCR were consistent with the RNAP results. Currently, we are examining our -175 and -195 HPFH mice for pancellular versus heterocellular distribution of HbF. To examine the molecular basis for the -175 and 195 HPFH phenotypes, fetal livers of these animals were collected on day E18 of gestation, after the fetal-to-adult β-like globin switch occurred, for chromatin immunoprecipitation (ChIP) analysis of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2 and LDB1. Previous unpublished DNA-protein array and ChIP data, comparing human primary erythroid cell cultures from normal donors and -195 HPFH individuals, showed a 6-fold enrichment of YY1 recruitment to the -195 region of the normal ^Aγ-globin promoter and a 5-fold enrichment of PAX1 recruitment to the HPFH mutant promoter, suggesting that YY1 may act as an ^Aγ-globin gene repressor and PAX1 may be an activator when the -195 mutation is present. Preliminary ChIP experiments in β-YAC mice showed a similar pattern with YY1 enriched 2-fold in wild-type mice and PAX1 enriched 2-fold in -195 HPFH animals. Regarding -175 HPFH and wild-type β-YAC samples, we found occupancy enrichment of LMO2, TAL1 and LDB1 proteins (1.5-fold, 9-fold and 2.5-fold, respectively) in the -175 region of the ^Aγ-globin gene promoter in -175 HPFH β-YAC mice. Recently published studies in cell lines have shown that these three proteins form a complex with GATA-1 to mediate long-range interactions between the LCR and β-like globin genes. These mouse models provide additional tools for studying the regulation of γ-globin gene expression and may reveal new targets for selectively activating HbF.