Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH

Abstract

Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the (A)γ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the (A)γ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of (A)γ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study γ-globin gene expression and may reveal new targets for selectively activating fetal hemoglobin.

Keywords: Globin gene; HPFH; fetal hemoglobin; hemoglobinopathies; sickle cell disease; transgenic mice.

Figure 1

Human β-globin locus 213 kb yeast artificial chromosome (β-YAC). Upstream of the HBE gene (ɛ) is the locus control region (LCR), defined as a set of four erythroid specific and developmentally stable DNase I-hypersensitive sites (5' HS). Another developmentally stable HS, termed 3' HS1, is present ∼20 kb 3' to the HBB gene (β). Also present in this construct are the yeast TRP1 gene, origin of replication, ARS1 (autonomously replicating sequence), a centromere, CEN1, the LYS2 gene, and a mammalian selectable marker cassette, MMTneo, encoding G418 resistance. Two restriction sites for EcoRI and four for SfiI relevant to generation and structural characterization of this YAC are indicated below the line. In the expanded square below the YAC map, the position and nucleotide changes of the three HPFH mutations included in this study are shown

Figure 2

RPA results of the human β-globin and ^Aγ-globin and mouse α-globin mRNA in the -175 #4, -175 #27, -175 #57, -195 #9, and -195 #18 HPFH mouse lines compared to the wild-type β-YAC line (negative control) and the -117 line (positive control)

Figure 3

Flow cytometry analysis of cells isolated from adult mouse blood samples labeled with HbF antibody conjugated with FITC. Each panel shows a comparison between a HPFH line (in blue) and the non-transgenic control mice sample (in red). The percentage shown in each graphic is the HbF-expressing cell population after subtraction of the non-transgenic (non-HbF-expressing) background. Data representative of duplicate experiments are shown

Figure 4

Chromatin Immunoprecipitation (ChIP) analysis using fetal liver samples of wild-type β-YAC transgenic mice (wild-YAC) and β-YAC transgenic mice carrying the HPFH -175 T > C mutation (HPFH -175 T > C), using anti-TAL1, anti-LMO2, and anti-LDB1 antibodies. For these three transcription factors, significant enrichment of occupancy (P < 0.05) in the HBG1 gene promoter region was verified only in the samples with the -175 T > C mutation. Data are shown as mean ± standard deviation for at least four experiments. * = P-value < 0.05; ** = P-value < 0.01; *** = P-value < 0.001