Transcription level of Ey directly regulates βh0. (A) Representative gel of βh0 expression in e10.5 wild-type and TR2/TR4 transgenic yolk sacs. Genotype of each sample is marked above the gel, and 2 TR2/TR4 transgenic lines are noted as line 1s and 2. All samples were digested with BslI. (B) Quantitation of βh0 expression in e10.5 wild-type and 2 different TR2/TR4 transgenic lines. Error bars represent SD of at least 3 embryos of that genotype.

HPLC assays identify a new peak in peripheral primitive erythroid cells from ΔEyΔβh1 embryos. (A) HPLC of cell lysate of e10.5 circulating blood from wild-type D/S embryo shows identified elution peaks from the wild-type D and S alleles. Ey1 is from the S allele, and Ey2 is from the D allele. The βh1 peak includes protein from D and S, and alpha and zeta are α-like globin chains. (B) HPLC of cell lysate of d10.5 circulating blood from ΔEyΔβh1/S embryo. Identified elution peaks include alpha and zeta and from the S allele, Ey1, and βh1. The Ey2 peak from D is missing. A peak represents β-major and β-minor (beta), and a new peak (?) represents a novel β-like globin chain. (C) HPLC of cell lysate of mixed e10.5 circulating blood from wild-type D/S and ΔEyΔβh1/S embryos shows that the new peak (?) is distinguished from Ey1 and Ey2.

Activation of βh0 transcription is influenced by multiple parameters in mice with reduced Ey transcription. (A) Schematic map of ΔEyhygro allele. (▨) Selectable marker gene PGK-Hygro. (arrow) Its transcriptional direction. Representative gel and quantitation of βh0 expression in e10.5 wild-type D/S and ΔEyhygro/S yolk sacs. Error bars represent SD of at least 3 littermates with the genotype marked throughout. (B) Schematic map of ΔEy(inv)Δβh1 allele. (■) βh0 gene that was relocated 5 kb further away from the LCR on the mutant allele compared with the wild-type allele. (arrow) Inversion also changed the transcription direction of the βh0 gene on this mutant allele. Representative gel and quantitation of βh0 expression in e10.5 wild-type D/S and ΔEyΔβh1(inv)/S yolk sacs. (C) Comparative summary of βh0 transcription in various mutants in which it has increased transcription.

βh0 is activated exclusively during primitive erythropoiesis in embryos with the promoter of Ey deleted. (A) Steady state RT-PCR strategy to detect and quantitate transcription from βh0. PCR primers were perfect matches for βh0 and βh1 cDNA. (asterisk) The amplified region contained one restriction enzyme site in βh0 (BslI) that was not present in βh1 cDNA. Amplified cDNA from e10.5 yolk sacs of wild-type D/S and (B) ΔEyΔβh1/S, (C) ΔEy/S, and (D) Δβh1/S. All samples were digested with BslI. (E) Quantitation of βh0 expression in each mutant genotype comparing the ratio of βh1 (uncut) and βh0 (cut), adjusted for the number of active βh1 alleles. Error bars represent SD of at least 3 embryos of that genotype in a litter.

Deletion of either or both embryonic gene promoter(s) increased transcription of β-major/minor in primitive erythroid cells. (A) Representative RT-PCR/RFLP assay of β-major/minor transcription in e10.5 ΔEy/S, Δβh1/S, and ΔEyΔβh1/S yolk sac primitive erythroid cells. Sample genotypes are marked above the gel. (B) Quantitation of β-major/minor transcription in yolk sac of promoter deletion mutants. Each pair of bars represents data from 2 litters, with a total of at least 12 embryos with error bars denoting SD. Mutations assayed and days of gestation are indicated below the graph. Wild-type ratios from e10.5 and e11.5 were close to 1 and were normalized to 1, and the mutant ratio was similarly adjusted. Wild-type ratios from e9.5 were not normalized and are represented as simple D/S ratios from each genotype. All mutant ratios are statistically different from wild-type ratios (P > .05) for e10.5 and e11.5 but not e9.5. (C) Representative RT-PCR/RFLP assay and (D) quantitation of β-major/minor transcription in ΔEy/S, Δβh1/S, and ΔEyΔβh1/S adult peripheral blood. Genotype of each sample is marked above the gel. Each pair of bars represents data from least 5 adult mice. Wild-type D/S ratios were normalized to 1.

βh2 and βh3 are not expressed in any of the promoter-deletion embryos. (A) Schematic map of the D allele with the highly expressed β-like globin genes as dark bars and genes potentially expressed at low levels as white bars. (B) RT-PCR analysis of βh2 transcription in cDNA from e10.5 yolk sac. Lanes with genomic DNA (50 ng) are labeled G, marker lanes are labeled M, and genotypes of yolk sac samples are labeled. Expected sizes of PCR products from the βh2 cDNA and genomic DNA are marked. Genomic bands verify the ability to amplify the βh2 cDNA if it was present. (C) RT-PCR analysis of βh3 transcription in cDNA from e10.5 wild-type D/S (lanes 1-3), Δβh1/S (lanes 4, 5), ΔEy/S (lanes 6, 7), and ΔEyΔβh1/S (lanes 8, 9) yolk sac. G represents genomic DNA. PCR primers preferentially amplified cDNA from βh3 and, less efficiently, from Ey (2 mismatches on both primers). Uncut bands from any gene measured 96 bp. βh3 can be distinguished from Ey because the βh3 amplicon cut with NcoI to 64 bp but not with PvuII and the Ey amplicons cut with PvuII to 75 bp but not with NcoI.

Deletion and Southern blot strategies to generate ΔEyΔβh1 promoter deletion mice. (A, I) Schematic map of the Hbb(d) allele. (arrows) LCR hypersensitive sites. (■) β-like globin genes. (II) Ey and βh1 genes with exons marked. Deleted regions from each gene are indicated by solid lines under the gene names. (III) Ey promoter was replaced by PGK-hygro, and βh1 promoter was replaced by PGK-neo. (▴) Selectable markers were each flanked by 2 convergent lox P sites and were removed by Cre recombinase-mediated deletion. (arrows) Selectable marker transcriptional start and orientation (IV). Cre recombinase also mediated an additional inversion of DNA between the 2 LoxP sites left from the deletion of the selectable markers and resulted in 2 double-promoter deletion alleles, ΔEyΔβh1 and ΔEy(inv)Δβh1. (arrows) Transcriptional orientation of βh0 on each allele. (B) Southern blot strategies and representative Southern blot used to screen for ΔEyhygroΔβh1neo ES clones modified in cis after homologous recombination. Restriction sites for SpeI are marked as S. The probe is denoted as a short line under each map. Note that the expected band for wild-type Hbb(d) measures 42.2 kb, which is not shown on the gel. Each lane represents a single ES clone. (C) Southern blot strategies and representative Southern blot to screen for ΔEyΔβh1 and ΔEy(inv)Δβh1 promoter deletion alleles in mice after Cre-mediated selectable marker deletion in vivo. Restriction sites for NsiI are marked as N, and the probe used is a line underneath each allele. Expected band sizes for each genotype are marked accordingly.