Alterations in expression and chromatin configuration of the alpha hemoglobin-stabilizing protein gene in erythroid Kruppel-like factor-deficient mice

Abstract

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the beta-globin promoter, where it establishes local chromatin structure permitting beta-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the beta-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and beta-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

FIG. 1.

Target gene expression in wild-type and EKLF-deficient fetal livers. (A) RPA of wild-type and EKLF-deficient fetal liver RNA demonstrated a 95% ± 6.2% decrease in AHSP mRNA in EKLF-deficient cells. (B) Western blot analysis of fetal liver proteins with an anti-AHSP monoclonal antibody. Recombinant AHSP (rAHSP) was added as a positive control. Virtually no AHSP was found in EKLF-deficient cells.

FIG. 2.

Hypersensitive site mapping across the AHSP locus. (Upper panel) In chromatin from E13.5 wild-type fetal liver nuclei digested with DNase I and BamHI (B), a strong DNase I HS was found in the 5′ flanking DNA of the AHSP gene generated from a 2.9-kb parent band (PB). (Lower panel) This HS site was absent in chromatin from EKLF-deficient fetal liver.

FIG. 3.

Histone acetylation across the murine AHSP locus in vivo. (A) Locations of primers used for quantitative PCR amplification after chromatin immunoprecipitation of wild-type and EKLF-deficient fetal liver chromatin. The DNase I hypersensitive site in the core promoter region is denoted by the arrow. (B) Pattern of acetylation of diacetylated histone H3. (C) Pattern of acetylation of tetraacetylated histone H4. In panels B and C, differences between wild-type and EKLF-deficient chromatin with P values of <0.15 are denoted by asterisks and the values are provided in Table 5.

FIG. 4.

Gel mobility shift assays of the EKLF consensus binding sites in the 3′ region of the AHSP gene. Gel mobility shift assays using oligonucleotide probes corresponding to the EKLF consensus binding sites in the 3′ flanking region of the murine AHSP gene were performed using recombinant EKLF protein (rEKLF). Results with site 1 are shown. A β-globin promoter-proximal CACCC box probe was used as a positive control. Excess, unlabeled probe or EKLF antibody was added where indicated. +, present; −, absent.

FIG. 5.

EKLF occupancy across the murine AHSP locus in vivo. Quantitative chromatin immunoprecipitation across the AHSP locus was performed with fetal liver chromatin from mice with an HA-EKLF-TAP-tagged knock-in allele. Quantitative PCR amplification was performed with the primers shown in Fig. 3A.

FIG. 6.

Gel mobility shift assays of the AHSP promoter CACCC box. Gel mobility shift assays using oligonucleotide probes were performed using recombinant EKLF protein (rEKLF). Excess, unlabeled probe or EKLF antibody was added where indicated. (A) Probes corresponding to the AHSP promoter CACCC box and the β-globin promoter-proximal CACCC box. (B) A β-globin CACCC box probe with position 1 mutated from C to T to mimic the AHSP CACCC box. (C) Probes corresponding to the AHSP promoter CACCC box, the wild-type β-globin CACCC box, the β-globin promoter CACCC box with position 1 of the EKLF consensus sequence mutated to the other 3 possible nucleotides, and the γ-globin promoter CACCC box. +, present; −, absent.

FIG. 7.

Quantitative electrophoretic mobility shift assays of the AHSP promoter CACCC box. (A) Competitive electrophoretic mobility shift assays were performed with the β-globin promoter CACCC box as a probe. Different unlabeled probes are assayed for their ability to compete the EKLF-CACCC complex. The amount of complex without competitor is defined as 100%. The points where curves cross the 50% line of the percent signal remaining was used as estimate the competitive ability of each oligonucleotide probe for binding to EKLF relative to the wild-type β-globin promoter CACCC box. β-thal, β-thalassemia. (B) EKLF protein titrations with β-globin and AHSP CACCC boxes. To determine the K_D for the interaction between EKLF and each CACCC box, EKLF protein titrations were performed, gels scanned, and K_D calculated with the rearranged mass action equation, PD/D_t = 1/(1 + K_DP), using nonlinear least-square analyses. A sample gel with a wild-type β-globin promoter CACCC box probe is shown.

FIG. 8.

EKLF transactivates the AHSP promoter in K562 cells. HS2-AHSP promoter, HS2-γ-globin promoter, HS2-β-globin gene promoter, or mutant HS2-β-globin gene promoter/luciferase reporter plasmids were cotransfected into K562 cells with an EKLF cDNA expression plasmid. Luciferase activity was assayed 24 h after transfection and normalized to β-galactosidase to control for transfection efficiency. Thal, thalassemia.