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Previous Article | Next Article 
Molecular and Cellular Biology, June 2006, p. 4368-4377, Vol. 26, No. 11
0270-7306/06/$08.00+0 doi:10.1128/MCB.02216-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Hematopoiesis Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-4442,1 Department of Biochemistry and Molecular Genetics, School of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294,2 Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-80213
Received 16 November 2005/ Returned for modification 6 December 2005/ Accepted 6 March 2006
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ABSTRACT |
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INTRODUCTION |
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The ß-thalassemia syndromes are marked by clinical heterogeneity, and numerous modifier genes have been proposed (66). Recently, 
-hemoglobin stabilizing protein (AHSP, also known as erythroid differentiation-related factor and erythroid-associated factor) has been proposed as a ß-thalassemia modifier gene (19, 37, 38, 46, 68). AHSP binds and stabilizes free -hemoglobin, inhibiting the production of reactive oxygen species from -hemoglobin and preventing the precipitation of unstable, cytotoxic free -globin chains. Free -globin chains aggregate in erythroid precursors, damaging the membrane and triggering cell death. When ß-thalassemic mice were bred to AHSP-deficient mice, AHSP deficiency worsened the thalassemic phenotype, leading to the suggestion that AHSP could be a modifier gene in human ß-thalassemia syndromes (38).
EKLF-deficient mice die at embryonic day 14.5 (E14.5) to E15 from severe anemia due to defective definitive erythropoiesis (44, 50, 57). There is a marked decrease in ß-globin mRNA and protein levels in EKLF-deficient erythroid cells. In addition, there are alterations in chromatin configuration at the ß-globin gene promoter, including loss of a DNase I hypersensitive site at the proximal CACCC box as well as diminution of another HS site over 50 kb away in the ß-globin locus control region (HS3) (26, 69). Large amounts of iron accumulate in the reticuloendothelial system of EKLF-deficient mice, consistent with ineffective erythropoiesis and hemolysis. These observations led to the suggestion that the fatal anemia experienced by these EKLF-deficient mice was due entirely to deficient ß-globin expression.
Several observations have suggested that there are additional erythroid cell defects in EKLF-deficient mice. Fetal liver-derived circulating erythroid cells in EKLF-deficient embryos exhibit a greater degree of hemolysis than typically occurs in ß-thalassemia major (50, 57). Disruption of the ßmajor and ßminor genes in mice leads to anemia and death, but unlike the EKLF-deficient embryos, erythroid morphology in these animals closely mirrors that seen in human patients with ß-thalassemia major (16, 61, 63, 71). Finally, in rescue experiments with EKLF-deficient mice, overexpression of a human 
-globin transgene improved globin chain imbalance, but hemolysis persisted and survival was not improved (44, 49, 58).
We hypothesized that the expression of erythroid genes other than ß-globin was also affected by EKLF deficiency, contributing to the anemia experienced by EKLF-deficient mice. Subtractive hybridization with fetal liver RNA of wild-type and EKLF-deficient mice identified several potential EKLF target genes, including AHSP. Levels of AHSP mRNA were reduced 95 to 99.9%, and AHSP was undetectable in the EKLF-deficient fetal liver. In the AHSP promoter region, chromatin from a wild-type fetal liver demonstrated a DNase I hypersensitive site that was absent in the EKLF-deficient fetal liver. ChIP analyses identified two regions of histone hyperacetylation in wild-type chromatin, one corresponding to the location of the hypersensitive site and the other in the 3' end of the gene. In EKLF-deficient chromatin, histones across the AHSP locus were hypoacetylated. Two regions of EKLF binding were found in wild-type chromatin that corresponded to the regions of histone hyperacetylation, one at the hypersensitive site, peaking over an AHSP promoter CACCC box, and the other in the 3' end of the gene. In mobility shift assays, the AHSP promoter CACCC box, which differs from the EKLF consensus by 1 nucleotide, bound EKLF in a manner comparable to the ß-globin promoter CACCC box. Additional studies demonstrated that this nucleotide was not critical for EKLF binding. When combined with recent studies of finger one of the closely related transcription factor Sp1, these data indicate a broader recognition sequence for the EKLF consensus binding site. In K562 cells, the AHSP promoter was transactivated by EKLF. These results demonstrate that EKLF acts as a transcription factor and a chromatin modulator for both ß-globin and AHSP and may perform the same function for other erythroid genes as well.
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MATERIALS AND METHODS |
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Analysis of differentially expressed genes.
A dual riboprobe containing sequences for both exon 2 of the murine -globin gene and the last exon of the gene of interest was created for use in RNase protection assay (RPA) quantitation. This riboprobe ensures that both sequences are labeled to equal specific activity, allowing direct comparison of mRNA levels of target genes and -globin mRNA levels. Quantitative, real-time PCR confirmation of differential mRNA expression was performed using primers for individual target genes that amplify the penultimate exon to the 3' untranslated region (UTR) or the 5' UTR to exon 2 (to decrease amplification of potentially homologous genes and detect genomic DNA contamination) and an internal control, ß-actin, and SYBR green for detection using an iCycler instrument (Bio-Rad) (Table 1). Reverse transcription (RT)-PCR was performed with Thermus thermophilus polymerase (BD Clontech), a polymerase with reverse transcriptase and polymerase activities. Values obtained for target gene expression were normalized to ß-actin and were expressed relative to the expression in control samples. For calculations, the 2–
CT formula was used, with CT = (CT, target – CT, GAPDH)experimental sample – (CT, target – CT, GAPDH)control samples, where CT is cycle threshold.
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DNase I hypersensitive site mapping. DNase I HS mapping was performed as described previously (45, 70), with minor modifications, with 107 murine fetal liver cell nuclei. For HS mapping, DNA from embryonic stem cells was digested with BamHI for Southern blot analysis using a 211-bp fragment containing exons 1 and 2 of the AHSP cDNA as a probe. For fine mapping, the migration of the band generated by DNase I and BamHI enzyme digestion was compared with the migration of bands generated by the digestion of high-molecular-weight embryonic stem cell DNA digested with BamHI and AflIII, BlnI, HindIII, NsiI, or SmlI, respectively.
Quantitative ChIP assay.
ChIP analysis of diacetylated histone H3 and tetraacetylated histone H4 was performed with antibodies from Upstate Biotechnology (06-599 and 06-866; Lake Placid, NY) as described previously (33). After elution and extraction, immunoprecipitated DNA was analyzed by quantitative real-time PCR (iCycler; Bio-Rad) as described previously using primers in Table 2 (33). Signals of test genes were normalized to a region from the murine -globin promoter with a region from the keratin gene promoter utilized as a nonerythroid internal control. SYBR green fluorescence in 25-µl PCR mixtures was determined, and the amount of product was determined relative to a standard curve generated from a titration of input chromatin. Amplification of a single amplification product was confirmed by dissociation curve analysis and acrylamide gel electrophoresis. Samples from at least three independent immunoprecipitations were analyzed. Parallel controls for each experiment included samples of no chromatin, no antibody, preimmune serum, and nonimmune rabbit immunoglobulin G.
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EMSA. Recombinant GST-EKLF fusion protein was prepared using a GST-EKLF plasmid (clone C10) as described previously (48). Binding reactions, electrophoresis, and autoradiography were carried out as described previously (70). Oligonucleotide probes are shown in Table 3. Unlabeled competitor oligonucleotides were added at various molar excesses as described previously (22). EKLF antibody 6B3 was obtained from James Bieker. Quantitative electrophoretic mobility shift analyses (EMSA) was performed as described (27, 34, 73). Specifically, the fraction of free DNA, D/Dt, was determined by measuring the ratio of free DNA signal analyzed at each protein concentration at the DNA signal in a control lane containing no protein. The fraction of DNA in complex with protein, PD/Dt, was derived from the relationship PD/Dt = 1 – D/Dt. To derive the equilibrium dissociation constant (KD) with standard error, the data were fit to the rearranged mass action equation, PD/Dt = 1/(1 + KDP), using nonlinear least-square analyses. Multiple analyses (three or more) were performed with the same range of protein concentrations to provide mean and standard error values for each point.
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-globin gene promoter-luc plasmids were used as positive and negative controls, respectively, in transactivation assays (4, 32). The ß-globin gene promoter was removed from the HS2-ß-globin-luc plasmid and replaced with either a promoter fragment –170/+269 from the human AHSP gene (21) or a ß-globin promoter with a thalassemia-associated CACCC box mutation known to perturb EKLF binding (22). Integrity of all test plasmids was confirmed by sequencing. Transient K562 cell (ATCC, CCL 243) transfections were performed as described previously with 20 µg of test plasmid, 10 µg of an EKLF expression plasmid, (48), and 0.5 µg of pCMVß, a mammalian reporter plasmid expressing ß-galactosidase driven by the human cytomegalovirus immediate-early gene promoter (Clontech) as described previously (70, 74). At least two preparations of each plasmid were tested in triplicate. |
RESULTS |
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175 differentially expressed clones were identified and subjected to sequence analysis. Validating the subtractive approach, the most abundant clone isolated corresponded to ß-globin (n = 122). The second most abundant clone isolated was AHSP (n = 22). No other single clone except G protein Gi2a (n = 8) was represented by more than 5 clones. Results of subtraction were compared to results of differential gene expression in EKLF-deficient cells identified by analysis of a noncommercial microarray (20). Both techniques identified ß-globin, AHSP, and the membrane protein gene protein 4.9 (3 clones in the subtraction). Compared to the array, subtraction did not identify hemogen, hemoglobin Z, any enzymes involved in heme biosynthesis, and several membrane-associated genes, such as those for Kell, CD24a, Icam4, and Kcnn4. It did identify other membrane-associated proteins, such as aquaporin, Rh30, p55, protein 4.2, and ERMAP. When subtraction was performed using EKLF-deficient RNA as the tester population and wild-type fetal liver RNA as the driver, only 12 clones were obtained. No clone was represented more than once, even though EKLF has been shown to have a repressor function (14, 15).
Reduced expression of potential target genes in EKLF-deficient fetal liver RNA was confirmed using either quantitative, real-time RT-PCR (Table 4) or RPA. The most frequently isolated clone after ß-globin, AHSP, had nearly undetectable mRNA levels in EKLF-deficient RNA (Table 4; Fig. 1A). Western blot analysis with a rat anti-mouse monoclonal antibody against AHSP demonstrated that there was no AHSP in day 13.5 fetal livers from EKLF-deficient mice (Fig. 1B). Other genes with significantly decreased mRNA levels in EKLF-deficient mRNA included protein 4.9, which was also nearly undetectable, and aquaporin, reduced to less than 50% of the wild-type level (Table 4). Because AHSP was the most significantly altered gene in the differential subtraction, we selected it for further study.
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In chromatin from E13.5 WT fetal liver nuclei, a strong DNase I HS was demonstrated in the immediate 5' flanking DNA in the core promoter region in a 162-bp HindIII/SmlI fragment (Fig. 2, upper panel) corresponding to coordinates 2187 to 2349 of a 6,472-bp murine AHSP fragment (GenBank accession no. AF485327) (59). Fine mapping of the HS localized it to a 43-bp HindIII/SmaI fragment corresponding to the core AHSP promoter containing a CACCC box (not shown). This HS site was absent in chromatin from EKLF-deficient fetal livers (Fig. 2, lower panel).
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-globin gene promoter (Fig. 3B and C; Table 5). The 5' region corresponded to the 5' flanking DNA and promoter region of the AHSP gene, and the second region mapped 3' of the region of the AHSP cDNA in proximity to the polyadenylation signal (Fig. 3). Compared to the control, histones H3 and H4 were also acetylated in the interval between these peaks of hyperacetylation. In chromatin immunoprecipitated from EKLF-deficient fetal liver cells, there was hypoacetylation between primer pairs 4 through 8 (Fig. 3B and C; Table 5). This encompasses the core AHSP promoter and coding region, correlating with the severe reduction in AHSP gene expression. Significant H3 acetylation extends 5' of the AHSP core promoter region, potentially indicating the interaction of EKLF with other regulatory elements in this region.
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A CACCC box in the AHSP gene promoter binds EKLF in vitro. The AHSP promoter CACCC box, located in the region of the DNase I hypersensitive site, histone hyperacetylation, and EKLF binding, did not match the EKLF binding consensus, CCNCNCCCN, as it had a single mismatch at position 1, ACCCACCCT. To determine if EKLF could bind the imperfect EKLF site in the AHSP gene promoter in vitro, double-stranded oligonucleotide probes containing the AHSP 5' flanking DNA imperfect EKLF site or a ß-globin promoter EKLF binding site probe were prepared and used in EMSA with recombinant EKLF protein. The AHSP probe yielded a single complex that migrated identically to a complex formed by the control ß-globin promoter probe (Fig. 6A). These complexes were effectively competed both by an excess of unlabeled AHSP probe, an excess of unlabeled control ß-globin probe, and a monoclonal antibody against EKLF protein (Fig. 6A). These data indicate that EKLF binds to a region of the 5' flanking DNA region of the AHSP gene in vitro.
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Competitive electrophoretic mobility shift assays were performed to compare the ß-globin promoter CACCC site to the AHSP CACCC site. In this assay, the wild-type ß-globin promoter CACCC site is used as a probe and different unlabeled probes are assayed for their ability to compete the EKLF-CACCC complex. Approximately 50 nM unlabeled wild-type ß-globin CACCC probe was required to compete the EKLF-CACCC complex by 50% (Fig. 7A). Approximately 150 nM unlabeled wild-type AHSP probe or ß-globin CACCC probe with position 2 C to T was required for similar competition (Fig. 7A). An unlabeled ß-globin CACCC probe with a thalassemia mutation known to perturb EKLF binding (CACCC to CACGC) did not compete the EKLF-CACCC complex at the amounts added in these experiments. Quantitative EMSA were performed to further assess binding of the AHSP CACCC site. Recombinant EKLF-GST expressed in E. coli and purified on glutathione-Sepharose beads was used in DNA titration experiments to determine the concentration of protein capable of binding DNA (not shown). Active protein concentrations were then used in protein titrations to determine the KD for the various CACCC-containing oligonucleotide probes of interest (Fig. 7B). The highest-affinity site was the ß-globin promoter CACCC box, with a KD of 3.98 x 10–4 ± 0.91 x 10–4 nM. The AHSP promoter CACCC box and the ß-globin promoter CACCC boxes with position 2 mutated had affinities 30- to 40-fold less than the ß-globin promoter CACCC box: AHSP promoter CACCC box, 1.1 x 10–3 ± 0.90 x 10–3 nM; ß-globin C to T, 1.1 x 10–3 ± 0.18 x 10–3 nM; ß-globin C to A, 1.3 x 10–3 ± 0.46 x 10–3 nM; and ß-globin C to G, 1.2 x 10–3 ± 0.36 x 10–3 nM. The ß-globin thalassemia mutant demonstrated a significantly lower binding affinity than the wild-type ß-globin promoter CACCC box, 4.6 x 10–2 + 0.39 x 10–2. Together, these data indicate that the C at position 2 of the EKLF consensus site influences, but is not absolutely essential for, EKLF binding to the ß-globin proximal CACCC box in vitro.
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-globin promoter fragment as a negative control (4, 32) were transiently cotransfected into K562 cells with an EKLF cDNA expression plasmid. The AHSP promoter and the positive control ß-globin promoter plasmids were transactivated by EKLF, whereas the negative control -globin promoter and thalassemia-mutant ß-globin promoter plasmids were not (Fig. 8).
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DISCUSSION |
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Like the ß-globin locus, the AHSP locus demonstrates significant alterations in chromatin configuration in EKLF-deficient cells. Alterations in chromatin were not only found in the core AHSP promoter region at a site of EKLF-DNA binding but across the locus where there was global histone hypoacetylation. EKLF plays an important role in chromatin remodeling at the ß-globin locus (1, 26, 36, 42, 47, 69, 75). EKLF is acetylated by CBP and p300, coactivator proteins that posses histone acetyltransferase activity (9, 75). It also interacts with other proteins, including those of the SWI/SNF chromatin remodeling complex (1, 10, 36, 42, 76). Recent evidence demonstrates that EKLF plays an important role in formation of an active chromatin hub in erythroid cells (21). Together, these data demonstrate support for the hypothesis that chromatin remodeling of the AHSP locus requires EKLF and demonstrate that EKLF may act as a transcription factor and a chromatin modulator for genes other than ß-globin.
These data also demonstrate the value of an HA-EKLF-TAP-tagged knock-in allele mouse. In vivo histone acetylation data demonstrated two regions of hyperacetylation in wild-type mice that were absent in EKLF-deficient mice, one over the promoter and the other in the 3' region of the gene. In vitro EMSA studies suggested that EKLF binding was occurring in the 3' region. Generation of in vivo data from the HA-EKLF-TAP-tagged mouse permitted identification of EKLF binding to the core promoter CACCC box, which based on the published EKLF consensus sequence, was not initially considered.
The EKLF consensus sequence CCNCNCCCN was generated from data obtained from modeling of the crystal structure of DNA zinc finger contacts of Zif268 at 2.1 Å, as well as in vivo footprinting and methylation interference studies of the murine ß-globin promoter (22, 54, 60). These studies demonstrated that ß-thalassemia mutations associated with the ß-globin promoter CACCC box disrupt specific contacts between guanine on the G-rich strand and arginine or histidine of the XYZ of fingers 2 or 3 (22). Identification of an EKLF binding site with a substitution in finger 1, where no thalassemia-associated mutations have been identified, suggests some relaxed freedom for DNA binding of finger 1 by EKLF. Finger 1 of EKLF is similar to another member of the C2H2-type zinc finger protein family, the ubiquitous transcription factor Sp1. Compared to fingers 2 and 3, finger 1 of Sp1 has more relaxed sequence and site specificity and contributes less to its DNA binding affinity (51, 67, 72).
Recent nuclear magnetic resonance structure of Sp1 revealed that, compared to fingers 2 and 3, which recognize four DNA base pairs by residues –1, 2, 3, and 6 of the recognition helix, finger 1 uses only residues –1 and 3 for DNA recognition (51). Differences in 3 amino acids of Sp1 finger 1 contribute to a broader recognition sequence than Zif268 and other C2H2-type zinc finger proteins. EKLF has a sequence identical to that of Sp1 at these 3 residues (see Fig. S1 in the supplemental material). Based on these data, it is possible that the EKLF consensus is more similar to the GGGCCG consensus of Sp1, e.g., NCNCCC, and that other factors, such as sequence context, composition of basal transcription machinery recruited to the promoter, and/or other EKLF-protein interactions, determine specificity and activity (4, 8). Several reports support this hypothesis, demonstrating that the function of the ß-globin CACCC box is context dependent (4, 18, 29, 43, 64).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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