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Molecular and Cellular Biology, May 2008, p. 3101-3113, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.01858-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Silencing of A{gamma}-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the –566 GATA Site{triangledown} ,{dagger}

Susanna Harju-Baker,1,§,{ddagger} Flávia C. Costa,1,{ddagger} Halyna Fedosyuk,1 Renee Neades,1 and Kenneth R. Peterson1,2*

Departments of Biochemistry and Molecular Biology,1 Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 661602

Received 11 October 2007/ Returned for modification 20 November 2007/ Accepted 8 March 2008


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ABSTRACT
 
Autonomous silencing of {gamma}-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the A{gamma}-globin gene was identified between –730 and –378 relative to the mRNA start site. A marked copy of the A{gamma}-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the {varepsilon}-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the –566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low {gamma}-globin levels, whereas only a weak signal was detected when {gamma}-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the A{gamma}-globin –566 or G{gamma}-globin –567 GATA site when {gamma}-globin expression was low (day 18) but not when {gamma}-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, {gamma}-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the –566/–567 GATA sites of the proximal {gamma}-globin promoters.


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INTRODUCTION
 
The human β-globin locus consists of five functional genes, arranged spatially as 5'-{varepsilon}-G{gamma}-A{gamma}-{delta}-β-3' in the order in which they are expressed during development. Globin gene expression is regulated, in part, by the locus control region (LCR), which physically consists of at least five DNase I-hypersensitive site (HSs) located 6 to 22 kb upstream to the {varepsilon}-globin gene. The embryonic {varepsilon}-globin gene is transcribed during the first 6 weeks of primitive erythropoiesis in the embryonic yolk sac. During the switch to fetal definitive erythropoiesis in the liver, the {varepsilon}-globin gene is silenced and the G{gamma}- and A{gamma}-globin genes are activated. Around the time of birth, a second switch occurs, to adult definitive erythropoiesis. The site of hematopoiesis moves to the bone marrow, the {gamma}-globin genes are silenced, and the {delta}- and β-globin genes are activated (67). Previous studies have demonstrated that two nonexclusive mechanisms are involved in the {gamma}- to β-globin gene switch, competition between the two genes for interaction with the LCR and autonomous silencing of the {gamma}-globin genes (6, 13, 20). In the competition model, the gene closer to the LCR has a higher probability of interaction with the LCR and is more abundantly transcribed, unless it is autonomously silenced (28, 29, 53, 70). Autonomous silencing plays a major role in repressing the embryonic {varepsilon}- and fetal {gamma}-globin genes during definitive erythropoiesis (17, 59), and competition plays a major role in silencing the adult β-globin gene during primitive and fetal definitive erythropoiesis (70). An understanding of the molecular basis underlying interaction of these regulatory motifs is not complete. Although autonomous silencing of {gamma}-globin transcription during development has been documented (17, 19, 29, 39, 66), the sequences encoding the silencer element(s) and the mechanism(s) of repression have only recently been explored (64, 68, 69).

Silencers bind repressor protein complexes that interfere with promoter activity, thereby down-regulating gene expression (61). Recent data demonstrate that two direct repeat (DR1) elements located in the proximal {varepsilon}-globin promoter bind a novel repressor activity in definitive erythroid cells called direct repeat erythroid-definitive protein (DRED) (71). A similar DR1 sequence exists 5' to the A{gamma}-globin gene (49). The nondeletional –117 Greek hereditary persistence of fetal hemoglobin (HPFH) point mutation (25) alters this sequence, resulting in continued expression of A{gamma}-globin in adult humans and human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice bearing this mutation (51). Purification of DRED binding activity revealed that it is a multiprotein complex composed of at least five proteins. DR1-specific binding activity consists of two nuclear orphan receptors, testicular receptor 2 (TR2) and TR4, which are expressed in both embryonic and adult erythroid cells (68). These two proteins (and others) collaboratively repress transcription of the {varepsilon}- and {gamma}-globin genes. Recent evidence demonstrates that the DR sequence element autonomously mediates definitive stage-specific {gamma}-globin gene silencing (49, 69). COUP-TFII (NF-E3) is an orphan receptor that has both repressor and activator properties and may be involved in globin gene switching by repressing {varepsilon}-globin expression in fetal erythroid cells (21). In mice, COUP-TFII binds to the same direct repeats of the {varepsilon}- and {gamma}-globin promoters as DRED, possibly assisting in repression of expression from these genes. SSP, an activator of {gamma}-globin gene expression, is a heteromeric complex consisting of CP2 and an erythroid-specific protein, NF-E4 (24, 32, 77, 80). This complex binds the stage selector element in the proximal promoter of the {gamma}-globin genes (32). A truncated form of NF-E4, p14 NF-E4, may interact with CP2 later in development, inhibiting binding of the SSP complex to the stage selector element, resulting in repression of {gamma}-globin gene expression (79). Finally, DNA methylation by an MBD2-containing complex maintains {gamma}-globin suppression in adult erythroid cells (64).

Genetic analyses showed that {gamma}-globin gene-flanking sequences encompass numerous regions with either positive or negative regulatory function (27, 66). When a marked A{gamma}-globin gene (A{gamma}m) was inserted between LCR 5' HS1 and the {varepsilon}-globin gene in β-YAC transgenic mice (A{gamma}m 5' {varepsilon} β-YAC), it was silenced during adult definitive erythropoiesis (29). In contrast, when a marked β-globin gene (βm) was inserted in this same location, it was expressed throughout ontogeny. Competition with the β-globin gene for interaction with the LCR does not account for the A{gamma}m-globin silencing, because the gene was silenced even in the absence of the β-globin gene (29). The nonnative location of the A{gamma}m-globin gene also does not explain the down-regulation, since the βm-globin gene inserted in the same location was expressed throughout ontogeny. Therefore, the A{gamma}m-globin gene-proximal sequences must be responsible for the developmental silencing of this gene.

To identify the A{gamma}m-globin gene silencer, a series of truncations was designed based on previous {gamma}-globin promoter analysis (66). A 352-bp deletion ({Delta}1s) encompassing sequences from –730 to –378 relative to the A{gamma}-globin mRNA CAP site was introduced into the A{gamma}m-globin gene of the A{gamma}m 5' {varepsilon} β-YAC, and the resultant {Delta}1s A{gamma}m 5' {varepsilon} β-YAC was used to establish transgenic mice. Our data demonstrate that A{gamma}m-globin was expressed during adult definitive erythropoiesis. Further analysis of the this region revealed that a GATA site located at –566 was bound by GATA-1 in non-{gamma}-globin-expressing cells but lost in cells expressing {gamma}-globin at a high level. Thus, the A{gamma}m-globin gene promoter region from –730 to –378 functions as a {gamma}-globin gene silencer in adult definitive erythropoiesis, and this silencing is mediated by GATA-1.

GATA-1 is a member of a family of zinc finger transcription factors that plays a seminal role in the development and differentiation of several cell types, including erythrocytes, megakaryocytes, eosinophils, and mast cells (55, 63). β-Globin locus transcriptional activation can be resolved into discrete molecular steps involving the formation of distinct GATA-1-cofactor assemblies at the promoters and the LCR (33). Recent data suggest that GATA-1 can act both as an activator and as a repressor of gene transcription. Interaction between GATA-1 and the YY1 protein is involved in the silencing of the {varepsilon}-globin gene in primate and nonprimate species (58). More recently, binding of a GATA-1-FOG-1-NuRD complex was shown to silence hematopoietic genes in erythroid cells (31, 63). FOG-1 serves as the bridging factor between GATA-1 and the NuRD chromatin-remodeling complex (31). The NuRD complex was originally identified in mammalian and Xenopus cells, and some subunits of this complex were also found in Drosophila, Caenorhabditis elegans, and Arabidopsis, suggesting that it is widely conserved during evolution (2, 78). The NuRD complex is a 2-MDa complex and is comprised of at least seven polypeptides, including HDAC1, HDAC2, MBD3, Mi2 (CHD), MTA, RbAp46, and RbAp48 (15, 78). Functions of the complex include nucleosome remodeling and histone deacetylase activities (2, 9, 36, 73). It has been associated with epigenetic mechanisms of transcriptional repression during development. Based on these data, we hypothesized that NuRD and FOG-1 interact with GATA-1 to form a complex that may be involved in remodeling {gamma}-globin gene-proximal chromatin into a repressed state, leading to silencing of transcription during the adult stage of erythropoiesis. Chromatin immunoprecipitation analysis supports this hypothesis. We show that GATA-1, FOG-1, and Mi2 are recruited to the –566 A{gamma}-globin and analogous –567 G{gamma}-globin GATA sites when {gamma}-globin is not expressed or is expressed at low levels but not when {gamma}-globin is transcribed at high levels.


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MATERIALS AND METHODS
 
{Delta}1s A{gamma}m 5' {varepsilon} β-YAC construct. A 213-kb YAC construct carrying the human β-globin locus with a marked A{gamma}-globin gene (A{gamma}m 5' {varepsilon} β-YAC) was used to develop the {Delta}1s A{gamma}m 5' {varepsilon} β-YAC (Fig. 1A) (29). This YAC was originally estimated to be 248 kb in size (46, 47, 52, 74), until a nearly complete sequence of the β-globin locus region was made available on the globin gene server (http://globin.cse.psu.edu) (10). The β-YAC contains approximately 187 kb of the human β-globin locus flanked by YAC arms. The A{gamma}m-globin gene is a 5.4-kb SspI fragment (Fig. 1B) (GenBank file U01317 coordinates 38683 to 44077) inserted into an EcoRV site at GenBank coordinate 15185 (29). The mark in the A{gamma}m-globin gene is a 6-bp deletion at +21 to +26 relative to the A{gamma}-globin transcription start site utilized to differentiate between transcripts originating from the normally located {gamma}-globin genes and the A{gamma}m-globin (62).


Figure 1
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  		FIG. 1. Constructs. (A) Schematic diagram of the 213-kb β-YAC. A 187-kb EcoRI genomic fragment containing the human β-globin locus is shown as a line. YAC vector sequences and β-like globin gene sequences ({varepsilon}, G{gamma}, A{gamma}, {psi}β, {delta}, and β) are shown as open boxes. The location of the LCR is noted; arrows above the line denote HSs. The location of the A{gamma}m/{Delta}1s A{gamma}m-globin gene insertion is indicated by an arrow below the line. SfiI restriction sites are displayed below the locus; the line above the locus delineates the 115-kb SfiI fragment used in assessment of β-YAC transgene integrity. YAC components are represented by black boxes: TRP1 and LYS2, YAC selectable markers for tryptophan and lysine prototrophy, respectively; ARS1, yeast autonomous replicating sequence (origin of replication); CEN1, centromere; MMTneo, mammalian cell selectable marker for G418 resistance. The sequence of the YAC insert (containing a 6-kb gap) is available on the Globin Gene Server (http://globin.cse.psu.edu/). (B) 5.4-kb SspI A{gamma}m-globin gene. The 5.4-kb SspI A{gamma}m-globin gene utilized for modifying the β-YAC contains approximately 0.7 kb of 5' flanking sequences and 3.2 kb of 3' flanking sequences. The transcription start site is indicated by an arrow. Restriction enzyme sites used in previous experiments for promoter truncations are shown below the line (66). The numbers indicate distance from the A{gamma}m-globin gene 5' CAP site. Canonical binding sites for erythroid-specific transcription factors with their approximate locations are shown above the line. Two predicted GATA binding sites in the {Delta}1s region are shown by arrows, with the footprinted site marked with an asterisk. Previously suggested positive and negative regulatory regions are indicated by (+) and (–), respectively (66). The 352-bp {Delta}1s deletion from –730 to –378 is indicated at the bottom.

To create a {Delta}1s A{gamma}m 5' {varepsilon} β-YAC, the A{gamma}m 5' {varepsilon} β-YAC was modified using the "pop-in, pop-out" homologous recombination method in Saccharomyces cerevisiae (5). A 352-bp deletion was introduced in the 5'-distal promoter of the A{gamma}m-globin gene (–730 to –378; GenBank coordinates 38683 to 39050). Plasmid pHS1(H3/RV)/A{gamma}m(StuI/RI), a gift from Qiliang Li, was utilized for the yeast mutagenesis. This plasmid contains a fragment of the β-LCR 5' HS1 from HindIII to EcoRV (GenBank coordinates 13769 to 15185) subcloned into the HindIII site of pRS406 (Stratagene, Carlsbad, CA) and a fragment of the A{gamma}m-globin gene from StuI to EcoRI (GenBank coordinates 39050 to 40836). Thus, this construct lacked the 352-bp SspI-StuI distal promoter region of the A{gamma}m-globin gene but contained the region downstream of 5' HS1 that the A{gamma}m-globin gene was originally integrated into. The plasmid was linearized with SphI (GenBank coordinate 14100). Yeast carrying the A{gamma}m 5' {varepsilon} β-YAC was spheroplasted and transformed with 4 µg of the linearized plasmid. YAC transformation, screening of positive clones, purification, and mouse transgenesis were performed as described previously (26, 29, 46, 51, 54).

Structural analysis. Transgene integrity and copy number structural analyses of F2-generation animals were performed as described elsewhere (see Materials and Methods in the supplemental material) (23, 26, 29, 46, 51, 54). A PCR-based approach was also utilized to confirm the {Delta}1s A{gamma}m 5' {varepsilon} region structure. The primer sequences and expected size fragments are shown in Table S1 in the supplemental material.

RNase protection assays. Total mRNA was isolated from developmentally staged mouse fetuses, K562 human erythroleukemia cells, and MEL585 mouse erythroleukemia cells using the Promega RNagents RNA isolation kit. RNase protection assays (RPAs) were performed as described previously (29, 30).

Antibody detection of {gamma}- and β-globin protein chains. Blood from adult mice was used to prepare slides to assess {gamma}- and β-globin chain synthesis. Details are provided in materials and methods in the supplemental material.

Cell culture. Murine erythroleukemia (MEL) cells (76) and K562 cells were cultured in RPMI medium (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum, 100 µM nonessential amino acid mix, 2 mM L-glutamine, and 1 mM sodium pyruvate at 37°C in 5% CO2. Induction of K562 cells was performed by culturing with 3 mM hexamethylene bisacetamide and 10 µM hemin for 3 days (14) prior to nuclear extract preparation or RNA extraction.

Phenylhydrazine treatment of mice. Adult C57BL/6 mice at least 6 weeks old were given 60 mg/kg of body weight of phenylhydrazine (10 mg/ml in phosphate-buffered saline; P-6926; Sigma-Aldrich, St. Louis, MO) via intraperitoneal injection for three consecutive days. Mice were sacrificed 4 days posttreatment, and spleens were harvested and processed for nuclear extract preparation as described elsewhere (see materials and methods in the supplemental material).

Nuclear extract preparation. Nuclear extracts were prepared as described elsewhere (see materials and methods in the supplemental material) (5). Protein concentrations were determined using the Bio-Rad (Hercules, CA) quick Bradford assay.

DNase I footprinting. DNase I footprinting assays were performed essentially as described elsewhere (see materials and methods in the supplemental material) (12). The primers employed for the DNase I footprinting assay were as follows: forward Delta 1S, 5'-GCGAGCTCTATTTTTCTAAGATGA-3'; reverse Delta 1S, 5'-TAGCTAGCTGTCTGTAGCTCCAG-3'. The footprinting experiments were performed at least three times.

Electromobility gel shift assays. Electromobility shift assays (EMSAs) were performed as described elsewhere (see Materials and Methods in the supplemental material) (30, 37). The oligonucleotides utilized for EMSAs were as follows: –679 5' double GATA, 5'-TTCAAATAGGTACGGATAAGTAGATATTGAGGTAAGCATT-3' (GenBank coordinates 38715 to 38754); –566 3' single GATA, 5'-ACTATTGAGAAATTAAGAGATAATGGCAAAAGTCACAAAG-3' (GenBank coordinates 38828 to 38867). Potential GATA-binding sites are in bold. The mutated oligonucleotides were the same, except the underlined T was changed to G. As a control, the Sp1 and Egr oligonucleotides (sc-2502 and sc-2529, respectively) were used (Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies against murine GATA-1 and GATA-2 were used in shift inhibition experiments (Santa Cruz Biotechnology).

Transient-transfection assays. A series of pGL2 plasmids (Promega, Madison, WI) was created for transient-transfection assays. PCR primers were designed to amplify the 352-bp {Delta}1s region from an A{gamma}-globin gene plasmid and to add restriction enzyme sites to both ends of the fragment. The same primers utilized for the DNase I footprinting assay were employed for subcloning the {Delta}1s insert into the pGL2 vectors. The 5' end of the sense-strand primer contained a SacI site, whereas the 5' end of the antisense primer carried a NheI site. The amplified 5'-SacI-{Delta}1s-NheI-3' fragment was ligated to SacI/NheI-digested pGL2 plasmids (pGL2-promoter and pGL2-control). Correct clones were identified by restriction enzyme digestion analysis, followed by DNA sequencing. The sequencing primers utilized were as follows: pGL2primer1, 5'-TGTATCTTATGGTACTGTAACTG-3'; pGL2primer2, 5'-CTTTATGTTTTTGGCGTCTTCCA-3' (Promega).

K562 cells (1 x 107) were cotransfected with 1 µg pSVβ-Gal plasmid (Promega) and 17.5 µg of the pGL2 plasmids in 0.4 ml Dulbecco's modified Eagle medium using a Bio-Rad Gene Pulser electroporator set at 950-µF capacitance and 300 V. After electroporation, the cells were directly cultured for 24 h as described above. Cells were collected, washed with phosphate-buffered saline, and lysed with β-galactosidase (β-Gal) assay lysis reagent (Promega). Lysates were analyzed for protein concentration using the Bio-Rad quick Bradford assay, β-Gal activity was assayed using the Promega β-Gal assay, and luciferase activity was measured using the Promega LARII reagent, all as described by the respective manufacturers. β-Gal and luciferase signals were normalized to the protein concentration. Luciferase signals were further normalized to the β-Gal signals.

ChIP assay. Chromatin immunoprecipitation (ChIP) assays were performed as described previously (38) with the some modifications (see materials and methods in the supplemental material). Fetal liver from wild-type β-YAC transgenic mice at 12 and 18 days postconception (E12 and E18) were utilized. Cross-linking of protein-protein and protein-DNA complexes within the cells was performed with 1% formaldehyde (fresh paraformaldehyde). Chromatin was sonicated to a size range between 200 and 1,000 bp. The samples were precleared with species-matched normal serum. Immunoprecipitations were carried out with GATA-1-, FOG-1-, and Mi2-specific antibodies or isotype-matched immunoglobulin G (IgG) (rabbit, mouse, or goat) and protein G conjugated to agarose beads containing salmon sperm DNA (Upstate, Temecula, CA). The immunoprecipitate was washed, the cross-links were reversed, and the genomic DNA was purified. Recruitment of GATA-1, FOG-1, and Mi2 proteins was measured by real-time quantitative PCR, using gene-specific primers (see Table S2 in the supplemental material).

Real-time PCR. ChIP samples were analyzed in duplicate by real-time PCR with SYBR Green dye using a Roche LightCycler (Roche Applied Science, Palo Alto, CA). To allow comparison among primer sets, input samples from each condition were diluted serially from 1:10 to 1:10,000 and used as standards for all PCR samples. Enrichment of a protein binding to a specific DNA sequence was calculated using the crossing-point method (35). The data shown are the averages of duplicate results from at least two independent experiments for each sample. Error bars represent standard deviations from the mean. PCR primer sequences are provided in Table S2 in the supplemental material.

Preparation of nuclear lysates and Western blotting analysis. Nuclear extracts were prepared from K562 and MEL cell lines as described elsewhere (see materials and methods in the supplemental material) (34). Western blotting was performed according to standard procedures (see materials and methods in the supplemental material) (5).

Antibodies. See materials and methods in the supplemental material for information on antibodies employed and sources.


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RESULTS
 
{Delta}1s A{gamma}m 5' {varepsilon} β-YAC transgene structure determination and copy number analysis. To identify potential A{gamma}-globin silencer elements, a series of deletions in the A{gamma}-globin gene promoter and 5' and 3' flanking regions was planned for introduction into the A{gamma}m 5' {varepsilon} β-YAC (Fig. 1A). The first deletion, from –730 to –378 relative to the mRNA start site of the A{gamma}m-globin gene, was introduced into the A{gamma}m 5' {varepsilon} β-YAC by homologous recombination in yeast (Fig. 1B). The resultant {Delta}1s A{gamma}m 5' {varepsilon} β-YAC was used to produce transgenic mice. Five transgenic lines were established after identification of founders by PCR of tail biopsy DNAs.

Long-range restriction enzyme mapping structural analysis was performed using 11 radioactively labeled DNA probes spanning the locus from 5' HS3 through the HPFH6 breakpoint on Southern blots of pulsed-field gels (see Figure S1 in the supplemental material) (47, 51). Transgene copy numbers were determined as previously published (23, 46). It is important to determine both the copy number and continuity of the YAC sequences to accurately calculate quantitative copy number-corrected gene expression levels. Only genes that are linked to the LCR will be expressed at physiological levels (52). Line 1 carried four different-size copies of the locus extending from the LCR through the HPFH3 breakpoint. Line 2 had two copies of the locus extending from the LCR through the HPFH3 region. Line 3 carried four copies of the YAC extending from the LCR through the HPHF6 breakpoint. However, in addition, this line bore two copies of the YAC with a deletion of the {delta}-globin gene and downstream region. Therefore, the copy numbers utilized for calculating per-copy expression levels for this line were six for the globin genes upstream of the breakpoint and four for the β-globin gene. Line 4 had two copies of two different sizes extending from the LCR through the HPFH6 breakpoint. Line 5 had four copies of the complete locus extending from 5' HS3 through the β-globin gene; at least one of these transgenes extended through the H500 HPFH breakpoint.

To confirm that the individual wild-type A{gamma} (A{gamma}wt)-, A{gamma}m-, and {Delta}1s A{gamma}m-globin gene structures were intact within these mouse lines, Southern blot hybridization and PCR analyses were performed to assess transgene integrity. Southern blots of SphI- or HpaI-digested genomic DNAs were hybridized with a 0.5-kb SmaI-XmnI 5' HS1 probe to detect the β-globin locus fragments (data not shown). Wild-type β-YAC DNA samples showed the expected 23.7-kb HpaI and 10.4-kb StuI fragments for the 5' HS1 region. The A{gamma}m-globin gene insertion introduced additional HpaI and StuI recognition sites into the 5' HS1-{varepsilon}-globin region. The predicted 5.2-kb HpaI and 6.4-kb StuI A{gamma}m-globin gene fragments were observed. The {Delta}1s deletion in the A{gamma}m-globin gene removed 0.4 kb of the 5' region of the A{gamma}m-globin gene insert. Therefore, the expected HpaI fragment for {Delta}1s was 4.8 kb instead of 5.2 kb. All {Delta}1s lines had this fragment. Similarly, the {Delta}1s deletion removed a StuI site from the A{gamma}m-globin gene, resulting in a 15-kb fragment that was observed in these lines. These data indicated that the A{gamma}m- and {Delta}1s A{gamma}m-globin gene inserts were in the correct location.

A PCR-based approach was also utilized to confirm the {Delta}1s A{gamma}m 5' {varepsilon} region structure (see Fig. S2A and B in the supplemental material). Primer set B (see Table S1 in the supplemental material) was designed to anneal to the promoter and exon 2 of the A{gamma}m-, {Delta}1s A{gamma}m-, and A{gamma}wt-globin genes (see Fig. S2A in the supplemental material). The mark in the A{gamma}m-globin gene (and {Delta}1s A{gamma}m) is a 6-bp deletion in the 5' untranslated region (UTR) of the A{gamma}-globin gene (62), which removes a DdeI restriction enzyme site. Digestion of a wild-type {gamma}-globin gene PCR product with DdeI results in three fragments, 175 bp, 107 bp, and 20 bp in size, whereas the A{gamma}m-globin gene PCR product gives only two fragments, 195 bp and 107 bp in size. Additionally, two sets of primers that flank the 5' and 3' junctions of the A{gamma}m- and {Delta}1s A{gamma}m-globin gene inserts were designed. The 5' junction primer set A (see Table S1 in the supplemental material) produced a 793-bp fragment for the A{gamma}m-globin gene and a 426-bp fragment for the {Delta}1s A{gamma}m-globin gene due to the 352-bp {Delta}1s deletion (see Fig. S2B, top panel, in the supplemental material). The 3' junction is common to both A{gamma}m- and {Delta}1s A{gamma}m-globin genes, and thus, PCR with primer set C (see Table S1 in the supplemental material) generated a 629-bp fragment for both genes (see Fig. S2B, bottom panel, in the supplemental material). Wild-type β-YAC did not give a product with either primer set A or C, as expected. The PCR analysis resulted in the predicted fragments for all the {Delta}1s A{gamma}m 5' {varepsilon} β-YAC lines. Together, these data demonstrate that the {Delta}1s A{gamma}m-globin gene inserts are in the correct location within the {Delta}1s A{gamma}m 5' {varepsilon} β-YACs.

A{gamma}m-Globin is expressed in adult blood of {Delta}1s A{gamma}m 5' {varepsilon} β-YAC transgenic lines. Analysis of the expression of human β-like globins in the {Delta}1s A{gamma}m 5' {varepsilon} β-YAC transgenic mice was performed by RPAs as described in Materials and Methods. Samples of hematopoietic tissues and blood were collected from developmentally staged embryos, fetuses, and adults and analyzed using antisense RNA probes for human {varepsilon}-, {gamma}-, and β-globins and for the murine {alpha}- and {zeta}-globins. The mouse {alpha}-like globins served as internal controls to quantitate human β-like globin transgene expression levels. The {gamma}-globin riboprobe was synthesized from an A{gamma}m-globin gene DNA template, pT7A{gamma}m (170) (4, 62). When this probe anneals with the wild-type A{gamma}-globin (A{gamma}wt-globin) gene transcript, double-stranded regions are formed at exon 2 and exon 1/5' UTR, except where the mark is located in the 5' UTR. Therefore, RNase digestion will generate a 170-bp band for exon 2, which is common for both the A{gamma}m- and A{gamma}wt-globin genes. However, the A{gamma}wt-globin exon 1/5' UTR protected fragment is digested into two bands, 118 bp plus 26 bp. The smaller band is not usually visualized on an RPA image. The A{gamma}m-globin exon 1/5' UTR protected fragment is 138 bp in size, since it is not cleaved by RNase. All of the {Delta}1s lines expressed {gamma}-globin, whereas the positive control wild-type β-YAC adult blood did not show any {gamma}-globin expression (Fig. 2). Wild-type β-YAC E12 fetal liver expressed a high level of A{gamma}wt-globin and a smaller amount of β-globin. Although the β-globin band intensities look variable and lower in one instance in the wild-type β-YAC adult blood control than in the {Delta}1s lines, the average β-globin expression in the {Delta}1s lines was 25.3%, corrected for transgene copy number and normalized to per-copy mouse {alpha}-globin expression, compared to 56.4% for the wild-type β-YAC control (Fig. 2).


Figure 2
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  		FIG. 2. Human {gamma}- and β-globin transcription in adult blood of {Delta}1s A{gamma}m 5' {varepsilon} β-YAC transgenic mouse lines. Total RNA isolated from the blood of adults and an E12 fetal liver (FL; wild-type β-YAC control) was subjected to RNase protection assays. Antisense RNA probes for human {gamma}- and β-globin and mouse {alpha}-globin (Mo {alpha}) were utilized. Protected fragments are displayed on the right: β, β exon 2, 205 bp; {gamma} ex 2, A{gamma} exon 2, 170 bp; A{gamma}m ex 1, A{gamma}m exon 1, 138 bp; A{gamma}wt ex 1, A{gamma}wt exon 1, 118 bp; Mo {alpha}, 129 bp. The constructs are shown above the panel: numbered lanes, {Delta}1s A{gamma}m 5' {varepsilon} β-YAC lines; (+), wild-type β-YAC controls; M, pBR322 MspI molecular weight marker.

Antibody detection experiments with adult transgenic mouse blood smears demonstrated expression of both {gamma}- and β-globin proteins in erythroid cells of the {Delta}1s A{gamma}m 5' {varepsilon} β-YAC lines and in the positive-control –117 A{gamma}m Greek HPFH β-YAC line but not in wild-type β-YAC transgenics (Fig. 3). Thus, protein expression reflected the mRNA synthesis profile for each of these lines during the adult stage of development.


Figure 3
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  		FIG. 3. {gamma}- and β-globin chain expression in transgenic mouse adult blood. Adult blood smears from transgenic mice carrying the wild-type β-YAC, –117 A{gamma}m Greek HPFH β-YAC, or {Delta}1s A{gamma}m 5' {varepsilon} β-YAC were stained for {gamma}- and β-globin protein chains (top and bottom panels, respectively) using indirect immunolabeling.

A representative developmental profile RPA for {Delta}1s line 1 hematopoietic tissues is shown in Fig. 4A. {varepsilon}- and A{gamma}wt-globin expression were not detected at any developmental stage, which was similar to {varepsilon}- and A{gamma}wt-globin expression patterns measured for the A{gamma}m 5' {varepsilon} β-YAC mice (29). Levels of human transgene expression corrected for copy number and normalized to copy number-corrected murine {alpha}-like globin gene expression are shown graphically in Fig. 4B and C. β-Globin gene expression was normal during early fetal definitive erythropoiesis, but the level in adult blood was decreased compared to that for the control transgenics (Fig. 4B). In fact, the level of β-globin expression in these lines was even lower than that in the A{gamma}m 5' {varepsilon} β-YAC lines (29); expression was 38% in the former versus 59% in the latter (Fig. 4B). Expression of the {gamma}-globin gene in the adult blood may repress transcription from the adult β-globin gene, presumably because the LCR is engaging the {gamma}-globin gene promoter and interacting less with the β-globin gene promoter. The A{gamma}m-globin gene was strongly expressed during fetal definitive erythropoiesis and in adult blood (Fig. 4C). These results suggest that the –730 to –378 region contains an A{gamma}-globin-specific silencer, since deletion of this region allows A{gamma}-globin expression during adult definitive erythropoiesis.


Figure 4
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  		FIG. 4. (A) Human β-like globin expression in {Delta}1s A{gamma}m 5' {varepsilon} β-YAC mouse tissues. Total RNA isolated from the hematopoietic tissues of staged embryos, fetuses, and adults was subjected to RNase protection assays. Antisense RNA probes for human {varepsilon}-, {gamma}-, and β-globin and mouse (Mo) {alpha}- and {xi}-globin were utilized. Protected fragments are as described in the legend to Fig. 2 with the following additions: {varepsilon}, {varepsilon} exon 2, 188 bp; Mo {xi}, 151 bp. Developmental days postconception are indicated above the panels. Different tissues are indicated above the lanes: YS, yolk sac; FL, fetal liver; Bl, blood. The numbers denote multiple samples from the same line. (B) Quantitation of human β-globin expression in {Delta}1s A{gamma}m 5' {varepsilon} β-YAC lines. Per-copy β-globin transgene expression was calculated as a percentage of murine {alpha}-like globin gene expression, also corrected for endogenous copy number. Average and standard deviation are shown for three different lines of wild-type β-YAC mice (white bars) and A{gamma}m 5' {varepsilon} β-YAC mice (gray bars) and for all {Delta}1s A{gamma}m 5'{varepsilon} β-YAC mice (black bars). (C) Quantitation of human {gamma}-globin expression in {Delta}1s A{gamma}m 5' {varepsilon} β-YAC lines. {gamma}-Globin expression was calculated and is represented as described in the legend for panel B.

A GATA site in the 352-bp {Delta}1s fragment is footprinted in vitro in cells expressing low levels of {gamma}-globin mRNA. In vitro DNase I-footprinting studies were done utilizing probes covering the 352-bp {Delta}1s fragment from –730 to –378 and nuclear extracts from CCRF-CEM cells, MEL 585 mouse erythroleukemia cells, and uninduced or induced K562 cells. CCRF-CEM is a human T-cell-lymphoblast-like cell line that does not express any globin genes (22). MEL 585 cells are murine erythroleukemia cells that express predominantly β-globin but also some {gamma}-globin when they carry the β-YAC (8, 54). K562 cells express predominantly {varepsilon}-globin and some {gamma}-globin but no β-globin (1, 14, 48, 65). Following induction, K562 cells display increased {gamma}-globin production (1, 14, 48, 65). We also utilized nuclear extract prepared from the spleens of phenylhydrazine-treated transgenic mice, which express high levels of {gamma}-globin due to the reticulocytosis resulting from hemolytic anemia (18). Only a single footprint was observed in cells that do not express any {gamma}-globin or have a low expression level (CCRF-CEM, MEL 585, and uninduced K562 cells) (Fig. 5; see also Fig. S3 in the supplemental material). The footprint was not seen in cells that express {gamma}-globin (induced K562 cells or phenylhydrazine-treated mouse spleen) (Fig. 5). The most striking difference in the footprint intensity was observed between induced and uninduced K562 cells. Uninduced K562 cell nuclear extracts produced a clear footprint at the –566 GATA site, which disappeared upon induction. The footprinted sequence (5'-aagaGATAatggc-3') was predicted to be a strong, canonical GATA-1 binding site (WGATAR) by MatInspector analysis (57). This site was also present in the G{gamma}-globin gene promoter; however, we did not utilize primers to test this region of the G{gamma}-globin gene promoter. A second pair of noncanonical GATA sites further upstream of the promoter centered at –679 was also predicted by MatInspector, but it did not appear to be bound by proteins in these experiments (see Fig. S3 in the supplemental material). Therefore, we conclude that the WGATAR sequence located at –566 binds a repressor in cells that do not express A{gamma}-globin or that express it at low levels.


Figure 5
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  		FIG. 5. In vitro DNase I footprinting of the 352-bp –730 to –378 {Delta}1s fragment. The 352-bp –730 to –378 fragment was PCR amplified using an end-labeled sense-strand primer. Protein binding experiments were performed using labeled fragment and various nuclear extracts (labeled above the figure). The sequence of the footprint (FP-1) is shown to the right of the autoradiograph with the –566 GATA site bases underlined.

GATA-1 binds to the distal A{gamma}-globin gene promoter when {gamma}-globin gene expression is repressed. Since a GATA binding site was found to be footprinted in cells in which {gamma}-globin gene transcripts were at a low level or not present, we wanted to confirm whether this site was bound by GATA-1 or GATA-2 proteins under conditions of {gamma}-globin repression or expression. We performed EMSAs with 40-mer double-stranded DNA fragments which contained the –566 (footprinted) or –679 (not footprinted) GATA sites. We also utilized DNA oligonucleotide probes in which the putative GATA binding site was ablated by changing the T to a G (GATA -> GAGA). The data showed that only the wild-type GATA site at –566 was shifted using uninduced MEL 585 and K562 extracts (conditions of no or low {gamma}-globin expression) (Fig. 6A). Although this is a perfect GATA site, the induced K562 nuclear extracts did not show significant GATA-1 binding activity. Interestingly, Western blot analysis demonstrated a decrease in GATA-1 protein in induced K562 cells, although β-actin and Sp1 protein levels were not decreased (see Fig. S4 in the supplemental material). The similar levels of these two proteins in uninduced and induced K562 cells in equal total protein aliquots as measured by the Bradford assay (see Materials and Methods) indicate that these extracts are qualitatively similar. Within a population of K562 cells, {gamma}-globin transcription is generally low but measurable in uninduced cells and increases approximately 1.4-fold in induced cells (data not shown) (65). Thus, the higher GATA-1 concentration in uninduced cells may aid in occupation of silencer GATA sites, and following induction, only activator GATA sites are bound by the more limited level of GATA-1. The MEL 585 data corroborate the uninduced K562 data, since {gamma}-globin is poorly expressed in transgenic derivatives of this adult erythroid phenotype cell line (54), and the extracts from phenylhydrazine-treated mouse spleens support the induced K562 data, since {gamma}-globin is highly induced in these cells. The –679 GATA sites also appeared to weakly shift, but use of a mutant GAGA oligonucleotide did not ablate this shift, indicating that the observed shift was due to nonspecific binding of protein. In addition, a GATA competitor or GATA antibodies did not affect the shift at this site (data not shown). A single base pair mutation in the –566 GATA site (T -> G) ablated the shift. Competition assays with cold competitors or antibodies confirmed that GATA-1 binds to the –566 GATA site in uninduced cells (Fig. 6B). Addition of oligonucleotides with Sp1/Egr protein binding sites or a cold GAGA competitor did not affect the shift, whereas a cold –566 GATA oligonucleotide efficiently inhibited the shift. Preincubation with a GATA-1 antibody abolished the shift, whereas a GATA-2 antibody had no effect, indicating that GATA-1 was the protein in the nuclear lysate responsible for the shift of the –566 GATA site-containing oligonucleotides (Fig. 6B). These data suggest that the –566 GATA site is bound by GATA-1 and that GATA-1 is involved in repression of {gamma}-globin gene expression.


Figure 6
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  		FIG. 6. (A) EMSA of GATA factor binding sites within the silencer sequence. Four 40-mer double-stranded DNA probes prepared by end labeling were incubated with various nuclear extracts. The probes and nuclear extract sources are indicated above the figure. The GAGA probes have a single nucleotide change, from GATA to GAGA. The nuclear extracts were as follows: (–), no nuclear extract; M–, uninduced MEL585; K–, uninduced K562; K+, HMBA-hemin-induced K562; P, phenylhydrazine-treated mouse spleen. (B) Competition and antibody shift ablation EMSAs of the –566 GATA site. Uninduced MEL585 and K562 nuclear extracts were incubated with the –566 GATA probe. Various competitors or antibodies (Ab) were added to the mixture (labeled above the figure).

The {Delta}1s fragment is a silencer of gene expression. K562 cells were electroporated with pSVβ-Gal and a series of pGL-2 plasmids. The pGL-2-series reporter constructs (Promega) carry a luciferase gene linked to simian virus 40 (SV40) promoter and enhancer fragments. The pGL-2 promoter construct has an SV-40 promoter driving the luciferase gene, whereas the pGL-2 control vector has in addition an SV40 enhancer downstream to the luciferase reporter gene. The 352-bp {Delta}1s fragment was inserted 5' to the promoter of these two constructs (Fig. 7A). Luciferase activity was corrected to the protein concentration and normalized to β-galactosidase activity to control for electroporation efficiency. Expression was increased approximately fivefold whether or not the SV40 enhancer was present (Fig. 7B). Previous data have shown that this enhancer does not increase the level of gene expression in K562 cells but only the probability of expression (75). Our data revealed that the –730 to –378 {Delta}1s fragment functions as a silencer following transient transfection and cell culture of uninduced K562 cells (Fig. 7B). In support of these data, we recently produced β-YAC transgenic mice containing a point mutation in the –566 A{gamma}-globin GATA site. The founders showed a weak HPFH phenotype (data not shown), demonstrating that the –566 GATA site within this fragment is where the repressor complex binds.


Figure 7
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  		FIG. 7. Transient transfection of K562 cells with pGL2-{Delta}1s constructs. The constructs are depicted by drawings at the top (A), and the relative luciferase units corresponding to each construct are shown at the bottom (B). Luciferase activity was corrected for total protein concentration and transfection efficiency as measured by cotransfection with a β-Gal reporter construct (pSVβ-Gal). The pGL2 promoter (pGL2-p) contains only the luciferase reporter gene driven by the SV40 promoter, denoted by white and medium-gray boxes, respectively. The pGL2 control (pGL2-c) carries in addition a SV40 enhancer, shown as a light-gray box. pGL2-p-{Delta}1s is the same as pGL2-p with the –730 to –378 fragment (dark-gray box) ligated into the SacI and NheI sites of pGL2-p. pGL2-c-{Delta}1s is the same as pGL2-c with the {Delta}1s fragment ligated into the SacI and NheI sites of pGL2-c. The results are the average and standard deviation for three separate experiments with two to three replicates per sample.

The GATA-1 protein binds to the –566 GATA site of the A{gamma}-globin gene in vivo when {gamma}-globin is not expressed. To confirm that the GATA-1 protein binds to the –566 GATA site of A{gamma}-globin in vivo, we employed ChIP assays using E12 and E18 fetal liver samples from wild-type β-YAC transgenic mice ({gamma}-globin expressed and not expressed, respectively). Anti-GATA-1 and normal rat IgG (control) antibodies were used in the immunoprecipitations. Figure 8A shows that the –566 region of the A{gamma}-globin gene was enriched for binding of the GATA-1 protein in E18 fetal liver samples ({gamma}-globin repressed) compared with results in the rat IgG control samples. Binding of the GATA-1 protein was not observed in E12 fetal liver samples, where {gamma}-globin was expressed. A negative control included in all experiments, mouse glyceraldehyde-3-phosphate dehydrogenase real-time PCR with the same ChIP samples, showed an absence of binding by GATA-1 in E12 and E18 samples (data not shown). Similar results were observed for FOG-1 and Mi2 (data not shown; discussed below). As a positive control, we included ChIP data that demonstrated recruitment of GATA-1 to the –2.8 GATA site upstream of the GATA-2 gene using E12 samples (see Fig. S5 in the supplemental material), a well-documented region where GATA-1 binds as an activator (45). The signal range was similar to that measured at the –566 A{gamma}-globin GATA site with E18 fetal liver samples (Fig. 8A). Finally, a region 2.3 kb downstream of the –566 A{gamma}-globin GATA site was included as an additional negative control (see Fig. S5 in the supplemental material). The data showed an absence of GATA-1 (and FOG-1 and Mi2) binding in the same E18 samples where we demonstrated GATA-1 (and FOG-1 and Mi2) recruitment to the –566 A{gamma}-globin GATA site. These findings demonstrate that GATA-1 is recruited to this region only when {gamma}-globin expression is silenced.


Figure 8
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  		FIG. 8. ChIP analysis of the –566 A{gamma}-globin GATA site (left column) and the –567 G{gamma}-globin GATA site (right column) in fetal liver samples from day E12 and E18 conceptuses of wild-type β-YAC transgenic mice. The relative occupancy of the –566 region of the A{gamma}-globin gene (GenBank coordinates 38772 to 38937 from accession file GI455025) or the –567 region of the G{gamma}-globin gene (GenBank coordinates 33992 to 33845 from accession file GI455025) by the GATA-1 (A), FOG-1 (B), or Mi2 (C) protein (gray bars) is shown in comparison to the IgG (control) samples (white bars). ChIP was carried out using isotype-matched IgG, GATA-1, FOG-1, and Mi2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The results are the averages for at least two experiments, and each experiment was performed in duplicate. Mouse glyceraldehyde-3-phosphate dehydrogenase primers were used as a negative control for all real-time PCR (data not shown). The specificity of the primers for A{gamma}-globin was confirmed by restriction enzyme digestion of the PCR products. The asterisk indicates a Student t test value (P) of ≤0.025.

FOG-1 interacts with the GATA-1 protein in repressing transcription of the A{gamma}-globin gene. Previous studies showed that FOG-1 is required for repression mediated by GATA-1 (31). In this work we showed that the GATA-1 protein binds to the A{gamma}-globin gene at the –566 GATA site. Using the ChIP assay, we next examined whether FOG-1 was recruited to the –566 GATA site in the absence of {gamma}-globin expression. Our analysis with anti-FOG-1 and normal goat IgG antibodies in E12 fetal liver samples revealed no significant occupancy of FOG-1 at the –566 site when {gamma}-globin was expressed (Fig. 8B). In contrast, in E18 fetal liver where {gamma}-globin was silent, recruitment of FOG-1 was observed (Fig. 8B).

The Mi2 protein associates with GATA-1 and FOG-1 at the A{gamma}-globin gene –566 GATA binding site. The Mi2 protein, which is one of the protein subunits of NuRD or MeCP1 complexes, has been shown to associate with GATA-1 (31, 63). Thus, we hypothesized that Mi2 might interact with GATA-1 at the A{gamma}-globin –566 GATA silencer. To first assess the presence of the Mi2 protein in erythroid cell lines, Western blot analysis was performed with nuclear extracts of MEL and K562 cells using anti-Mi2 antibody (see Fig. S4 in the supplemental material). K562 and MEL cells were treated with hemin and HMBA to induce {gamma}-globin expression or left untreated. Mi2 protein showed a stronger signal in nuclear extracts of cells where the level of {gamma}-globin expression was low or absent (uninduced MEL and K562 cells) than in cells where {gamma}-globin expression was induced (hemin- and HMBA-treated K562 cells; see Fig. S4 in the supplemental material). Although we detected the presence of the Mi2 protein in the induced erythroid cell line, the level was increased in cells with low or no {gamma}-globin expression, suggesting that the presence of the Mi2 protein might be associated with the repression of {gamma}-globin gene expression.

We next utilized ChIP to test whether the Mi2 protein binds in vivo to the –566 GATA site of the A{gamma}-globin gene and if this binding correlated with the transcriptional repression or activation of the A{gamma}-globin gene. Immunoprecipitation was carried out using antibodies against Mi2 and normal rabbit IgG as a control. Specific recruitment of the Mi2 protein to the –566 GATA site of the A{gamma}-globin gene was observed only when {gamma}-globin expression was repressed (E18 fetal liver) (Fig. 8C). No recruitment of Mi2 was observed in those samples where {gamma}-globin expression was active (E12 fetal liver, Fig. 8C). These results provide strong evidence that Mi2 is associated with the transcriptional repression of A{gamma}-globin gene expression beginning during late fetal liver definitive erythropoiesis in vivo, presumably through recruitment to the –566 GATA site by colocalization with GATA-1. Since Mi2 is a well-characterized subunit of the NuRD or MeCP1 repressor complexes, its presence indicates that other components of these repressor complexes may function at this newly identified silencer region. Together our findings strongly suggest that FOG-1 mediates the binding of the Mi2 protein to the GATA-1 protein and that this complex binds to the –566 GATA site of the A{gamma}-globin gene. The binding of a GATA-1-FOG-1-Mi2 (NuRD or MeCP1?) repressor complex to this silencer region plays an important role in the transcriptional repression of {gamma}-globin expression during adult definitive erythropoiesis.

Recruitment of the GATA-1-FOG-1-Mi2 repressor complex to the analogous G{gamma}-globin –567 GATA site silencer. To analyze whether the same repressor complex was recruited to the analogous –567 GATA site of the G{gamma}-globin gene, ChIP assays using GATA-1, FOG-1, and Mi2 antibodies and specific primers was carried out as described above (Fig. 8). Our results showed that the GATA-1, FOG-1, and Mi2 proteins were recruited to this GATA site only when {gamma}-globin gene expression was silent (E18 fetal liver samples from wild-type β-YAC transgenic lines) (Fig. 8) and not when the {gamma}-globin gene was expressed (E12 fetal liver samples from wild-type β-YAC transgenic lines), in a parallel manner to the –566 GATA site of the A{gamma}-globin gene. Interestingly, the binding of the GATA-1, FOG-1, and Mi2 proteins to the G{gamma}-globin gene –567 GATA site was weaker than that observed for the A{gamma}-globin gene, suggesting that different mechanisms are involved in the regulation of the expression of the two {gamma}-globin genes.


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DISCUSSION
 
A number of regulatory mechanisms operate to coordinate temporal and tissue-specific expression of the β-like globin genes, including chromatin modification, DNA methylation, and dynamic changes in the transcriptional milieu as development proceeds. Gene-proximal regulatory regions of the {varepsilon}-, {gamma}-, and β-globin genes have been extensively analyzed using various small gene constructs and by deletion analysis using larger β-globin locus transgenes (3, 40, 56, 60, 66, 72). The {varepsilon}-globin gene has a complex promoter structure, with a stage-specific silencer (11, 41, 60), whereas the β-globin gene has a stage-specific enhancer 3' to the gene, which is required for high-level β-globin expression during adult definitive erythropoiesis (40, 72). {gamma}-Globin gene flanking sequences contain both positive and negative cis-regulatory elements (3, 66), and previous evidence suggested that the A{gamma}-globin gene may be autonomously silenced (7, 29, 49, 68, 69). A region located between –730 and –378 relative to the A{gamma}-globin mRNA CAP site was implicated as a silencer, although inclusion of the sequence in small transgene constructs did not completely repress {gamma}-globin expression (66). This result may have been due to the character of small µLCR transgene constructs, in which distances between the LCR HSs and linked transgenes are shortened and the transgenes are juxtaposed to the strong LCR enhancer, which may override any silencing effect. Our data with the A{gamma}m 5' {varepsilon} β-YAC mice (Fig. 1) clearly demonstrated that the A{gamma}m-globin gene was autonomously silenced during ontogeny (29). The A{gamma}m-globin gene was silenced even in transgenic lines carrying β-YACs with a deletion of the β-globin gene, suggesting that gene competition was not the exclusive determinant of {gamma}-globin gene silencing in transgenic mice. Silencing of the A{gamma}m-globin gene was not a result of its location within the β-YAC, because a βm-globin gene in exactly the same location was strongly expressed throughout development. Therefore, we hypothesized that the 5.4-kb SspI A{gamma}m-globin gene fragment integrated at this location within the β-YAC must contain the regions responsible for autonomous silencing of the {gamma}-globin gene.

To identify the A{gamma}m-globin gene silencer, a series of truncations was designed based on previous {gamma}-globin promoter analysis by Stamatoyannopoulos et al. (66). The first deletion construct, encompassing sequences from –730 to –378 relative to the {gamma}-globin mRNA CAP site, was introduced into the A{gamma}m 5' {varepsilon} β-YAC by homologous recombination. Five transgenic mouse lines were obtained containing at least one full-length β-globin locus transgene. Expression of {gamma}-globin was observed throughout development in all the {Delta}1s A{gamma}m 5' {varepsilon} β-YAC lines (Fig. 1). Normalized, copy number-corrected {gamma}-globin expression indicated that the A{gamma}m-globin gene was expressed at approximately 25% of level of the murine {alpha}-globin gene in adult blood, in contrast to the case for wild-type mice and the parental A{gamma}m 5' {varepsilon} β-YAC mice, which did not express {gamma}-globin during adult definitive erythropoiesis. Thus, our results indicated that we had deleted a silencer element within this 352-bp region of the A{gamma}m-globin gene promoter.

During adult erythropoiesis, {gamma}-globin and β-globin gene expression was low in all of the lines analyzed. Although {gamma}-globin was expressed in the adult blood of {Delta}1s A{gamma}m 5' {varepsilon} β-YAC transgenic mice, transcription levels were reduced and did not approach those observed during primitive erythropoiesis. Perhaps deletion of the negative region from –730 to –378 in the A{gamma}m 5' {varepsilon} β-YAC enhanced the ability of the LCR to interact with the A{gamma}m-globin gene, effectively shifting competition between the {gamma}- and β-globin genes for LCR interaction away from the β-globin gene. However, the transcriptional milieu would favor β-globin transcription during adult definitive erythropoiesis; the {gamma}-globin promoter would be inefficiently transcribed due to the loss of transcription factors present only during embryonic and fetal erythropoiesis that are necessary for {gamma}-globin gene activation. The net result would be a decrease in both {gamma}- and β-globin expression, with {gamma}-globin affected by a suboptimal transcription environment and β-globin affected by an unfavorable LCR-{gamma}-globin interaction. Subnuclear localization of this portion of the locus in a nonexpressing domain, methylation of the promoter, silencing via other transcriptional repression mechanisms active along the locus, such as DRED, or a combination of some or all of these mechanisms could also play a role in the observed gene expression levels.

GATA-1 is predominantly an activator of β-like globin gene expression and erythroid differentiation (54) but can also function as part of repressor complexes (31, 63). FOG-1 and GATA-1 have been shown to function as repressors in mast cells (44). ChIP assays demonstrated that a GATA-1-FOG-1-Mi2 complex binds to the –566 GATA site of the A{gamma}-globin gene and the analogous –567 GATA site of the G{gamma}-globin gene silencer elements, likely functioning as a repressor. In support of our data, a T->G mutation in the –567 GATA site of the G{gamma}-globin gene in an Iranian-American family was recently associated with an HPFH phenotype by David Chui (personal communication; see reference 42). His patient data validate the presence of a silencer element at these sequences of the {gamma}-globin genes. We propose that the binding of GATA-1 and recruitment of FOG-1, Mi2, and the NuRD complex to the {gamma}-globin promoter result in chromatin remodeling actions. In 12-day fetal liver, where {gamma}-globin is expressed, relative occupancy of GATA-1, FOG-1, and Mi2 was the same as that of the IgG control, indicating that these proteins did not bind at the –566 GATA site. The same is true for the G{gamma}-globin promoter –567 GATA site. In contrast, at 18 days, when erythropoiesis has moved to the bone marrow, GATA-1, FOG-1, and Mi2 bind the –566 GATA site in fetal liver. The Mi2/NuRD complex may interact with other proteins that bind to this region or that are part of this repressor complex. The binding of GATA-1-FOG-1-Mi2 is weaker at the G{gamma}-globin gene, suggesting one possible explanation for the slightly different regulation of the two {gamma}-globin genes.

The question remains as to what modulates the function of GATA-1 as an activator or a repressor. Perhaps posttranslational modification of the protein plays a role. Partington et al. studied the effect of induction of K562 and MEL cells and the relationship to DNA binding activity of the endogenous GATA-1 protein for an {alpha}G2-GATA site (50). GATA-1 was phosphorylated following induction of these cells, and phosphorylation resulted in increased GATA-1 binding to the {alpha}G2 fragment. In addition, GATA-1 phosphorylation also changes upon induction in MEL cells (16). We observed decreased binding of GATA-1 to the –566 GATA site at developmental stages where {gamma}-globin is expressed, likely reflecting the difference in GATA-1 binding as an activator versus that as a repressor. It will be interesting to determine whether the phosphorylation status of GATA-1 influences its binding at the –566 GATA site. Different factors may interact with phosphorylated and nonphosphorylated GATA-1. Alternately, the developmental stage may dictate the availability of other protein factors or the modification of already-present factors, which in turn may affect not only GATA-1 binding but also the recruitment of GATA-1 cofactors with specific functions.

We propose that the low level of {gamma}-globin expression in the adult is a result of the {gamma}-globin gene residing in a nonpermissive chromatin domain that is not likely to engage the LCR. It is likely that other epigenetic modifications, such as DNA methylation and histone acetylation, are involved in the GATA-1-mediated silencing of the {gamma}-globin genes. The NuRD complex has chromosome remodeling and histone deacetylase activity (15, 78). Mi2 coprecipitates with the MBD2 methyl-CpG binding protein complex in a repressed chicken β-like globin gene locus, suggesting that repression is linked to a closed chromatin structure and increased methylation of the genes (34). The recruitment of the repressive NuRD complex to the –566 silencer region of the {gamma}-globin genes may change the acetylation/methylation pattern of the {gamma}-globin genes, leading to a transcriptionally repressed state. Mabaera et al. demonstrated that there is a temporal and transient hypomethylation of the {gamma}-globin gene promoter in cell culture of bone marrow-derived cells, in addition to a strictly progressive repressive methylation along the locus (43). Thus, histone acetylation and DNA methylation likely work together in the locus.

Further study of the entire repressor complex that is associated with the silencer region described herein will provide additional insights into the mechanism by which GATA-1 operates to recruit this complex. It is provocative to suggest a mechanism of progressive repression of {gamma}-globin gene expression, where this and other repressor complexes are initially recruited late in fetal development to reversibly silence {gamma}-globin gene expression. As development proceeds into the adult stage of definitive erythropoiesis, the chromatin-remodeling repressor complexes, including the one bound at the –566/–567 {gamma}-globin GATA sites, would convert the surrounding chromatin into a more permanent transcriptionally closed structure, likely by affecting changes in the methylation and acetylation patterns of histones and DNA.


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ACKNOWLEDGMENTS
 
We thank Raymond Camahort, Patrick Sturdivant, Lesya Zelenchuk, and Taras Zelenchuk for excellent technical assistance.

This work was supported by PHS NIH grants R01 DK053510 (NIDDK), R01 HL067336 (NHLBI), R01 DK061804 (NIDDK), and P20 RR016475 (INBRE Program of the NCRR) and a Self Faculty Scholar Award to K.R.P. and University of Kansas Medical Center Biomedical Training Grants awarded to S.H.-B. and F.C.C.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, MS 3030, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160. Phone: (913) 588-6907. Fax: (913) 588-7440. E-mail: kpeterson{at}kumc.edu Back

{triangledown} Published ahead of print on 17 March 2008. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back

§ Present address: Center for Lung Biology, University of Washington School of Medicine, Seattle, WA 98109. Back

{ddagger} Co-first authors: these authors contributed equally to this work. Back


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Molecular and Cellular Biology, May 2008, p. 3101-3113, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.01858-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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