INTRODUCTION

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Hemoglobin switching describes the changes that occur in the sites of erythropoiesis during development as well as the changes in the globin genes that are expressed (51). The human -like globin genes (5'^GA3') are located in an ~75-kb region on the short arm of chromosome 11 and are arranged 5' to 3' in the order in which they are expressed during development (9, 58). The earliest human erythroid cells express -globin and are derived from the embryonic yolk sac. At 16 weeks of gestation, the fetal liver is the site of erythropoiesis. Fetal liver-derived erythroid cells express the duplicated -globin genes, while the -globin gene is silenced (45). By birth, the major site of erythropoiesis is the bone marrow, which expresses the - and -globin genes only (for reviews, see references 9, 23, 31, 51, and 58).

The locus control region (LCR), a group of DNase I-hypersensitive sites located upstream of the globin cluster, is the most important cis-regulatory element for the -like globin cluster (14, 19). Many studies have shown that the LCR is essential for high-level expression of globin genes in transgenic mice (15, 19, 47). In the absence of the LCR, individual human -like globin genes are expressed in transgenic mice at low levels but in a developmentally appropriate manner (7, 26, 29, 52, 53).

The effects of the LCR on the developmental stage-specific expression of the human -like globin genes are complex. In transgenic mice with the LCR and a single human - or -globin gene, no developmental stage-specific expression has been observed (23, 31). The globin genes are expressed in yolk sac, fetal liver, and adult erythroid cells. However, when the LCR was linked to both a - and a -globin gene, the developmental changes in gene expression were restored; the -globin genes were expressed in yolk sac- and fetal liver-derived erythroid cells, and the -globin genes were expressed in fetal liver and adult erythroid cells (4, 12). Other studies made use of marked -globin genes whose mRNAs could be distinguished from those of unmarked -globin genes. Marked -globin genes were inserted at different positions within a -globin locus cosmid. These studies showed that both the position of the marked -globin gene and the distance between the LCR and the genes in the cosmid construct determined the levels of both the marked and unmarked -globin mRNAs (10, 39).

These and other studies of hemoglobin switching have suggested several models for the developmental regulation of the -like globin genes which are not mutually exclusive. One model suggests that the gene order and distance of a gene from the LCR (polarity) determine the correct developmental expression pattern (20, 39). A second model proposes that the LCR serves only as an enhancer element, allowing high-level expression of the globin genes which are activated by developmental stage-specific elements (31). The competition model proposes that erythroid cells of each developmental stage contain stage-specific elements which compete for and stabilize interactions between globin genes and the LCR (8, 23).

All of these models suggest that elements near or within the individual globin genes are required for developmental activation and/or suppression of the -like globin locus. Physical evidence for interactions between the LCR and individual globin genes has been shown by in situ hybridization analysis of erythroid cells from transgenic mice with a complete -globin locus. Analysis of individual nuclei labeled with LCR, -globin, and -globin probes have shown that the LCR interacts with only "one gene at a time" (56).

Other studies have shown that transgenic mice containing an LCR, a -globin gene with a promoter deletion, and the -globin gene do not express the -globin gene at any stage of development, while the -globin gene is expressed throughout development (3). Similarly, transient transfection studies of K562 cells have demonstrated that deletion of the proximal -globin promoter allows expression from a downstream -globin promoter (24, 25). Both groups concluded that the -globin promoter sequences were responsible for the silencing of the downstream -globin gene, but they could not exclude the possibility that -globin gene expression prevented -globin gene expression in embryonic erythroid cells.

One prediction of the competition model for hemoglobin switching is that a heterologous promoter attached to a -globin gene would not compete for stage-specific elements or the LCR and would therefore permit the expression of a downstream -globin gene in embryonic erythroid cells. To test this hypothesis, we fused a 576-bp fragment of the human -spectrin promoter characterized by Gallagher et al. (18) to a human -globin gene and generated transgenic mice containing the -spectrin/^A-globin gene. We chose the promoter from the -spectrin gene because -spectrin is expressed at relatively high levels in erythroid tissues and does not exhibit changes in expression during development (44, 57).

Analysis of transgenic mice containing only -spectrin/^A-globin genes demonstrated that the human -spectrin promoter directed high levels of -globin mRNA in the absence of the LCR at all stages of development. In transgenic mice in which the human -spectrin promoter was substituted for the human ^A-globin promoter in a cosmid construct containing a micro-LCR (µLCR) and ^A-, -, -, and -globin genes, both the -spectrin/^A-globin and -globin genes are expressed throughout development. We conclude from these data that sequences within the -globin promoter, and not -globin transcription or intragenic sequences, are necessary and sufficient to silence the downstream -globin gene.

(This work was performed by Denise E. Sabatino in partial fulfillment of the doctoral degree requirements in the Graduate Genetics Program of the Columbian School of Arts and Sciences at George Washington University.)

	MATERIALS AND METHODS

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Plasmid and cosmid constructs. A 576-bp fragment containing the -spectrin promoter (sp) was excised from pGL2B (18) as a KpnI/HindIII fragment and cloned into the KpnI/HindIII sites of pSP72. The HindIII site in plasmid pSP72 sp was destroyed by digestion with HindIII, filling in the ends, and religation. A 1,909-bp BsaHI/HindIII fragment containing the coding region of the human ^A-globin gene was cloned into the ClaI/HindIII sites of pSP72. A 2,614-bp AatII/PvuII fragment from the sp plasmid was ligated to a 2,266-bp EcoRV/AatII fragment from the ^A-globin plasmid to create pSP72 sp/^A. The construct was confirmed by sequencing across the sp/^A exon 1 region. The 2,483-bp sp/^A gene was excised from this plasmid with EcoRV and HindIII for microinjection.

Generation of transgenic mice. Transgenic mice were generated as described by Hogan et al. (21). Fertilized eggs were collected from superovulating FVB/N female mice approximately 9 h after mating to CB6 F₁ male mice. Fragments for microinjection were separated on agarose gels and concentrated by using a DNA Geneclean isolation system according to the manufacturer's instructions. The fragments were diluted to a concentration of 1 µg/ml in 7.5 mM Tris-HCl-0.25 mM EDTA (pH 7.5), and 1 to 3 pl was injected into the male pronuclei of fertilized eggs. The injected eggs were transferred to pseudopregnant CB6 F₁ foster mothers. Founder animals were identified by Southern blotting (30) of DNA extracted from tail biopsies. Southern blots were probed with human -spectrin and/or human -globin probes. Copy number was determined by comparing transgenic mouse DNA to K562 DNA on a Southern blot and analysis using a Molecular Dynamics PhosphorImager. Founder animals were crossed to FVB/N mice for propagation and developmental studies.

Cellulose acetate electrophoresis. Blood samples were collected from 10.5-day embryos, 13.5-day embryos, and adult mice. Samples were lysed in cystamine as described by Whitney (55). Samples were run at 300 V for 20 min in a Titan electrophoresis chamber (Helena Laboratories), stained with Ponceau S (in 5% trichloroacetic acid) for 10 min, and destained in 7% acetic acid for 10 min (twice). The gels were fixed in methanol for 5 min (twice), cleared in a solution of 15 ml of glacial acetic acid, 35 ml of methanol, and 2 ml of Clear Aid for 7.5 min, and dried at 50 to 60°C for 15 min. The globin chain composing each hemoglobin tetramer was confirmed by acid-urea gel electrophoresis (1) of hemoglobins separated by cellulose acetate electrophoresis.

RNase protection assays. Total cellular RNA was extracted from 10.5-day embryo blood cells, 13.5-day fetal livers, and adult reticulocytes, using TRIZOL reagent (Gibco BRL, Life Technologies, Inc., Grand Island, N.Y.). Linear DNA templates for the RNase protection assay were prepared by EcoRI (sp/^A), HindIII (mouse ), and HindIII (human ) digestion of cesium chloride-purified plasmid preparations. The templates were purified by agarose gel electrophoresis and purified by using a Geneclean II kit (Bio 101, Inc., Vista, Calif.). ³²P-labeled RNA probes were transcribed by using a MAXIscript in vitro transcription kit (Ambion, Inc., Austin, Tex.). Hybridization of the probe and the RNA (1 µg) was carried out overnight according to the standard procedure for the RPA II RNase protection assay kit (Ambion). RNase digestion was performed with an RNase A-RNase T₁ mixture in RNase digestion buffer (Ambion), and the protected fragments were separated on an 8% nondenaturing polyacrylamide gel (SEQUAGEL-8; National Diagnostics, Atlanta, Ga.).

Immunofluorescence analysis. Fetal liver cells from transgenic mice and control littermates were stained with two monoclonal antibodies. One antibody, directed against human -globin, was directly conjugated to fluorescein isothiocyanate (FITC); the second monoclonal antibody, against human -globin, was directly conjugated to rhodamine. The same field of cells was photographed under different exposure conditions: with an FITC filter (for detection of -globin), with a rhodamine filter (for detection of -globin), and with first an FITC filter and then a rhodamine filter for a double exposure.

	RESULTS

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Transgenic mice with human -globin single-gene constructs. We created two single-gene constructs containing the -spectrin promoter fragment linked to the human ^A-globin gene, sp/^A and HS2 sp/^A (6, 15, 27, 33, 42, 50, 54) (Fig. 1; see Materials and Methods). Three transgenic mouse lines and two 13.5-day fetal livers containing sp/^A were analyzed. RNase protection demonstrated that two of three sp/^A transgenic lines and one of two fetal livers expressed the sp/^A transgene in erythroid cells. No sp/^A mRNA was detected in erythroid cells of the third transgenic line and the other fetal liver. Analysis of RNA extracted from a variety of organs and tissues of adult sp/^A transgenic mice revealed high levels of sp/^A mRNA in reticulocytes, bone marrow, and spleen and lower levels in thymus and other tissues (Fig. 2) (18). Much of the globin mRNA in nonerythroid cells can be attributed to the high reticulocyte counts (~15%) associated with a mild thalassemia caused by the excess of -like globin chains in these and all other transgenic mouse lines described in this study (18).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   -Spectrin/-globin single-gene constructs used to generate transgenic mice. The -spectrin promoter fragment (576 bp) is fused to the A coding sequence at position 3 (A). A 712-bp fragment containing HS2 of the LCR is linked to the -spectrin/-globin gene (B). Transgenic mice were identified by Southern blot analysis using a 576-bp -spectrin promoter probe.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   RNase protection analysis of sp/A mRNA expression in transgenic mice. (A) RNase protection of 10 µg of K562 RNA and 1 µg of adult reticulocyte mRNA from strain sp/A A. 32P-labeled 1,493-bp RNA containing sp/A gene sequences from exon 2, intron 1, and the sp/A exon 1 fusion region was used as a probe. The positions of 32P-labeled, protected RNA fragments corresponding to A-globin exon 2, sp/A exon 1, and A-globin exon 1 are indicated. (B) RNase protection of 1 µg of bone marrow (BM) and spleen RNA and 10 µg of thymus RNA from strain sp/A A. Labeled RNA fragments corresponding to the sp/A (exon 2 shown), mouse -globin (exon 2), and mouse actin (control) mRNAs were used as probes.

View this table: [in this window] [in a new window]   TABLE 1.   Expression of sp/A mRNA in transgenic mouse lines

View larger version (45K): [in this window] [in a new window]   FIG. 3.   RNase protection of 1 µg of 10.5-day embryonic blood, 13.5-day fetal liver, and adult reticulocyte RNAs from strains sp/A A and C (A) and from strains HS2 sp/A B and C (B). Labeled RNA fragments corresponding to the sp/A (exon 2 shown) and mouse -globin (exon 2) genes were used as probes.

View larger version (46K): [in this window] [in a new window]   FIG. 4.   Cellulose acetate electrophoresis of 14.5-day (A) and adult (B) peripheral blood lysates from strain sp/A A. Positions of the endogenous mouse hemoglobins and the mouse -2/human -2 hemoglobin tetramer are shown. +, blood lysate from transgenic animal; , blood lysate from littermate control.

Transgenic mice with cosmid gene constructs. A 37-kb µLCR^A cosmid construct (Fig. 5) containing a 4-kb µLCR linked to the ^A-, -, -, and -globin genes was modified by replacing 381 bp of the ^A-globin promoter with the 576-bp -spectrin promoter (Fig. 5). Three transgenic founder animals were generated with the resulting µLCRsp^A cosmid. High levels of sp/^A mRNA were detected in RNA from reticulocytes, bone marrow, and spleen, and no sp/^A mRNA was detected in RNA from thymus (Fig. 6). Southern blot analysis showed that these lines contained between 8 and 40 copies of the cosmid. One transgenic founder animal (strain C) did not transmit the transgene to F₁ progeny and was concluded to be mosaic for the transgene. Transgene expression in this line was studied only in the founder animal. Three additional transgenic lines containing the control µLCR^A cosmid construct were generated for comparison.

View larger version (9K): [in this window] [in a new window]   FIG. 5.   Control and sp/A cosmid constructs used to generate transgenic mice. The control µLCRA cosmid originally described by Enver et al. (12) (A) was modified to replace the A promoter region from 381 to 1 with a 576-bp fragment of the human -spectrin promoter (B). Transgenic mice were identified by Southern blot analysis using a 576-bp fragment containing the human -spectrin promoter region and a 960-bp fragment containing exon 2 of the human -globin gene as probes.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   Analysis of sp/A mRNA expression in transgenic mice containing the µLCRA cosmid by RNase protection assay of 1 µg of bone marrow (BM) and spleen RNA and 10 µg of thymus RNA from strain µLCRspA A. Labeled RNA fragments corresponding to the sp/A (exon 2 shown), mouse -globin (exon 2), and mouse actin (control) genes were used as probes.

View larger version (51K): [in this window] [in a new window]   FIG. 7.   RNase protection of 1 µg of 10.5-day embryonic blood, 13.5-day fetal liver, and adult reticulocyte RNA from control cosmid strain C (A) and from strain µLCRspA A (B). Labeled RNA fragments corresponding to the sp/A (exon 2 shown), human -globin (exon 3), and mouse -globin (exon 2) genes were used as probes.

View this table: [in this window] [in a new window]   TABLE 2.   Expression of sp/A mRNA in µLCRspA and µLCRA transgenic mouse lines

View larger version (34K): [in this window] [in a new window]   FIG. 8.   Cellulose acetate electrophoresis of 14.5-day (14.5d) and adult peripheral blood lysates from control µLCRA C (left) and µLCRspA A (right) transgenic mice. The positions of the endogenous mouse hemoglobins, the mouse -2/human -2 hemoglobin tetramer, and the mouse -2/human -2 hemoglobin tetramer are shown. +, blood lysate from transgenic animal; , blood lysate from littermate control.

Immunofluorescence analysis of 14.5-dpc fetal livers from µLCRsp^A embryos. Immunofluorescence analysis of human - and -globin gene expression was done on 14.5-dpc fetal livers from µLCRsp^A transgenic embryos and control µLCR^A cosmid-containing embryos. As previously described, fetal liver cells from control µLCR^A cosmid-containing mice expressed either human -globin or human -globin (12). Fetal liver cells from µLCRsp^A cosmid-containing mice all expressed both human -globin and human -globin (Fig. 9).

View larger version (84K): [in this window] [in a new window]   FIG. 9.   Immunofluorescence analysis of sp/A and human -globin chains in fetal liver cells from µLCRspA transgenic mice. The fetal liver cells were fixed and stained with conjugated monoclonal antibodies against human -globin (FITC) and human -globin (rhodamine). The identical field was photographed with an FITC filter (), a rhodamine filter (), and both filters (+).

	DISCUSSION

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Analysis of transgenic mice containing either cosmids or yeast artificial chromosomes (YACs) has shown that appropriate developmental regulation requires the presence of an active globin gene or genes upstream of the -globin gene (4, 11, 12, 16, 17, 28, 37, 40, 43). However, these studies have not identified the specific sequences involved. Analysis of deletion constructs in transgenic mice or cultured cells demonstrated that deletion of the -globin promoter allowed expression of the downstream -globin gene (3, 24). However, the same deletions which allow -globin gene expression abolished -globin gene expression. Our data support the competition model for hemoglobin switching, which posits that the individual globin genes compete for stage-specific elements to stabilize transcription of individual globin genes. By replacing the -globin promoter with the -spectrin promoter, the -globin gene is expressed at all stages of development in the presence of -globin gene expression. We conclude that the sequences between 381 and +1 of the -globin promoter, and not -globin transcription or intragenic sequences, are necessary and sufficient to silence the downstream -globin gene in the embryonic stage of development.

The observation that the -spectrin promoter is an enhancer-dependent and erythroid cell-specific promoter makes it an ideal promoter for the studies described here. Previous analyses of transgenic mice carrying phosphoglycerate kinase-Neo gene constructs inserted into either the mouse -globin locus (49) or the mouse (13, 22) and human (5, 38) LCRs had strikingly negative effects on globin gene expression. It is clear from these studies that the phosphoglycerate kinase gene promoter or Neo sequences are not neutral substitutions and that they may be strong competitors for stage-specific elements and/or the LCR (41).

Our data also support models in which specific sequences in the -globin promoter capture the LCR in the yolk sac erythroid cells and prevent -globin gene expression (2, 8, 24, 25). The testing of this model, and the importance of gene order and polarity, would require substitution of the -spectrin promoter for the ^A-globin promoter in a YAC construct, which would place two globin promoters upstream of the -spectrin promoter. If the LCR specifically interacts with these promoters, we hypothesize that expression of the -globin gene will be suppressed in yolk sac erythroid cells.

Our data are not consistent with some aspects of the models in which stage-specific elements alone are responsible for hemoglobin switching (31). These models posit that the presence of an enhancer-dependent -globin gene might be sufficient to suppress -globin expression in the yolk sac, in contrast to what we observed. The possibility that the -spectrin promoter does not compete with the -globin promoter for the same stage-specific factors which interact with the -globin promoter can be tested in the YAC experiments as well.

Since the sp/^A globin gene is expressed at relatively high levels in erythroid cells at all stages of development, sp/^A transgenic mice may be useful to test the effects of -globin on the amelioration of sickling in the sickle cell disease mouse model (35, 48). Previous in vitro studies have demonstrated that the amount of hemoglobin S (HbS) polymers decreases with increasing proportions of HbF, reaching a maximum inhibition of polymerization at 20% HbF (34). Other studies have found that as the HbF concentration increases, the severity of sickle cell disease decreases, with significantly less severe disease with >10% HbF (32, 36, 46). Generating sickle cell disease mice carrying the sp/^A gene would test whether the ^A-globin expressed from the -spectrin promoter is sufficient to reduce HbS polymerization in the mouse model.