Autonomous Silencing as Well as Competition Controls {gamma}-Globin Gene Expression during Development

To investigate the control of the ${gamma}$ -globin gene during development,we produced transgenic mice in which sequences of the ß-genepromoter were replaced by equivalent sequences of the ${gamma}$ -genepromoter in the context of a human ß-globin locusyeast artificial chromosome (ßYAC) and analyzed theeffects on globin gene expression during development. Replacementof 1,077 nucleotides (nt) of the ß-gene promoter by1,359 nt of the ${gamma}$ promoter resulted in striking inhibition ofthe ${gamma}$ -promoter/ß-gene expression in the adult stageof development, providing direct evidence that the expressionof the ${gamma}$ gene in the adult is mainly controlled by autonomoussilencing. Measurements of the expression of the ${gamma}$ promoter/ß-globingene as well as the wild ${gamma}$ genes showed that gene competitionis also involved in the control of ${gamma}$ -gene expression in the fetalstage of development. We conclude that autonomous silencingis the main mechanism controlling ${gamma}$ -gene expression in the adult,while autonomous silencing as well as competition between ${gamma}$ andß genes contributes to the control of ${gamma}$ to ßswitching during fetal development.

INTRODUCTION

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Introduction

The ß-globin locus spans at least 100 kb on chromosome11 and consists of a distal enhancer, called the locus controlregion (LCR), and five functional genes, ${varepsilon}$ , ^G ${gamma}$ , ^A ${gamma}$ , ${delta}$ , and ß,organized in the order of their developmental expression (reviewedin reference 23). The ${varepsilon}$ -globin gene is the first gene expressedduring ontogeny, and its expression is restricted to the bloodislands of the yolk sac. At approximately 6 to 8 weeks of gestation,the ${varepsilon}$ -globin gene is silenced and the ${gamma}$ -globin genes are activated.Around birth, ß- and ${delta}$ -globin gene activity dramaticallyincreases and the ${gamma}$ -globin gene expression progressively declinesto about 0.5% of the total globin expression. These developmentalchanges in human globin gene activity are recapitulated in transgenicmice carrying human ß-locus yeast artificial chromosome(ßYAC) constructs, except for the fact that in thismurine model the human ${gamma}$ -globin genes display both embryonicand fetal expression (12, 18, 20, 24).

Currently, the silencing of the ${gamma}$ gene is considered to be autonomousand independent of the presence of other globin genes in cis(4). The molecular mechanism of autonomous silencing is unclear,but it is likely that it is complex. Early transgenic studieshave shown that a DNA fragment containing the entire ${gamma}$ -globingene, but no sequences of the LCR, expresses the ${gamma}$ gene onlyin the embryonic erythroid cells (2, 13). Although the ${gamma}$ genewas not expressed in the fetal erythroid cells where it is normallyexpressed, these results have been considered evidence thatthe ${gamma}$ gene itself contains the information required for silencingin definitive erythropoiesis. The level of ${gamma}$ -gene transcriptionin these transgenic mice was extremely low (2, 13). When enhancedby a truncated LCR (designated µLCR), the ${gamma}$ gene is expressedin the embryonic but also the fetal and adult stages (7). The ${gamma}$ gene is partially silenced in adult transgenic mice carryingone or two copies of a cosmid construct containing the entireLCR and the ${gamma}$ gene but not in transgenic mice carrying multiplecopies of that construct (4). The gene is completely silencedin human ß-locus YAC in adult transgenic mice (18).Sequences involved in activation as well as in ${gamma}$ gene silencingare located in the ${gamma}$ -gene promoter. The minimal promoter of thegalago ${gamma}$ gene, which is expressed exclusively in embryonic erythroidcells, suppresses the expression of the human ${gamma}$ gene in the definitiveerythropoiesis of transgenic mice (27). The elements responsiblefor this silencing activity have been localized in the CACCCand CCAAT boxes of the galago ${gamma}$ -gene promoter (14). However,mutations of the human ${gamma}$ -gene promoter have shown that each ofthese boxes also is indispensable for ${gamma}$ -gene activation in theadult (5, 9, 22). In addition to the globin gene promoters,the LCR has a role in developmental regulation of the globingenes, and individual DNase I-hypersensitive sites of this regulatoryelement appear to have gene specificity (11, 15). The hypersensitivesite 3 (HS3) of the LCR preferentially enhances the expressionof the ${gamma}$ gene, and deletion of the core sequences of the HS3abolishes ${gamma}$ -gene expression in the fetal stage of erythropoiesis(16).

To investigate the control of ${gamma}$ -globin gene during development,we replaced the ß-gene promoter with its ${gamma}$ -gene counterpart,in the context of ß-globin locus YAC constructs, andanalyzed the effects on globin gene expression during developmentof transgenic mice. We obtained evidence that the silencingof the ${gamma}$ gene in the adult stage of development is controlledthrough sequences residing in the ${gamma}$ -gene promoter. Measurementsof the expression of the hybrid ${gamma}$ promoter/ß gene aswell as of the expression of the wild ${gamma}$ genes located in thesame YAC provided evidence that gene competition is also involvedin the control of ${gamma}$ -gene expression during the fetal stage ofdevelopment. We conclude that autonomous silencing is the mainmechanism that controls ${gamma}$ -gene expression in the adult, and autonomoussilencing as well as competition between the ${gamma}$ - and the ß-globingenes controls the ${gamma}$ to ß switch during fetal development.

MATERIALS AND METHODS

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Materials and Methods

ßYAC constructs. A yeast interacting plasmid (YIP) containing a BglII/RcaI fragment(coordinates 60577 to 64029; GenBank humhbb accession numberU01317) with 1,080 bp of the ß-globin gene promoter(coordinates 61110 to 62189) replaced with 1,404 bp of the ${gamma}$ -globingene promoter (coordinates 38066 to 39469) was made by standardmolecular cloning techniques. After linearizing with PmlI, theplasmid was transformed into the yeast containing 155 kb ofßYAC using the Clontech yeast transformation kit followingthe manufacturer's protocol. The transformants were selectedon uracil dropout media and PCRs were performed to select thecorrect clones, which were confirmed by Southern hybridizationanalysis. Spontaneous excision of the YIP was induced by overnightgrowth in nonselective rich medium (yeast peptone dextrose).The yeast cells were plated on the 5-fluoroorotic acid plateto select for loss of a Ura3 gene residing on the YIP vector,which results in 5-fluoroorotic acid resistance. The replacementof 1,080 bp of the ß-gene promoter with the 1,359-bp ${gamma}$ -gene promoter was confirmed by Southern hybridization. ThisYAC was designated 1.36kb ${gamma}$ pr-ßYAC.

To delete the ^G ${gamma}$ and ^A ${gamma}$ genes from 1.36kb ${gamma}$ pr-ßYAC, aloxP-ura3-loxP deletion cassette was prepared. A 348-bp fragment5' to the ^G ${gamma}$ gene (coordinates 33170 to 33517) was inserted upstreamof the first loxP site, and a 861-bp fragment 3' to the ^A ${gamma}$ gene(coordinates 41308 to 42168) was inserted downstream of thesecond loxP site. The construct sequence was released from theplasmid backbone with restriction enzymes and transformed intothe yeast containing 1.36kb ${gamma}$ pr-ßYAC as described above.The transformants were selected on the dropout media lackinguracil, and PCR was performed to check the replacement of the7.8 kb of the ${gamma}$ genes by the loxP-Ura3-loxP cassette. The positiveclones were then transformed with a cytomegalovirus-Cre plasmid.The transformants were plated on 5-fluoroototic acid platesand further selected on dropout media lacking lysine and tryptophan.PCR was performed to screen the correct clones in which excisionhad happened between the loxP sites. The deletion was furtherconfirmed by sequencing. This YAC was designated (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ )YAC.

Transgenic mice. YACs were separated from the yeast chromosomes by pulsed-fieldgel electrophoresis. The agarose gel block containing the YACwas cut into small pieces, equilibrated with the ß-agarasebuffer, and digested with ß-agarase (New England BioLabs).Undigested agarose was removed by centrifugation. Purified DNAfragments were injected into fertilized mouse eggs (B6/C3F1)and then transferred to pseudopregnant foster mothers (B6/D2F1).Founder animals were identified by slot blotting with an HS3probe. F₁ progeny were obtained by breeding founder animalswith nontransgenic mice (B6/D2F1) and were used for structuralanalysis. To study the developmental pattern of human globingene expression, staged pregnancies were interrupted at day12, 14, or 16, and samples from blood, yolk sac, and fetal liverwere collected.

Structural analysis of YAC transgenes. Intactness of YAC transgenes in transgenic mice was examinedas previously described (16). Briefly, agarose plugs preparedfrom mouse liver cell suspensions were digested overnight withSfiI, and the DNA was fractionated by pulsed-field gel electrophoresis.The gel was capillary blotted overnight to nylon membranes.Individual lanes were cut out, and each one was hybridized overnightto a different probe. After washing, the membrane was reassembledand an autoradiograph or a phosphorimage was made.

Copy number measurement. DNA from fetal brain or carcass of F₂ progeny in each line wasisolated by standard procedures. At least three DNA sampleswere obtained from each line. Individual samples were then restrictedwith HindIII. Ten micrograms of DNA from each enzyme reactionperformed on samples from a given line was loaded onto a 0.8%agarose gel, and DNA fragments were resolved by electrophoresis.Southern blots were consecutively hybridized with four probesencompassing the HS3 or ${varepsilon}$ -, ${gamma}$ -, or ß-globin gene sequence.Signals were quantitated with a PhosphorImager. Copy numberswere determined by comparing the signal from a given transgenicline with those of human genomic DNA. In cases in which thecomputed value was not an integer, the copy number was roundedto the nearest integer in standard fashion.

RNA analysis. Total RNA was prepared from the yolk sacs, embryonic and fetalblood, fetal liver, and adult blood. RNA samples were separatelyisolated from three or more transgenic individuals for eachtime point. The human ${varepsilon}$ -, ${gamma}$ -, and ß-globin mRNAs andmurine ${alpha}$ - and ${zeta}$ -globin mRNAs were detected by RNase protectionassay and quantified by a PhosphorImager. To minimize experimentalerror, samples from individual animals were quantified independentlyand multiple measurements were performed by RNase protection.Copy number-corrected globin mRNA levels were expressed as human ${gamma}$ mRNA/ ${gamma}$ copy number/[(murine ${zeta}$ -globin mRNA/2) + (murine ${alpha}$ -globinmRNA/4)]. Mean values and standard deviations are shown in Tables1 to 5. For t test calculations, the mean values of expressioncalculated from multiple measurements of multiple individualswere used for each transgenic line.

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TABLE 1. ${varepsilon}$ -Globin gene expression in 1.36kb ${gamma}$ pr-ßYAC transgenic mice^a

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TABLE 5. Expression of 1.36kb ${gamma}$ pr-ß gene in (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC transgenic mice^a

RESULTS

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Results

${gamma}$ -Gene promoter sequences control ${gamma}$ -globin gene silencing in the adult stage of development. To investigate the role of the ${gamma}$ -globin gene promoter on ${gamma}$ -genesilencing, we replaced the 1,077-bp ß-gene promotersequence 5' to the translation initiation codon with the corresponding1,359-bp sequence (subsequently referred to as 1.36kb) of the^A ${gamma}$ promoter in the context of a 155-kb ß-globin locusYAC. The transitional point from the ${gamma}$ -gene sequence to the ß-genesequence was at the NcoI restriction site that contains theATG starting codon in both genes. Figure 1 shows the structuralanalysis of 1.36kb ${gamma}$ pr-ßYAC in three established transgeniclines. The continuity of the transgene integrated in the mousegenome was determined by pulsed-field gel electrophoresis (Fig.1A). Single-cell suspensions of transgenic livers were embeddedin agarose gel and digested with SfiI. SfiI sites are locatedupstream of 5'HS5, between 5'HS3 and 5'HS4, and downstream of3'HS1. After electrophoresis and Southern blotting, each blotwas hybridized with 12 DNA probes spanning from the HOR2 gene(which is located upstream of the 5'HS6) to the downstream DNaseI-hypersensitive site, 3'HS1 (which is located about 22 kb 3'to the ß-globin gene). All lines contained continuousSfiI fragments from 5'HS3 to 3'HS1. The next upstream SfiI fragment,which contains 5'HS4 to 5'HS6, was also intact. Two of the fourcopies of the transgene in line A and one of the two copiesin line C lacked the 3'HS1 hybridization band; however, theabsence of the 3'HS1 sequences is not expected to affect properregulation of the globin genes in transgenic mice (4). The copynumbers of the transgenes were determined by conventional Southernhybridization analysis. The blots of HindIII-restricted mousegenomic DNA were hybridized with four DNA probes (HS3, ${varepsilon}$ , ${gamma}$ , andß), and as shown in Fig. 1B, each probe produced theexpected fragment with the correct sizes. Copy numbers wereestimated to be 4, 2, and 2 for lines A, B, and C, respectively.

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FIG. 1. Structural analysis of the 1.36kb ${gamma}$ pr-ßYAC transgene. (A) Examination of continuity of the ßYAC transgene integrated in the mouse genome. The line diagram on the top represents the human ß-globin locus. The five vertical arrows are DNase I-hypersensitive sites of the LCR. 3'HS1 is bracketed because it is not formed in transgenic mice. Five globin genes ( ${varepsilon}$ , ^G ${gamma}$ , ^A ${gamma}$ , ${delta}$ , and ß) are shown as filled boxes. Agarose-embedded mouse genomic DNA from lines A to C was restricted with SfiI, fractionated by pulsed-field gel electrophoresis, and Southern blotted onto nylon membrane. Blot strips were hybridized with 12 probes spanning from HOR2 (which is located upstream of the LCR) to 3'HS1. Reassembled blots are shown. The thin lines link the probes used in each strip to the corresponding positions in the ß-globin locus. (B) Measurement of copy number of the 1.36kb ${gamma}$ pr-ßYAC transgenes. Mouse genomic DNA was digested with HindIII and analyzed with Southern hybridization using four different probes encompassing HS3, ${varepsilon}$ , ${gamma}$ , and ß genes. Sizes of the hybridization bands are indicated on the right side of each blot. (C) Confirmation of the replacement of the ß-gene promoter with its ${gamma}$ -gene counterpart. The mouse genomic DNA was restricted with EcoRI and hybridized with a ß-gene probe. The right line diagram shows that the wild-type ßYAC produces a 5.5-kb band, while the 1.36kb ${gamma}$ pr-ßYAC produces a 2.6-kb band. The closed boxes indicate the coding sequence of the ß gene. The open box is the 1.36kb ${gamma}$ -gene promoter, which introduces a new EcoRI site. wt, wild type.

The replacement of the ß-gene promoter by the ${gamma}$ -genepromoter was verified by Southern hybridization analysis oftransgenic mouse DNA. In the wild-type ßYAC, the EcoRIfragment encompassing the ß gene is 5.5 kb long. Thereplacement of the ß-gene promoter with the 1.36-kb ${gamma}$ -gene promoter introduces an additional EcoRI site, generatinga new fragment 2.6 kb long. As shown in Fig. 1C, all three transgeniclines contained the new 2.6-kb fragment, indicating that thereplacement mutation was intact. Taken together, these analysessuggest that the 1.36kb ${gamma}$ pr-ßYAC constructs were integratedas intact copies in the mouse genome.

The expression of the 1.36kb ${gamma}$ pr-ß gene was measuredby RNase protection assays. A representative result is shownin Fig. 2. The protected mRNA fragments of the human ß-, ${gamma}$ -, and ${varepsilon}$ -globin gene along with the endogenous murine ${alpha}$ - and ${zeta}$ -globingenes (which serve as the internal controls) are shown. Thesamples from day 12 blood and day 12 yolk sac are composed almostexclusively of nucleated erythroid cells of the embryonic erythropoiesis;the day 12 fetal liver as well as days 14 and 16 blood and fetalliver contain enucleated erythroid cells of the definitive erythropoiesisof fetal liver; the adult blood contains enucleated erythroidcells from the adult erythropoiesis.

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FIG. 2. Globin gene expression in 1.36kb ${gamma}$ pr-ßYAC transgenic mice by RNase protection assay. Representative RNase protection assays (line A of Tables 1 to 3) showing the protected fragments of murine ${alpha}$ and ${zeta}$ as well as human ${varepsilon}$ , ${gamma}$ , and ß mRNA. The corresponding sizes of the protected band are shown on the right side. Bl, blood; YS, yolk sac; FL, fetal liver; wt, wild type.

Detailed results of globin gene expression at the differentstages of development in the three transgenic mouse lines arepresented in Tables 1 to 3. The mean expression levels of the1.36kb ${gamma}$ pr-ß transgene are compared, in Fig. 3A, withthe levels of ß-gene expression in the wild-type ßYACtransgenic mice. The wild-type ß gene is expressedin adult blood at 126.9% ± 23.0% of murine ${alpha}$ -like globinmRNA per copy; in contrast, the level of expression of the 1.36kb ${gamma}$ pr-ß gene in the adult transgenic mice is 1.7% ±1.1% per copy (range, 0.5% to 2.5%), providing evidence thatthe sequences responsible for ${gamma}$ -gene silencing in adult erythropoiesisare located in the 1,359-bp ${gamma}$ -gene promoter. The fact that the ${gamma}$ /ß hybrid construct is still expressed although atthese low levels at the adult stage suggests that remainingß-globin gene regulatory elements, like the 3' enhancerand intronic enhancer, are able to communicate with basal promoterelements in the gamma promoter to direct expression of the hybridgene.

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TABLE 3. Expression of the 1.36kb ${gamma}$ pr-ß gene in 1.36kb ${gamma}$ pr-ßYAC transgenic mice^a

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FIG. 3. Levels of globin mRNA species during the development of 1.36kb ${gamma}$ pr-ßYAC transgenic mice. (A) Mean mRNA levels of the 1.36kb ${gamma}$ pr-ß gene in transgenic lines A to C of Tables 1 to 3 (white columns) are compared to the expression levels of the ß gene in the wild-type ßYAC mice (black columns). Expression levels are corrected by the endogenous ${alpha}$ -like mRNA and copy number. (B) Mean mRNA levels of the 1.36kb ${gamma}$ pr-ß gene (white columns) are compared to the expression levels of the ${gamma}$ gene in the wild-type ßYAC mice (black columns). Ad Bl, adult blood; YS, yolk sac; FL, fetal liver; wt, wild type; d12, day 12.

Competition by the wild ${gamma}$ -globin genes inhibits the expression of the hybrid ${gamma}$ promoter/ß gene in embryonic erythropoiesis. For studies of embryonic erythropoiesis, RNase protection assayswere done using cells from day 12 yolk sacs and day 12 bloodof 1.36kb ${gamma}$ pr-ßYAC mice. As shown in Fig. 2 and 3A and B,the expression of the 1.36kb ${gamma}$ pr-ß gene was strikinglyreduced in the embryonic cells of the transgenic mice. The levelof ${gamma}$ mRNA of the wild-type ßYAC embryos was 29.4% inthe day 12 blood and 37.8% in the day-12 yolk sac. In contrast,the levels of the ß mRNA in the blood and the yolksac of day 12 embryos carrying the 1.36kb ${gamma}$ pr-ß genewere barely detectable, i.e., 1.5% and 1.7%, respectively. Thesefindings were unexpected, because the 1.36kb ${gamma}$ pr-ßtransgene was driven by a promoter identical to the wild ${gamma}$ -genepromoter. The most likely explanation of these results was thatcompetition by the upstream ^G ${gamma}$ and ^A ${gamma}$ genes was decreasing theprobability of interaction between the 1.36kb ${gamma}$ pr-ßgene and the LCR, resulting in the unexpectedly low expressionof that gene in embryonic cells. Previous studies have shownthat the two ${gamma}$ genes compete with each other in the yolk sacerythropoiesis (26).

To test this hypothesis, we deleted the ^G ${gamma}$ and ^A ${gamma}$ genes in thecontext of the 1.36kb ${gamma}$ pr-ßYAC construct, producingthe (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC. Five transgenic lines carryingintact copies of this construct were established. Structuralanalysis by pulsed-field gel electrophoresis showed that alllines contained intact YAC constructs (Fig. 4). Separate Southernhybridization analyses (data not shown) revealed that eitherone or two copies of the construct have integrated into thegenome of the five transgenic lines.

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FIG. 4. Structural analysis of the 1.36kb ${gamma}$ pr-ß transgene in the (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC transgenic mice. The data show continuity of the transgene integrated in the mouse genome in all five lines. The distance between LCR (starting from the middle between HS2 and HS3) and the ${gamma}$ -pr-ß gene was approximately 50 kb before the "^G ${gamma}$ -^A ${gamma}$ " deletion and 42 kb after the "^G ${gamma}$ -^A ${gamma}$ " deletion. See the legend to Fig. 1 for details.

Globin gene expression of the five lines is presented in Tables4 and 5. Notice that the expression of the ${varepsilon}$ gene of the (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC mice was about 50% higher than thatof the ${varepsilon}$ gene in the wild ßYAC mice, but the differencewas not statistically significant. Also notice that there wasrobust expression of the 1.36kb ${gamma}$ pr-ß gene in the yolksac and the embryonic blood (Tables 4 and 5). In Fig. 5A wecompare the levels of expression of the 1.36kb ${gamma}$ pr-ßgene in mice with or without the ^G ${gamma}$ and ^A ${gamma}$ gene deletion, andin Fig. 5B we compare the levels of expression of the 1.36kb ${gamma}$ pr-ß gene with the expression of the wild ${gamma}$ gene inwild ßYAC mice (Fig. 5B). In contrast to the barelydetectable expression when the ^G ${gamma}$ and ^A ${gamma}$ genes were present, the1.36kb ${gamma}$ pr-ß gene was expressed at levels of 24.5%and 36.8% of murine ${alpha}$ -like mRNA per copy in the embryonic cellswhen the ^G ${gamma}$ and ^A ${gamma}$ genes were deleted; these levels are very closeto the level of ${gamma}$ -gene expression in the embryonic cells of thewild-type ßYAC mice (Tables 4 and 5). Apparently,the suppression of transcription of the 1.36kb ${gamma}$ pr-ßgene in the embryonic stage shown in Fig. 2 and 3 is due tocompetition by the actively transcribed upstream ${gamma}$ -globin genes.When this competition is eliminated, as in the (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ )YAC mice, the ${gamma}$ -gene promoter of the 1.36kb ${gamma}$ pr-ß geneis able to drive that gene to the expected (for the embryonicerythropoiesis) ${gamma}$ -gene transcription level.

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TABLE 4. ${varepsilon}$ -Globin gene expressions in (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC transgenic mice^a

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FIG. 5. Levels of gene expression during the development of the (1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC mice. (A) Comparison of the mean expression of the 1.36kb ${gamma}$ pr-ß gene in the ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ transgenic mice (black columns) with that of the 1.36kb ${gamma}$ pr-ß gene in the 1.36kb ${gamma}$ pr-ßYAC transgenic mice (white columns). Notice that the level of 1.36kb ${gamma}$ pr-ß gene expression in embryonic cells is restored when the ^G ${gamma}$ ^A ${gamma}$ genes are deleted (black columns). (B) Comparisons of the expression of the 1.36kb ${gamma}$ pr-ß gene of the ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ transgenic mice to the expression of the wild ${gamma}$ gene in wild ßYAC mice (white columns). Ad Bl, adult blood; YS, yolk sac; FL, fetal liver; wt, wild type; d12, day 12.

Presence of the ß gene is required for silencing of the ${gamma}$ gene during fetal erythropoiesis. Globin gene expression in the fetal stage of erythropoiesiswas evaluated using cells from day 12 fetal liver and day 14and -16 fetal liver and blood. As shown in Fig. 3B, in the day12 fetal liver the expression of the 1.36kb ${gamma}$ pr-ß genewas very close to that of the ${gamma}$ gene of wild ßYAC.In day 16 fetal liver, the levels of ${gamma}$ -gene expression in thewild ßYAC mice was 1.2% ± 0.3%, while thatof the 1.36kb ${gamma}$ pr-ß gene was 8.8% ± 1.5%. Thus,the activity of the ${gamma}$ genes of the wild YAC declined by 11-foldbetween days 12 and 16, while the activity of the 1.36kb ${gamma}$ pr-ßgene declined by only 1.9-fold. The rate of the switch of thewild ${gamma}$ genes of the 1.36kb ${gamma}$ pr-ß YAC was also affected.As shown in Tables 1 to 3 and Fig. 6, the expression of thewild ${gamma}$ gene in these mice declined by 5-fold by day 16, comparedto the 11-fold decline of expression of the ${gamma}$ gene of the wildßYAC mice.

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FIG. 6. Comparison of levels of expression of the wild ${gamma}$ genes of the 1.36kb ${gamma}$ pr-ßYAC (black columns) to the wild ${gamma}$ genes of a wild ßYAC (white columns). Ad Bl, adult blood; YS, yolk sac; FL, fetal liver; wt, wild type; d12, day 12.

When the upstream ^G ${gamma}$ and ^A ${gamma}$ genes were absent, expression of the1.36kb ${gamma}$ pr-ß gene in the day-12 fetal liver was almostthree times higher than that of the ${gamma}$ gene of the wild ßYACmice (Fig. 5B). Importantly, the expression of the 1.36kb ${gamma}$ pr-ßgene remained high throughout fetal liver erythropoiesis (Fig.5) so that between days 12 and 16, the expression of the 1.36kb ${gamma}$ pr-ß gene declined only by 0.6-fold (from 36.7% to21.2%) compared to the 11-fold decline of the wild ${gamma}$ gene.

The common denominator of the 1.36kb ${gamma}$ pr-ßYAC and the(1.36kb ${gamma}$ pr-ß)( ${Delta}$ ^G ${gamma}$ ^A ${gamma}$ ) YAC transgenes is the absence ofa ß-globin gene promoter. We interpret these datato suggest that absence of competition by the ß promoterresults in the diminished turning off of the hybrid ${gamma}$ /ßgenes as well as the wild ${gamma}$ genes of the mutant YACs. These resultsindicate that competition between ${gamma}$ and ß genes playsa role in the control of the switch from ${gamma}$ - to ß-geneexpression during fetal erythropoiesis.

DISCUSSION

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Discussion

Our results provide new evidence that the primary mechanismof control of ${gamma}$ -globin gene expression in the adult is autonomoussilencing. Sequences of the ${gamma}$ -gene promoter replaced equivalentsequences of the ß-gene promoter in the context ofa ß-locus YAC, and the effect of this manipulationon the expression of the ${gamma}$ promoter/ß gene was assessed.The ${gamma}$ -gene promoter severely suppressed ß-gene expressionin adult erythropoiesis, providing new evidence that ${gamma}$ -gene silencingin the adult is an intrinsic property of the ${gamma}$ -gene promoter.Previously, hybrid genes containing sequences of the human ${gamma}$ -genepromoter as well as sequences of the galago ${gamma}$ -gene promoter havebeen used to investigate ${gamma}$ -gene silencing (14). This approachis informative because the galago ${gamma}$ gene, in contrast to thehuman ${gamma}$ gene, behaves as a strictly embryonic gene (25), andthe embryonic restriction of the galago ${gamma}$ gene is recapitulatedin transgenic mice (27). Expression studies in transgenic micecarrying hybrid galago ${gamma}$ /human ${gamma}$ promoters with various promotermutations showed that primarily the ${gamma}$ CACCC box, but also the ${gamma}$ CCAAT box, is the main contributor to ${gamma}$ -gene silencing in adulterythropoiesis (14). A direct repeat element which is collocatedwith the ${gamma}$ -gene CCAAT box binds a transcriptional complex thatcontributes to ${gamma}$ -gene silencing (17). The role of the ${gamma}$ -gene promoterin ${gamma}$ -globin silencing is also shown by the finding that silencingis severely impaired by the substitution of the ß-spectrinpromoter for the ${gamma}$ -gene promoter in a cosmid construct (21).

The decline of ${gamma}$ - to ß-globin gene expression duringfetal development was initially attributed solely to competitionbetween the ${gamma}$ - and ß-globin genes for interaction withthe LCR (1, 8). The initial experiments were done in transgenicmice carrying either LCR ${gamma}$ or LCRß constructs or LCRconstructs in which the ${gamma}$ and ß genes were in theirnatural location (1, 8). While the ${gamma}$ and the ß geneslose developmental control when each is alone in a construct,they display the expected developmental regulation when theyare placed together in LCR ${gamma}$ ß cosmids (1, 8). Subsequentstudies showing ${gamma}$ -gene silencing in adult transgenic mice (4)challenged the contribution of gene competition on the controlof ${gamma}$ - to ß-gene globin switching, and they were interpretedto indicate that autonomous silencing is the sole mechanismof ${gamma}$ to ß switching during development. As we showhere, autonomous silencing is the principal mechanism of thecontrol of ${gamma}$ -gene expression in the adult, but competition appearsto play a role in the switch from ${gamma}$ - to ß-gene expressionduring fetal development. This is surmised from the impairedrate of ${gamma}$ -gene switching during the fetal erythropoiesis of the ${gamma}$ pr-ß hybrid genes and of the wild ${gamma}$ genes in the ßYACtransgenics which lack a ß-globin gene. The most likelyexplanation for the essential lack of silencing (Fig. 5B) orthe impaired silencing (Fig. 3B) of the ${gamma}$ pr-ß genesand the impaired silencing (Fig. 6) of the wild ${gamma}$ gene containedin the mutant YACs is the lack of competition by the ßgene. A decreased rate of decline of ${gamma}$ -gene expression in thefetal stage has also been observed before in transgenic mouselines lacking ß-globin genes (19). Collectively, thesedata indicate that gene competition as well as autonomous silencingcontributes to the control of the ${gamma}$ genes during the fetal stageof development.

Competition may also contribute to a smaller degree to ${gamma}$ -genesilencing in the adult. As shown here, the 1.36kb ${gamma}$ -gene promoterdoes not completely silence ${gamma}$ -gene expression in the adult, butthe ${gamma}$ pr-ß gene is still expressed to 1.7% of the levelof murine ${alpha}$ mRNA in the adult blood. Significantly, thalassemiamutants carrying deletions of the ß-gene promoterare characterized by elevated ${gamma}$ -gene expression in the adultheterozygotes (23), which is expected if competition also contributesto ${gamma}$ -gene silencing in the adult.

Several studies have provided unequivocal evidence for the roleof gene competition in the control of gene activity during development.The gene competition hypothesis was first proposed by Engeland colleagues to interpret the mutually exclusive expressionof the chicken adult ß^A- and embryonic ${varepsilon}$ -globin geneswhich are developmentally regulated by a shared enhancer residingbetween the two genes (3). Strong evidence in support of thehypothesis was provided by the finding that expression of thehuman ß gene is suppressed when the upstream ${varepsilon}$ and ${gamma}$ genes are active during embryonic and fetal erythropoiesis(1, 6, 8, 10). Furthermore, experiments using RNA fluorescencein vivo hybridization have shown that the human ${gamma}$ and ßgenes are alternatively expressed in the fetal liver of transgenicmice, suggesting that the LCR is able to interact with onlyone of these genes at a given time (28). A striking exampleof the role of competition in the control of gene expressionduring development is provided in our study by the expressionof the hybrid ${gamma}$ promoter/ß gene in embryonic erythropoiesis.The expression of the wild ${gamma}$ -globin gene in the wild ßYACmice is very robust in embryonic erythroid cells. Surprisingly,the expression of the ${gamma}$ promoter/ß gene was very lowin the embryonic state in spite of the fact that this gene wasdriven by the ${gamma}$ -gene promoter. The most likely explanation ofthis finding is that competition by the upstream ^G ${gamma}$ and ^A ${gamma}$ genesdecreased the probability of interaction of the hybrid ${gamma}$ promoter/ßgene with the locus control region. Indeed, deletion from theßYAC of the upstream ^G ${gamma}$ and ^A ${gamma}$ genes restored the expressionof the ${gamma}$ promoter/ß gene in the embryonic cells, providinga clear-cut example of the contribution of gene competitionin the control of gene expression during development.

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TABLE 2. ${gamma}$ -Globin gene expression in 1.36kb ${gamma}$ pr-ßYAC transgenic mice^a

ACKNOWLEDGMENTS

We thank Xin Ye and Mary Stafford for skillful technical help.

This study was supported by National Institutes of Health grantsDK61805 and HL73439 to Q.L. and DK45365 to G.S.

FOOTNOTES

* Corresponding author. Mailing address: Medical Genetics, Box 357720, University of Washington, Seattle, WA 98195. Phone: (206) 543-3526. Fax: (206) 221-5112. E-mail: gstam{at}u.washington.edu.

REFERENCES

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