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J Biol Chem, Vol. 273, Issue 28, 17361-17367, July 10, 1998


Multiple epsilon -Promoter Elements Participate in the Developmental Control of epsilon -Globin Genes in Transgenic Mice*

Qiliang LiDagger , C. Anthony Blau§, Christopher H. Clegg, Alex RohdeDagger , and George StamatoyannopoulosDagger parallel

From the Divisions of Dagger  Medical Genetics and § Hematology, University of Washington, Seattle, Washington 98195 and  Bristol-Myers Squibb Pharmaceutical Research Institutes, Seattle, Washington 98121

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

To delineate the regulation of the human epsilon -globin gene, we investigated epsilon -gene expression during the development of transgenic mice carrying constructs with epsilon -promoter truncations linked to a micro-locus control region (µLCR). Expression levels were compared with those of µLCRepsilon mice carrying a 2 kilobase epsilon -promoter and beta YAC controls. epsilon  mRNA in the embryonic cells of µLCR (-179)epsilon mice were as high as in µLCRepsilon mice suggesting that the proximal epsilon -promoter contains most elements required for epsilon -gene activation. epsilon  mRNA in adult µLCR (-179) epsilon  mice was significantly lower than in the embryonic cells indicating that elements involved in epsilon -gene silencing are contained in the proximal epsilon -promoter. Extension of the promoter sequence to -463 epsilon  decreased epsilon -gene expression in the definitive erythroid cells, supporting previous evidence that the -179 to -463epsilon region contains an epsilon -gene silencer. However, the epsilon -gene of the µLCR(-463)epsilon mice was not silenced in the definitive cells of fetal and adult erythropoiesis indicating that additional silencing elements are located upstream of position -463epsilon . These results provide in vivo evidence that multiple elements of the distal as well as the proximal promoter contribute to epsilon -gene silencing.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

All animal species have different hemoglobins in the embryonic and definitive stages of development. These hemoglobins are synthesized under the control of globin genes whose expression is restricted to either the definitive or the primitive stages of erythropoiesis. In the mouse there are two embryonic genes, epsilon  and beta h1, and two adult genes beta major and beta minor; expression of embryonic genes is totally restricted to the yolk sac stage of erythropoiesis, whereas adult gene expression starts only after the onset of definitive hematopoiesis in the liver of the 11-day-old mouse fetus. In humans, the first gene of the beta  globin locus to be expressed is the embryonic (epsilon ) followed by the two fetal (gamma ) genes and the adult delta  and beta  genes (1). epsilon -globin synthesis occurs predominately in primitive yolk sac origin erythroblasts, where it accounts for over 80% of beta -like globins at 5 weeks of gestation, falling to 15% by week 7 (2-5). In humans, the epsilon -gene is totally and permanently silenced after the 7th week of gestation. The silencing of the epsilon -gene is controlled at the transcriptional level as shown by the total absence of a DNase I hypersensitive sites in the epsilon -gene promoter of erythroid cells of 54-day-old human embryos (6).

The absolute nature of epsilon -globin gene silencing is remarkable in view of the relative proximity of the epsilon -gene to the locus control region (LCR),1 residing 6-22 kb upstream. The LCR, characterized physically by five DNase I hypersensitive sites (HS), influences chromatin structure over the entire beta  globin domain (7), acts as a powerful erythroid-specific enhancer (8, 9), and protects linked globin genes from the effects of surrounding chromatin (9, 10). Expression of the epsilon -gene in transgenic mice requires the presence of the LCR (11, 12); in mice bearing a 2.5-kb micro LCR cassette linked to the epsilon -globin gene, epsilon -globin expression is restricted to the primitive, yolk sac origin erythroblasts (11). Transgenic mice bearing a fragment containing the LCR hypersensitive sites 1 and 2 and contiguous epsilon  5'-flanking sequence fused to the coding region of the gamma  globin gene display an embryonic pattern of transgene regulation (13), suggesting that the cis elements necessary for proper developmental control of epsilon -globin gene are contained within its promoter and 5'-flanking sequence (12-14). Several negative regulatory elements have been identified upstream of the epsilon -globin gene (14-18). Transient expression assays have localized a silencer between 392 and 177 base pairs upstream of the epsilon -globin cap site (15, 16). Transgenic mice carrying a 2-kb epsilon -gene promoter from which the -177 to -392 silencer has been deleted express the epsilon -globin gene in the adult stage of development (14).

Transgenic mice provide an excellent model for delineation of the sequences necessary for epsilon -gene silencing, because of the absolute restriction of epsilon -globin gene expression in the yolk sac cells of the mouse. If such sequences exist, their deletion or mutation is expected to be associated with loss of epsilon -gene silencing resulting in continuation of epsilon -gene expression in the adult stage of development. In the experiments described in this report developmental studies were performed using transgenic mice containing an LCR cassette linked to an epsilon -globin gene containing only 179 or 463 bp of promoter sequence. We found that although a significant level of developmental control is retained in the proximal promoter, the bulk of epsilon -gene silencing resides with sequences located upstream of -179 epsilon . These sequences include but are not limited to the -177 to -392 epsilon -silencer. These results provide in vivo evidence that elements located both in the proximal as well as in the distal epsilon -gene promoter are involved in epsilon -gene silencing.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

DNA Constructs (Fig. 1)-- pµLCR(-463)epsilon was produced from a modified version of pµLCRepsilon , which contains 2 kb of sequence upstream of the epsilon -globin gene transcription start site. The µLCR cassette of pµLCRepsilon was modified to provide an additional 0.6 kb of sequence 5' to HS4 of the original µLCR, producing 3.1-kb pµLCRepsilon (thereafter, the term µLCR used in the text represents this new LCR cassette except where otherwise indicated). pµLCRepsilon was linearized using SmaI, partially digested using BfrI, and the digestion products were blunted using Klenow enzyme. An 8.3-kb product was gel purified and religated to generate pµLCR(-463)epsilon . To generate pµLCR(-179)epsilon the construct pµLCRepsilon was linearized using SmaI, partially digested using BamHI, and the digestion products were blunted using Klenow enzyme. A 7.9-kb product was gel purified and religated to produce pµLCR(-179)epsilon .

All constructs were freed from vector sequences using restriction enzymes, gel purified, and resuspended in filtered Tris-EDTA before injection into fertilized oocytes.

Transgenic Mice-- Transgenic mice carrying the pµLCR(-179)epsilon and pµLCR(-463)epsilon constructs were produced as described previously (19). Founder animals were identified by Southern blotting with an epsilon -gene sequence probe. F1 progeny were obtained by breeding founder animals with nontransgenic mice and were screened for correct integration and to exclude the presence of mosaicism in the founders. To study the developmental pattern of human epsilon -gene expression, staged pregnancies were interrupted on days 9, 12, and 16 of development. Samples from blood and yolk sac were collected on day 9 embryos; blood, yolk sac and liver were collected on day 12 fetuses; and blood and liver were collected on day 16 fetuses.

epsilon mRNA Quantitation-- Total RNA was isolated from transgenic tissues by the method of Chomczynski and Sacchi (20). The epsilon  mRNA level was measured by the quantitative RNase protection assay described previously (21). Briefly, riboprobe for epsilon  mRNA was labeled by transcribing the linearized plasmid pT7epsilon (188) using T7 RNA polymerase (22). The epsilon  probe protects a 188-bp fragment in exon 2 of epsilon  mRNA. The mouse alpha  and zeta  riboprobes were used in RNase protection assays as internal globin mRNA controls. mRNA levels were determined in all transgenic siblings of each litter. RNA samples from different tissues were analyzed at least twice to reduce experimental error in mRNA quantitations. Human epsilon  and mouse alpha  and zeta  signals were quantitated with a PhosphorImager. Levels of human mRNA per transgene copy were expressed as percentages of mouse alpha -like mRNA levels per copy, taking into account that the mouse possesses four copies of the alpha -globin gene and two copies of the zeta -globin gene. In the adult stages of development when zeta  mRNA is absent, murine alpha  mRNA per copy was calculated by dividing the levels of murine alpha  mRNA by four.

Copy Number Determination-- Copy number determination was accomplished by the multiply redundant protocol described previously (21) to reduce experimental errors. Multiple DNA samples were obtained from each of at least three animals from each transgenic line. These samples were digested with restriction enzyme EcoRI and resolved by electrophoresis over 1% agarose gel. Southern blots were hybridized with a radiation-labeled epsilon  probe by using a 0.6-kb BamHI fragment of the epsilon -globin gene as template. The signals were quantitated on a PhosphorImager. Copy numbers were calculated by determining the relative intensity of signals from a given transgenic line compared with the signals obtained from diploid human genomic DNA.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Constructs (Fig. 1)-- The construct µLCR(-179)epsilon consists of a 1.9-kb epsilon -gene fragment that contains the epsilon -globin gene, 179 bp of sequences of the epsilon -gene promoter and 280 bp of 3'-nontranslated sequences. The -463 epsilon  construct contains epsilon  genomic sequences identical to those of the -179 epsilon  construct, but the promoter is extended to a BfrI site at position -463. This construct therefore contains the -177 to -392 sequence previously shown in transient assays (15) and transgenic mouse studies (14) to behave like an epsilon -gene silencer.


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  		Fig. 1.   epsilon -gene constructs used for production of transgenic mice. A, schematic representation of the human beta -globin locus. Numbers correspond to GenBankTM coordinates (Humhbb). The thick vertical arrows indicate the DNase I hypersensitive sites of the LCR. Filled boxes show the five transcribed globin genes, whereas the open box marks the position of the pseudo beta  gene. B, the left line shows the µLCR, a 3.1-kb truncated version of the LCR. The numbers above the line indicate the 5' and 3' ends of each HS fragment. The right line is a 3.7-kb EcoRI fragment encompassing the epsilon -globin gene spanning from -2025 to +1745 relative to the cap site. C, epsilon -globin gene promoter. Shown are the location of the conserved boxes and the binding motifs for various proteins. + and - correspond to the sites of positive and negative elements identified by transient transfection assays (43). The truncated positions of the two µLCRepsilon constructs used in this study are indicated by arrows (BamHI and BrfI).

The -179epsilon - or -463epsilon -fragments were linked to a 3.1-kb µLCR, which consists of 0.71 kb of HS1, 0.73 kb of HS2, 0.56 kb of HS3, and 1.1 kb of HS4. This 3.1 µLCR contains the core element of DNase I hypersensitive sites 1, 2, and 3, which are also present in the previously used 2.5 kb µLCR (23). The 3.1 µLCR also contains 600 additional bp of HS4, which includes the core element of HS4; this core of HS4 is missing from the 2.5 µLCR (23).

Control mice were of two kind. First, 3.1 µLCRepsilon and 2.5 µLCRepsilon mice in which the epsilon -gene promoter is extending 2-kb upstream of the cap site, i.e. in the EcoRI site at position -2040 epsilon . Second, three beta YAC lines that were produced using a 248-kb beta -locus YAC. These lines have been analyzed in detail for structural integrity using previously published protocols (24) and found to contain an intact beta -globin locus, from 5' HS4 to 3' HS1.

Analysis of epsilon -Globin Gene Expression in Cells of Embryonic and Definitive Murine Erythropoiesis-- For developmental studies, timed pregnancies were interrupted at 9, 12, and 16 days, and yolk sac, blood, and fetal liver samples were collected for measurement of human epsilon  and murine alpha and zeta  mRNA by RNase protection (Fig. 2). More than one tissue was analyzed in each gestational day to increase the accuracy of epsilon  mRNA measurements. Multiple members from each litter were used for measurements of globin mRNA. Data from each line and each day are presented in Tables I and II as means ± S.D.


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  		Fig. 2.   Representative RNase protection assay for transgenic mice carrying the human globin constructs. Protected fragment sizes are as follows: human epsilon -globin (Huepsilon ), 188 nucleotides (nt); mouse zeta -globin (Mozeta ), 151 nucleotides; mouse alpha -globin (Moalpha ), 128 nucleotides. ys = day 9 yolk sac; f/b = day 12 fetal blood; f/l = day 16 fetal liver; a/b = adult blood. Panel A, µLCRepsilon (-179) (lines A-F); panel B, µLCRepsilon (-463) (lines G-I); panel C, beta YAC (lines J-L), and µLCRepsilon (lines M and N)

                              
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Table I
Human epsilon  mRNA levels in the embryonic erythropoiesis of transgenic mice with truncated epsilon -gene promoter and beta YAC or µLCRepsilon controls

                              
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Table II
Human epsilon  mRNA levels in definitive erythropoiesis of transgenic mice with truncated epsilon -gene promoter and beta YAC or µLCRepsilon controls

The day 9 blood and yolk sac represent an early stage of embryonic erythropoiesis. We have previously observed that in µLCRepsilon or beta YAC transgenic mice the levels of human epsilon  mRNA peak at day 12 (22, 24-27). The day 12 yolk sac still contains large numbers of nucleated embryonic red cells, and the day 12 fetal blood is composed predominantly from nucleated erythrocytes of yolk sac origin. Therefore, blood and yolk sac samples from day 12 fetuses were used to assess epsilon -globin expression in day 12 embryonic erythropoiesis.

epsilon -gene expression in cells of definitive erythropoiesis was studied using day 12 fetal liver, day 16 fetal liver, day 16 fetal blood, and adult blood. The day 12 fetal liver is an organ of adult erythropoiesis, and it mostly consists of definitive erythroblasts that can be distinguished from the embryonic erythroblasts by their smaller size and small cytoplasmic nuclear ratio. There is no expression of murine embryonic epsilon y and beta h1 genes or human epsilon -transgenes in fetal liver erythropoiesis (11, 14, 28). Yolk sac origin erythroblasts that contaminate the fetal liver preparations (11) account for the small levels of epsilon  mRNA detected in fetal liver specimens from µLCRepsilon or beta YAC transgenic mice.

epsilon -Gene Expression in the Embryonic Cells of µLCR(-179)epsilon Transgenic Mice and Controls-- The µLCR(-179)epsilon construct contains all the transcriptional motifs of the proximal epsilon -gene promoter, i.e. the TATA box at -30, the CAAT box at -84, the CACC box at -113, and a GATA-1 site in position -165 shown before to be required for up-regulation of epsilon -gene expression (29). Although the proximal promoters of the beta -like globin genes share a basic organization, several differences also exist between promoters. Differences between the epsilon -and gamma gene promoters include the presence of a duplicated CAAT box in the gamma -promoter and divergence of sequences surrounding the TATA, CAAT, and CACC boxes. Presumably these structural differences contribute to the difference in the developmental regulation of epsilon -and gamma  genes in humans.

Using the µLCR(-179)epsilon recombinant we produced 8 transgenic lines, all of which expressed the epsilon -globin transgene. Two lines had more than 30 copies of the integrated transgene and were excluded from further analysis because we have found that such high transgene copies are frequently associated with position effects resulting in loss of the correlation between copy number of the integrated transgenes and level of globin gene expression. The 6 lines were used for developmental studies in F2 progeny. The percentage of epsilon -globin mRNA relative to mouse alpha  and zeta  mRNA was measured by quantitative RNase protections and corrected for the number of copies of the transgene.

Expression of the epsilon -globin gene ranged from 11.8 to 23.3% of murine alpha  (mean 16.2 ± 3.1) in the 9-day embryonic cells, and it was slightly higher (range 11.8-33.3%; mean 19.5 ± 6.6%) in the 12-day embryonic cells (Table I).

In Fig. 3 we compare levels of epsilon  mRNA in day 9 yolk sac and blood and day 12 yolk sac and blood of µLCR(-179)epsilon mice to those of µLCRepsilon and beta YAC controls. There is no significant difference in epsilon  mRNA levels between the µLCR(-179)epsilon and the µLCRepsilon lines. epsilon  mRNA in the µLCR(-179)epsilon and the µLCRepsilon embryos was significantly higher than in the beta YAC embryos. The lower levels of epsilon  mRNA in the beta YAC transgenic mice most likely represent the decreased chance of interaction between the epsilon -gene and the LCR when a gamma  globin gene is also present in the construct. gamma  mRNA in embryonic cells of beta YAC mice is considerably higher than epsilon  mRNA (24-27), suggesting that the murine embryonic trans acting environment favors the transcription of human gamma  gene.


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  		Fig. 3.   epsilon mRNA levels in cells of day 9 and day 12 embryonic (yolk sac) erythropoiesis. Constructs are indicated at the top of each column. At the bottom of each column the means ± S.D. of the measurements are shown. square , epsilon -expression in yolk sac; open circle , epsilon -expression in blood.

epsilon -Gene Expression in the Embryonic Erythropoiesis of -463 epsilon -Transgenic Mice-- The µLCR(-463)epsilon construct contains the sequence -177 to -392epsilon previously shown to harbor an epsilon -gene silencer (14, 15). This silencer contains sites that bind erythroid-specific as well as constitutive transcriptional factors (16-18, 28, 30), a GATA-1 binding site at -208, two GATA-1 binding sites that overlap with a YY1 site at -269; and a CCACC site at -379 that binds Sp-1 or a related factor.

Seven founder lines were produced but only three transmitted the transgene and were used for developmental studies (Fig. 2, panel B and Table I). Levels of epsilon  mRNA (corrected per copy of transgene) in the 9-day embryonic cells ranged from 7.7 to 12.4% (mean 9.7 ± 2.24%) and in the 12-day embryonic cells from 6.0 to 16.6% (mean 12.8 ± 3.6%). Therefore, epsilon -gene expression in the embryonic tissues of the µLCR(-463)epsilon -transgenic mice was considerably lower compared with the µLCR(-179)epsilon transgenic mice at both the day 9 and day 12 developmental stages (Fig. 3). The lower epsilon -gene expression in the µLCR(-463)epsilon mice cannot be attributed to the increased distance of the LCR from the proximal promoter because, as shown in Fig. 3, epsilon -gene expression in the µLCRepsilon mice (which contain a promoter extending 2 kb from the cap site) is higher than epsilon  in µLCR(-463)epsilon mice. The lower levels of epsilon  mRNA in the µLCR(-463)epsilon mice may reveal the presence in the -179epsilon to -463epsilon sequence of negative elements that can be detected only when other upstream sequences are deleted.

epsilon mRNA Levels in Cells of Definitive Erythropoiesis of the µLCRepsilon and beta YAC Mice-- As shown in Table II, the 12-day fetal liver samples of the beta YAC mice contain from 2 to 3.7% epsilon  mRNA deriving from contaminating embryonic erythroblasts. There is no epsilon  mRNA in the 16-day blood and liver samples or in adult blood of the beta YAC transgenic mice. In the 3.1 µLCRepsilon and 2.5 µLCRepsilon mice, traces of epsilon  mRNA are detected in the 16-day liver (0.11%) and adult blood (0.18-0.67%). Therefore, compared with the beta -locus YACs, the two µLCRepsilon constructs are "leaky" and allow synthesis of residual levels of epsilon  mRNA in the cells of definitive erythropoiesis.

epsilon mRNA Levels in Definitive Erythroid Cells of -179epsilon Mice-- Fig. 4 shows that epsilon -gene expression in the 12-day fetal liver of the six µLCR(-179)epsilon lines is significantly higher than in the 2.5 µLCRepsilon and 3.1 µLCRepsilon controls. The difference is even more striking in the 16-day fetal liver in which the levels of epsilon  mRNA in µLCR(-179)epsilon mice are 30-50-fold higher than in the 3.1 µLCRepsilon control (Table II). The µLCR(-179)epsilon construct, however, has not totally lost the ability to down-regulate epsilon -gene expression, because, as shown in Fig. 5A, there is a significant decline in epsilon  mRNA levels in the adult µLCR(-179)epsilon -transgenic mice. This is in contrast to a µLCRAgamma gene containing only the proximal gamma -promoter (µLCR(-201)Agamma ), which is not down-regulated in the adult and it directs similar levels of gamma  mRNA in the adult and in the embryonic cells of transgenic mice (19).


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  		Fig. 4.   epsilon -gene expression in cells of day 12 and day 16 fetal liver definitive erythropoiesis. Constructs are indicated at the top of each column, and the means ± S.D. of the measurements are shown at the bottom of each column . open circle , epsilon -expression in fetal liver; square , epsilon -expression in blood at the same days.


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  		Fig. 5.   Comparison of epsilon  mRNA levels in embryonic cells (a) and adult cells (b) of transgenic mice carrying the µLCR(-179)epsilon construct (A), the µLCR(-463)epsilon construct (B), and µLCRepsilon (black-square) or beta YAC (square ) constructs (C). The highest level of epsilon  mRNA in the embryonic cells of each line was used for the data plotted in a.

epsilon mRNA Level in Definitive Cells of the µLCR(-463)epsilon Mice-- Despite the presence of the -177 to -392 silencer in the µLCR(-463)epsilon construct, the 12-day definitive erythroid cells of these mice had epsilon  mRNA levels that were about 2-fold higher than the levels of the 3.1 µLCRepsilon or beta YAC controls (Table II and Fig. 4). Levels of epsilon  mRNA in the 16-day fetal tissues of µLCR(-463)epsilon mice are 20-40-fold higher than in the µLCRepsilon control (Fig. 4). Therefore the definitive erythroblasts of the µLCR(-463)epsilon mice synthesize significant amounts of epsilon  mRNA. The levels of epsilon  mRNA in adult µLCR (-463)epsilon mice, are significantly lower than in adult µLCR(-179)epsilon mice (Fig. 5) most likely reflecting the presence of the -179 to -392 epsilon -silencer. epsilon -Expression in the adult µLCR(-463)epsilon mice is 10-20-fold higher than in the adult 3.1 µLCRepsilon control (Table II), most likely reflecting the presence of silencing elements upstream of -463epsilon .

Expression of µLCR (-179)epsilon and µLCR (-463)epsilon Transgenes Is Independent of Position of Integration-- The results in Tables I and II show consistency of expression of the epsilon -gene in the mouse lines carrying the µLCR(-179)epsilon or the µLCR(-463)epsilon constructs. The mean epsilon -gene expression in the µLCR(-179)epsilon mice is 18.9 ± 5.5 in the day 12 embryonic blood and 5.3 ± 2.2 in adult blood. The coefficients of variation in per copy expression between the µLCR (-179) epsilon  lines are 0.29 for the fetal day-12 embryos and 0.41 for the adult stage, respectively. Small coefficients of variation (less than 0.5) indicate that the expression of a transgene is independent of the position of integration (21). Since epsilon -gene expression is not influenced by the position of integration of the transgenes, the developmental profiles shown in Tables I and II must reflect an inherent property of the µLCR(-179)epsilon construct. It is noteworthy that copy number-dependent expression was also previously observed in the µLCR(-201) Agamma transgenic mice containing only the proximal gamma  gene promoter (19). Copy number dependence of epsilon  expression is also characteristic of the construct µLCR(-463)epsilon . Thus, the coefficient of variation is 0.38 in the day-12 embryonic blood and 0.39 in adult blood of the µLCR(-463)epsilon -transgenic mice, indicating that the expression of this transgene is also independent of position of integration.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Several studies have shown that sequences in the epsilon -gene promoter as well as in the LCR participate in the developmental regulation of the epsilon -globin gene. Experiments in transgenic mice have shown that, in the absence of the LCR, the human epsilon -genes remain silent in the embryonic cells of transgenic mice, indicating that the LCR is necessary for in vivo epsilon -globin gene transcription (11, 12). The contribution of LCR sequences in the developmental control of epsilon -gene has been clearly shown in studies of beta YAC transgenic mice containing deletions of DNase I hypersensitive site 3; mice carrying a 2.5-kb deletion removing HS3 and the surrounding flanking sequences display decreased epsilon -globin expression in the embryonic erythropoiesis (26), whereas mice carrying deletions of the core element of HS3 display total absence of epsilon -expression in day-9 embryonic cells and severe reduction of epsilon -expression in day-12 embryonic cells (31). Such data suggest that sequences of the core element of HS3 are necessary for activation of epsilon -gene transcription. The contribution of elements of the epsilon -gene promoter have been investigated in vitro, with transient expression assays and in vivo, in transgenic mice. Studies in transiently transfected cell lines have revealed sequences that either positively (17, 29, 30, 32-34) or negatively (15-17, 35) influence transcription from a linked reporter gene. Several of these sequences have been evolutionarily conserved (36). Studies in transgenic mice have shown that all the cis elements required for epsilon -gene silencing are located in a 2.0-kb fragment that contains the epsilon -gene promoter (11, 28).

In this study we wished to examine to what degree the proximal and distal epsilon -gene promoter are involved in the developmental regulation of the epsilon -globin gene in vivo. We used control constructs containing the whole beta -locus or a 2-kb epsilon -promoter and constructs with an epsilon -promoter containing only the essential transcriptional motifs or these essential motifs as well as a previously identified upstream silencer. We found high levels of epsilon  mRNA in the adult cells of transgenic mice carrying only the proximal epsilon -promoter indicating that structural elements located in the distal epsilon -gene promoter are critical for epsilon -gene silencing. The level of epsilon  mRNA in the adult cells decreased when sequences containing the previously described silencer were added, providing further evidence for the in vivo function of this element. However, presence of this silencer did not totally suppress epsilon -gene expression in definitive cells suggesting that additional elements located in the distal epsilon -promoter are involved in epsilon -gene silencing. Although mice carrying only the proximal epsilon -promoter have high levels of epsilon -gene expression in the adult stage of development, the level of epsilon  mRNA in the adult cells is strikingly lower than in embryonic cells, suggesting that sequences located in the proximal epsilon -promoter participate in epsilon -gene silencing. Overall, our results provide in vivo evidence that multiple elements located in the distal as well as in the proximal epsilon -gene promoter are involved in epsilon -gene silencing.

Liu et al. (37) have recently reported that a sequence located between position -179 to -304 of the epsilon -gene promoter contains a positive regulatory element, the deletion of which in the context of the beta -locus YAC results in catastrophic reduction of epsilon -globin (as well as on gamma -globin) gene expression. This finding contrasts to the results of the present study, which shows that the levels of epsilon  mRNA in the embryonic cells of µLCR(-179)epsilon -transgenic mice are as high as in transgenic mice carrying an epsilon  promoter extending 2-kb upstream of the epsilon -gene cap site. Such findings imply that most of the cis elements necessary to interact with the LCR are located in the proximal epsilon -gene promoter. The results of Liu et al. also contrast to the results of a previous study in which a -179 to -463 epsilon  sequence was deleted from a 2-kb epsilon -promoter. Transgenic mice carrying this construct have normal epsilon  mRNA levels in embryonic erythroblasts (14), although they should have lacked epsilon  expression if a positive element with the characteristics described by Liu et al. (37) was located in the deleted sequence.

There are several explanations for these discrepant findings. First, it is known that YACs have a considerable tendency for rearrangements and YAC transgenic lines may carry several YAC integrants, each showing different structural rearrangements (24). The finding of Liu et al. (37) could be explained if such rearranged YACs were contained in the transgenic lines they studied, a possibility that has not been totally excluded with the structural analyses done. Second, as previous studies have shown, globin genes behave differently when they are individually linked to the LCR in short constructs or when they are present in constructs containing the whole beta -locus. Thus, when the beta  genes are linked to a 2.5-kb µLCR (38) or a 20-kb LCR (39), they are expressed in embryonic as well as adult cells, whereas they are totally silenced in embryonic cells when they are located in gamma delta beta cosmids (38, 39) or in beta YACs (27, 40). Similar behavior has been documented with gamma -globin genes (19, 38, 39). Such results do not invalidate the use of short constructs but they point to the different insights obtained when short globin gene-LCR constructs or constructs containing the whole beta -locus are used in transgenic mouse experiments. The short constructs are useful for delineating the functional role of specific sequences flanking the globin genes or elements contained in the HSs of the LCR. Such constructs have allowed the delineation of specific cis elements of the beta - (41) or gamma - (19) gene promoters or the HSs of the LCR (41-45). Since, in the intact beta -locus, the competition between globin gene promoters to interact with the LCR becomes the dominant determinant of gene expression, "whole locus" constructs are most useful for addressing questions on the control of globin gene switching. It is likely that certain regulatory elements have different functions in the different stages of development, and these functions could be identified by the use of the two types of constructs. The discrepancies between our results and those of Liu et al. (37) perhaps reveal that the -179 to -392 sequence has a dual function. In the presence of the transcriptional environment of definitive erythropoiesis this element may act as an epsilon -gene silencer, and this function was depicted when the LCR epsilon -gene constructs were used in transgenic mice. In the presence of an embryonic transcriptional environment and an intact beta -locus, this sequence may behave as an "anchor" that facilitates the interaction of the LCR with the epsilon -and gamma  genes of embryonic cells; this function was perhaps depicted when in the studies of Liu et al. (37) a beta -locus YAC with a deleted -179 to -392 epsilon  sequence was used.

    ACKNOWLEDGEMENTS

We thank Harold Haugen, Sara Shaw, and Heimei Han for expert technical help and Sherri Brenner for assistance in the preparation of the manuscript.

    FOOTNOTES

* This work was supported by Grants DK45365, HL20899, and HL53750 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Div. of Medical Genetics, Box 357720, University of Washington, Seattle, WA 98195. Tel.: 206-543-3526; Fax: 206-543-3050; E-mail: gstam{at}u.washington.edu.

1 The abbreviations used are: LCR, locus control region; kb, kilobase(s); HS, hypersensitive sites; bp, base pair(s).

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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