©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequences Located 3` to the Breakpoint of the Hereditary Persistence of Fetal Hemoglobin-3 Deletion Exhibit Enhancer Activity and Can Modify the Developmental Expression of the Human Fetal A-Globin Gene in Transgenic Mice (*)

Nicholas P. Anagnou (1)(§), Carlos Perez-Stable (2), Richard Gelinas (3), Frank Costantini (2), Katerina Liapaki (1), Mary Constantopoulou (1), Theodore Kosteas (1), Nicholas K. Moschonas (1), , and George Stamatoyannopoulos (3)

From the (1) Institute of Molecular Biology and Biotechnology, and University of Crete, Schools of Medicine and Biology, 711 10 Heraklion, Greece, the (2) Department of Human Genetics and Development, Columbia University, New York, New York 10032, and the (3) Department of Medicine, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Expression of fetal -globin genes in individuals with the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has been attributed either to enhancement by 3` regulatory elements juxtaposed to -globin genes or to deletion of -gene silencers normally residing within the -globin gene cluster. In the present study, we tested the hypothesis of imported enhancers downstream of -globin gene using the HPFH-3 deletion as a model. The abnormal bridging fragment of 13.6 kilobases (kb) containing the A-gene with its flanking sequences and 6.2 kb of the juxtaposed region was microinjected into fertilized mouse eggs. Twelve transgenic mice positive for the fragment were generated. Samples from 11.5-day yolk sacs, 16-day fetal liver, and adult blood were analyzed for A-mRNA using RNase protection assays. Three mice lacked A expression in the yolk sac indicating non-optimal integration site. Four expressed A-mRNA at the embryonic stage only, while two expressed A-mRNA in both embryonic and fetal liver erythroid cells. Since the A-gene with its normal flanking sequences and in the absence of the locus control region is expressed only in embryonic cells of transgenic mice, these data suggest that the juxtaposed sequences have altered the developmental specificity of the fetal -globin gene. These sequences were further tested for the presence of an enhancer element, by their ability to activate a fusion reporter gene consisting of the CAT gene linked to the -globin gene promoter, in erythroid (K562) and non-erythroid (HeLa) cells. A 0.7-kb region located immediately 3` to the breakpoint, enhanced chloramphenicol acetyltransferase activity by 3-fold in erythroid cells. The enhancer also activated the embryonic -globin gene promoter by 2-fold but not the adult - or -globin gene promoters. The enhancer represents a region of previously known complex tandem repeats; in this study we have completed the sequencing of the region encompassing the 0.7-kb enhancer element. Multiple areas of the enhancer region exhibit homology to the core element of the simian virus 40 enhancer and to the sequences of the human 3` A- and the chicken 3` -globin enhancers. A consensus binding site for the erythroid specific GATA-1 transcription factor and seven consensus sites for the ubiquitous CP1 transcription factor are also included within the enhancer. These data suggest that these sequences located immediately 3` to the breakpoint of the HPFH-3 deletion, exhibit both the structure and the function of an enhancer, and can modify the developmental specificity of the fetal -globin genes, resulting in their continued expression during adult life.


INTRODUCTION

In humans and in a few other species, two ontogenetic switches in globin gene expression occur within the -globin gene cluster: one from embryonic to fetal () early in development and a second one from fetal to adult () during the perinatal period. Experimental data so far suggest that these switches reflect temporal and tissue-specific interactions between cis and trans-acting regulatory elements of the -globin gene cluster.

A unique feature of hemoglobin switching in humans is the availability of many deletion mutants associated with continued fetal -gene expression in the adult life. The deletions in these mutants occur within the -globin gene cluster and lead to clinical syndromes characterized by increased production of fetal hemoglobin (HbF)() in the adult life. The two syndromes, i.e. -thalassemia and hereditary persistence of fetal hemoglobin (HPFH) exhibit discrete phenotypes regarding the levels of HbF and its distribution in the peripheral blood erythrocytes: HbF levels of 10-15% and heterocellular distribution in -thalassemia versus 20-30% HbF levels and pancellular distribution in HPFH (1, 2) .

The HPFH syndromes resulting from large deletions of the human -globin gene cluster provide a model to study the mechanisms of fetal -gene activation. So far, seven large deletions have been described that are all associated with increased and continued expression of fetal hemoglobin in adult life (1, 2, 3, 4) . Three major hypotheses have been proposed to explain this failure of normal switching; one suggests that the increased -gene expression reflects enhancement by elements imported into the -globin gene region (5) , the other considers that silencer sequences in the A to region are removed by these deletions (6) , while the third hypothesis considers that the increased and continued expression of the fetal -genes in adult life, reflects position effects on the fetal -genes by the juxtaposed downstream chromatin structure (7) . Recently, an enhancer element immediately 3` to the deletion breakpoint of HPFH-1 deletion has been detected, which may play a role in the continued expression of -genes in HPFH-1 and HPFH-2 mutations (8) .

In the present study we decided to test the validity of the first hypothesis using as a model the HPFH-3 deletion, whose 3` breakpoint is located 30 kb downstream of the -gene, removing 48.5 kb of DNA (9, 10) . Using bioassays such as transgenic mice and transient assays in erythroid and non-erythroid cells, we characterized an enhancer element 3` to breakpoint of HPFH-3 that can modify the developmental specificity of A-gene, resulting in persistence of fetal -globin gene expression in adult life.


EXPERIMENTAL PROCEDURES

DNA Construct Microinjections

The abnormal 13.6-kb bridging fragment containing the normal fetal A-globin gene, its flanking sequences, and 6.2 kb of juxtaposed sequences was reconstituted as follows: the cloned (10) 11.5-kb XbaI fragment (kindly provided by Dr. O. Smithies) was ligated with the 2.0-kb HindIII- XbaI fragment containing the 5` end of the A-globin gene (Fig. 1). Prior to ligation, the HindIII site had been destroyed and replaced by a NotI site, to facilitate cleavage of the 13.6-kb reconstituted insert as an intact NotI- SmaI fragment. The utilized SmaI site resides 11 bp from the 3` XbaI site in the pUC18 polylinker. This strategy also avoids the presence of plasmid sequences in the construct that are deleterious in the transgenic mouse bioassay.


Figure 1: Stages of the reconstitution of the abnormal 13.6-kb bridging fragment. This fragment contains the normal fetal A-globin gene and its flanking sequences and about 6.2 kb of juxtaposed sequences (shown as hatched bar). A, normal arrangement of the region of the human -globin gene locus containing the fetal A-gene and the -pseudogene. B, the 5` end breakpont of the HPFH-3 deletion occurs within the Alu repeat (shown as a horizontal arrow) just 5` of the -pseudogene and juxtaposes sequences (shown as a hatched bar) normally residing 30 kb from the 3` end of the -globin gene. C, ligation of the cloned 11.5-kb XbaI fragment with the 5` end of the A-globin gene. D, reconstitution of the abnormal 13.6-kb fragment used for microinjection into mouse fertilized eggs N, NotI; X, XbaI; H, HindIII. The normal HindIII site 5` of the A-globin gene has been destroyed and replaced by a NotI site.



Transgenic Embryos and Fetuses

The 13.6-kb DNA fragment following digestion with NotI- SmaI was excised from the pUC18 vector and was purified following cesium chloride gradient ultracentrifugation (11) . Following dialysis, the DNA was suspended in fresh TE buffer (10 mM Tris, 1 mM EDTA), extracted with phenol, phenol-chloroform, and chloroform, and then subjected to ethanol precipitation. DNA concentration was accurately measured using a Hoeffer TKO 100 DNA fluorometer. DNA fragments of the reconstituted insert (3-6 ng/µl) were microinjected into the pronuclei of (C57BL/6J CBA/J)F2 mouse zygotes as described previously (12, 13) . Transgenic embryos and fetuses were identified by Southern blot analysis.

RNA Analysis from Embryonic, Fetal, and Adult Tissues

Total cellular RNA was isolated from 11.5-day embryonic yolk sacs, 16-day fetal livers, and adult blood of the various transgenic lines, as described previously (12, 13, 14) . The levels of A- mRNA were measured with a P-labeled SP6 polymerase-synthesized antisense RNA probe (560 nt) specific for the 3` end of A-globin mRNA. The probe was hybridized and digested as described (11, 13) resulting in a 165-nt A-gene-specific protected fragment.

The levels of human globin mRNAs in the erythroid tissues of transgenic mice were estimated by comparison to the hybridization signals obtained with various amounts of hemin-induced human K562 cell RNA. The results are expressed as picograms of A-globin mRNA per microgram of total mRNA. In these cells, 16 pg of -mRNA/µg of total mRNA are detected (11, 15) , while the G/A ratio is >10; (see Fig. 2, K562 line). Only the 165-nt fully protected fragment was considered for the quantitation. Comparison of the A-mRNA levels to the endogenous mouse embryonic globin mRNA was performed with the same human probe, since cross-hybridization with endogenous embryonic mRNA results in a protected fragment of low molecular size.


Figure 2: Expression of the HPFH-3 transgene in embryonic blood, fetal liver, and adult blood measured by RNase protection analysis. A, an RNA probe specific for the 3` end of A-globin mRNA (560 nt) was hybridized and digested as described (11, 13) resulting in a 165-nt protected fragment ( pf). Lanes K562 1-6 contain 0, 0.03, 0.15, 0.75, 3, and 7.5 µg of total mRNA from hemin-induced K562 cells, respectively. The smaller <85-nt protected fragment corresponds to cross-hybridization with G-specific mRNA. The remaining lanes show the analysis of 15 µg each (except in 1-7, 1.5 µg) of 11.5-day mouse embryonic ( E) blood mRNA, 50 µg of 16-day fetal ( F) liver mRNA, and 20 µg of adult ( A) blood mRNA from the six HPFH-3 transgenic lines 1-1, 1-4, 1-5, 1-7, 2-2, and 3-5. Cross-hybridization with endogenous mouse embryonic globin mRNA results in low molecular size fragments seen at the bottom of the gel for the E lanes. Lane M contains P-labeled size markers (pBR322 digested with HpaII), some of whose sizes are indicated on the right. B, expression of the HPFH-3 transgene in 16-day fetal liver. Lanes K562 1-4 contain 0, 0.03, 0.15, and 0.375 µg of mRNA, respectively. For comparison, 15 µg of embryonic ( E) blood mRNA from lines 1-4 and 1-5 and 100 µg of fetal ( F) liver mRNA from lines 1-4, 1-5, and 3-5, respectively, were analyzed. Lanes 1, 2, 15, 42, 47, and 51 represent a total of 36, 66, 35, 32, 33 and 46 µg of total fetal liver mRNA from the corresponding transient HPFH-3 transgenes 1, 2, 15, 42, 47, and 51, respectively. Molecular size markers are as described in panel A.



-CAT-HPFH-3 Plasmid Constructions

Ten different contiguous (A, B, C, D, and F) or overlapping (F1, G, H, J, and I) fragments encompassing the 6.2 kb region of the 3` juxtaposed sequences were generated following digestion with appropriate restriction enzymes, as shown in Fig. 3. The F and F1 fragments differ in the sense that the latter contains purely sequences residing immediately 3` to the breakpoint of the deletion, while F contains 66 bp of the Alu repeats residing immediately upstream of the breakpoint (see Figs. 1 and 3). The isolated fragments were subcloned upon modification of their ends in both orientations either into the BamHI site (fragments A and B), SmaI site (fragments C, D, F1, H, J, and I), or the AvaI- BamHI sites (fragments F and G) of the polylinker region of the pUC18 and pUC19 plasmids. The resulting recombinant plasmids were then subjected to SalI digestion, and the 2.1-kb SalI fragment derived from plasmid -CAT (kindly provided by Dr. D. Bodine) was subcloned into the SalI site in both orientations in all the plasmids (Fig. 3). This fragment (16) contains the human G-globin promoter gene (384 to +36 relative to the cap site) fused with the CAT gene. By employing the above strategy, the fragments were subcloned into both orientations (5`3` and 3`5`) and in both positions (upstream and downstream) relative to -CAT gene ( i.e. pCATX1, pCATX2, pCATX3 and pCATX4) as shown in Fig. 3. For the analysis of the effects of the enhancer element on the heterologous globin and non-globin promoters, the individual promoter elements (17) from CAT, CAT, and CAT plasmids (kindly provided by Dr. A. Schechter) and from the DHFR-CAT plasmid (16) (kindly provided by Dr. D. Bodine) were utilized. The F fragment, containing the enhancer element was subcloned in both orientations, 3` to the promoter-CAT fusion genes, i.e. into the BamHI (CAT and CAT), HpaI (CAT), and AvaI (DHFR-CAT) sites, respectively.


Figure 3: Experimental strategy followed for the detection of enhancer-like elements within the 3` juxtaposed sequences of the HPFH-3 deletion mutation. A, the 10 different contiguous ( A-D and F) or overlapping ( F1, G, H, J, and I) fragments encompassing the 3` juxtaposed sequences (shown as a hatched bar) that were used to detect enhancer activity. The numbers above each bar indicate the length of each fragment in kb. B, the fragments (shown as black boxes) were subcloned into the -CAT plasmid containing the -promoter gene (384 to +36 from the cap site) fused with the CAT gene (16). A, AvaI; H, HphI; B, BamHI; Bg, BglII; Nc, NcoI; K, KpnI; X, XbaI.



Transfections and Assessment of CAT Activity

The cells used in this study (K562 and HeLa) were grown in RPMI 1640 and Dulbecco's modified Eagle's media (Life Technologies Inc.), respectively, supplemented with 10% fetal calf serum. Following growth in mid-log phase, the cells were plated in 10 ml of medium on 100-cmdishes. Twenty-four h after plating, the cells were transfected with equimolar amounts ( i.e. about 10 µg) of supercoiled DNA of the various constructs using DNA-calcium phosphate precipitation, as described previously (16, 18) . A standard amount (3 µg) of plasmid pCH110 (19) was added to each DNA-calcium phosphate preparation for normalization of the transfection efficiency using the -galactosidase assay (20) . The total amount of DNA (20 µg/plate) was kept constant by adding the appropriate amount of sheared salmon sperm DNA. The protein concentration of the cell extract was quantitated using a protein assay kit (Bio-Rad). Forty-eight h after transfection, the cells were harvested, lysed, and the protein concentration of the cleared lysate was determined as described (21) , while CAT activity was analyzed by the method of Gorman et al. (22) .

DNA Sequencing of the Enhancer Region

DNA sequencing of both strands was performed using the dideoxy method of Sanger et al. (23) . The DNA fragments (F, A, A3, and G1) containing the enhancer sequences 3` of the breakpoint were subcloned into the M13mp phage vector and were sequenced using the Sequenase kit (United States Biochemical Co., Cleveland, OH), as described previously (24) .


RESULTS

Expression of the Human Fetal A-Globin HPFH-3 Gene in Transgenic Mice

To investigate the pattern of expression of the human fetal A-globin gene fused with the 3` HPFH-3 juxtaposed sequences during development, we analyzed the mRNA of 11.5-day embryonic blood, 16-day fetal liver, and adult blood of 12 transgenic mice. Six mice were independent founders, and six mice were used as ``transient'' transgenes sacrificed at day 16, followed by analysis of their fetal liver mRNA, using an RNA protection assay. In the six founder lines three mice failed to express the A-gene in 11.5-day embryonic blood, probably due to integration into an inactive chromatin region ( Fig. 2and ). The other three mice expressed the A-gene in levels ranging from 4 to 0.02% of the endogenous mouse embryonic globin mRNA (). Of the last three mice, two (lines 1-5 and 1-4) failed to express further the A-gene, while mouse 3-5 expressed the A-gene in 16-day liver, which is the organ of adult erythropoiesis. An additional mouse (line 42) of the group of transient HPFH-3 transgenic mice, expressed also the A-gene in 16-day fetal liver ( and Fig. 2). Thus, the human A-gene in the two mice, when fused with the 3` juxtaposed sequences of the HPFH-3 deletion, was expressed beyond the stage that is normally expressed, i.e. the 11.5-day embryonic blood (11, 13, 25, 26) . These data suggested a putative regulatory role of these juxtaposed sequences on the developmental specificity of the A-gene.

Identification of an Enhancer 3` to Breakpoint of the HPFH-3 Deletion

To further investigate the role of the 3` juxtaposed sequences of the HPFH-3 deletion, 10 overlapping and contiguous fragments spanning the entire 6.2 kb region were subcloned in both orientations (5`3` and 3`5`) and in both positions (upstream and downstream) of the CAT hybrid reporter gene (Fig. 3) which has been shown to be enhancer-responsive in K562 cells (16) . A series of 26 constructs were individually transfected into uninduced K562 cells, hemin-induced K562 cells, and HeLa cells, which were cultured for 48 h and were then analyzed for CAT activity. I shows the results of the analysis for the detection of the enhancer element. The enhancerless CAT plasmid was used as the reference basic plasmid, and its relative CAT activity was considered as 1.0. The 11 CAT plasmid, carrying the 2.3-kb EcoRI fragment containing the 3` A enhancer (16) was used as a positive control. Of the 24 recombinant plasmids tested, only plasmids with the F1, F, and G fragments (and to a lesser extent the ones with the A fragment) demonstrated enhancer activity by increasing almost 3-fold the average relative CAT activity of the CAT plasmid in induced K562 cells (I). This activity was less pronounced in uninduced K562 cells. The increased CAT expression by the F1, F, and G fragments was independent of their orientation (5`3` or 3`5`) and position (upstream or downstream) relative to CAT, a feature consistent with other transcriptional enhancers (16, 27, 28, 29) . Analysis of the effects of the F1, F, G, A, and H fragments, on the activation of the CAT reporter gene, permitted the localization of the enhancer core element immediately 3` to the breakpoint, extending for about 700 bp downstream (Fig. 4). Furthermore, the comparable activity of the F1 and F fragments ensures that the whole enhancer effect is actually conferred by the 0.7 kb region and that there is no aberrant synergy between the enhancer element and the residual Alu sequences contained in fragment F. When the same recombinant CAT plasmids were used to transfect non-erythroid HeLa cells, both F and G fragments failed to increase the level of CAT activity, suggesting that the enhancer element is probably erythroid-specific (data not shown).


Figure 4: Identification and completion of the sequencing of the enhancer core element. A, analysis of the effects of the F1, F, G, A, and H fragments on the activation of the CAT gene localized the enhancer element ( hatched bar) immediately 3` from the breakpoint ( vertical arrow), extending downstream for about 700 bp. The region corresponding to the F fragment has been previously sequenced by Henthorn et al. (10). Sequence analysis revealed that the region immediately 3` to the breakpoint of the deletion contains a complex array of repeated sequences (10). The 3` end half of a perfect palindrome is indicated by the large open arrow, starting just 3 bp before the breakpoint. A set of seven short tandem repeats is shown as solid arrows. An interruption of 24 bases of unrelated sequences (shown as hatched bars) occurs at the fourth repeat. The CCAAT motif is shown as a dot. There are two CCAAT motifs on the sixth repeat and a seventh CCAAT motif ( boxed) was found at the 3` end of the enhancer (see panel B). At the 3` end of the enhancer region there is a consensus (A/T)GATA(A/G) motif for the binding of the erythroid-specific transcription factor GATA-1. B, sequencing of the novel 431 bp region encompassing the 3` end of the enhancer element. The novel sequences start from position 55 (corresponding to 466 nt from the breakpoint of the HPFH-3 deletion) and end up at position 485. The upstream 466 bp region starting from the 3` end of the breakpoint and terminating close to the BamHI site ( GGATCC, underlined by light broken line) has been sequenced previously by Henthorn et al. (10); these sequences just upstream of the BamHI site are highlighted. The motif for the SV40 core element ( GTGGTCTG) is boxed. The novel CCAAT motif is also boxed and the PstI site ( CTGCAG) is underlined with a heavy broken line. The HphI site ( GTAACACTCACC) marking the 3` end of the F1 fragment is underlined with a solid line.



Activation of Heterologous Promoters by the HPFH-3 Enhancer Element

To test the ability of the 3` enhancer element to increase transcription from other globin or non-globin promoters, fragment F (exhibiting the highest activity) was subcloned 3` to the CAT gene in both orientations (5`3` and 3`5`) into vectors containing several hybrid genes such as -globin-CAT, -globin-CAT, -globin-CAT, and DHFR-CAT (16, 17) . These plasmids along with the control CAT plasmid were used to transfect induced or uninduced K562 cells by DNA-calcium phosphate coprecipitation. After 48 h following transfection, the cells were assayed for CAT activity. Fragment F significantly increased the expression of -CAT plasmid carrying the embryonic -globin gene promoter by 2.3-fold (data not shown). On the contrary, no effect was documented on the transcriptional activity of the adult - and -globin genes, while there was a repression by 3.8-fold of the DHFR-CAT activity (data not shown). These data suggest that the effect of the 3` enhancer of the HPFH-3 deletion is restricted on fetal and embryonic globin gene promoters, while it has a repressing effect on non-globin promoters such as the housekeeping DHFR gene promoter.

Nucleotide Sequence Analysis of the 3` Enhancer of HPFH-3 Deletion

Based on the information of the CAT assays described above, the region of the enhancer element was localized immediately 3` to the breakpoint of the HPFH-3 deletion extending for about 700 bp downstream. The region encompassing most of the F fragment had been previously sequenced by Henthorn et al. (10) extending 466 bp from the 3` breakpoint. Since primarily fragments F, F1, and G and to a lesser extent the A fragment were activating the CAT fusion gene (I and Fig. 4), they provided evidence that the region of the enhancer extends further downstream for an additional 200 bp (Fig. 4 A). We therefore performed DNA sequencing starting from the 3` breakpoint of the deletion and extended it to an additional 431 bp downstream from the end of the 3` normal sequence previously characterized (10) as shown in Fig. 4B. The sequence analysis of the 3` enhancer (Fig. 4, A and B) revealed that this region contains a complex array of repeated sequences (10) . These include ( a) the 3` half end of a perfect palindrome (80 bp each) starting just 3 bp before the breakpoint, ( b) a set of seven short tandem repeats (shown as black solid arrows in Fig. 4A) consisting of a 41-bp sequence, repeated totally or partially with one interruption of 24 bp of unrelated sequences (shown as hatched bars in Fig. 4 A) at the fourth repeat (10) . At the 3` end of the first, second, third, and seventh repeat there is a CCAAT motif (shown as a dot in Fig. 4 A) for the binding of the ubiquitous CP1 transcription factor. Two CCAAT motifs exist on the sixth repeat. An additional CCAAT motif was found downstream near the 3` end of the newly sequenced DNA region (Fig. 4, A and B). Within each of these repeats there are five regions which exhibit 54-62% homology with a conserved region of 24 bp, shared extensively (17 of 21 bp) by both the chicken 3` -enhancer, residing about 410 bp 3` to the chicken -gene poly(A) addition site (30, 31) and by the human 3` A-enhancer residing about 480 bp 3` to the A-gene poly(A) site (16) . This conserved element between these two enhancers is not found elsewhere in the human -globin gene cluster (16) , although the short repeats of 41 bp where the conserved 24-bp sequence resides are repeated elsewhere in the human genome (10) . A sixth region of homology with these enhancers is found upstream of the breakpoint within the 5` portion of the 160-bp palindrome but in an inverted orientation (10) . At least 27 regions were found within the sequenced area of 897 bp which show homology (62-87%) with the SV40 enhancer core sequence GTGG(A/T) (A/T) (A/T)G (22) . Finally, at position +348 from the breakpoint, the enhancer region contains the consensus motif AGATAA for the binding of the erythroid-specific transcription factor GATA-1, which participates in the regulation of the majority of erythroid-specific genes and erythroid-specific enhancers (32, 33) .


DISCUSSION

In this study we have tested the validity of the hypothesis of imported enhancers (5) derived from the 3` end of the human -globin cluster, as a result of large deletions, to the vicinity of the fetal -globin genes, leading to their continued expression in the adult life. We have used as a model the HPFH-3 deletion which removes 48.5 kb of DNA of the -cluster including the - and -globin genes and results in high levels of expression of the -genes. We have reasoned that the 3` juxtaposed sequences 3` to the breakpoint might contain regulatory elements that can modify the developmental expression of the fetal -globin genes. To address this question we reconstituted the abnormal 13.6-kb bridging fragment as it is formed in the heterozygote individual with the HPFH-3 deletion (9, 10) containing the normal fetal A-globin gene, its flanking sequences, and 6.2 kb of juxtaposed sequences derived from a region normally located 30 kb downstream of the -globin gene. The bridging fragment was then microinjected into the fertilized mouse eggs, and the developmental expression of the -globin gene was assayed in the generated transgenic mice. Previous studies from several laboratories (11, 34) have established that the human -globin genes, when introduced as individual fragments containing the minimum of flanking sequences, display exclusively an embryonic pattern of expression, i.e. being expressed only in the 11.5-day yolk sac cells but not in definitive erythroid cells of later stages such as the fetal liver cells or the adult blood erythrocytes. Therefore, this system provides the opportunity to test the effect of additional sequences on the developmental program of the fetal -genes. When these 3` juxtaposed sequences were added as part of the bridging fragment in tandem with the individual A-globin fragment, the transgene was expressed in two mice beyond the embryonic stage, its activity being detectable in the 16-day fetal liver, the organ of adult erythropoiesis. These qualitative data are in marked contrast to previous transgenic mice studies using the A-gene fragment in isolation, where expression was restricted to yolk sac cells (11, 34) and suggest that the presence of the 3` juxtaposed sequences 3` to the A-gene was capable in altering partially the developmental specificity of the fetal gene. The human fetal genes when introduced individually into transgenic mouse exhibit exclusive embryonic pattern of expression; however, their level of expression represents only a fraction of that of the endogenous mouse genes (11, 34) . On the contrary, when the individual -globin genes are linked with sequences containing the locus control region (LCR), a position-independent, copy number-dependent, and high level of erythroid-specific expression is achieved (25, 35, 36, 37) . However, the temporal specificity of the -genes is lost in the presence of the LCR sequences, resulting in their expression in all stages of development: yolk sac, fetal liver, and adult erythroid cells (25, 35) . Our construct did not contain LCR sequences and therefore permitted us to assay directly solely the effects of the 3` juxtaposed sequences on the developmental expression of -genes. Our assay was quite sensitive in revealing the effect of these sequences on the developmental and/or transcriptional specificity of the 3` juxtaposed sequences.

The role of these sequences was further investigated by testing the effects of the several fragments of this region on the transcription of the fusion CAT reporter gene in erythroid (K562 cells) and non-erythroid HeLa cells. Fragments delineating a region of about 700 bp immediately 3` to the breakpoint, activated the CAT gene nearly 3-fold, regardless of their orientation or position in respect to the CAT genes, primarily in induced K562 cells but not in HeLa cells. Thus, these data revealed an enhancer element located 3` to the breakpoint of the HPFH-3 deletion exhibiting an erythroid specificity. The levels of activation of CAT gene by the HPFH-3 enhancer were comparable (although slightly lower) to those of HPFH-1 enhancer detected previously using the same CAT plasmids in K562 cells (8) . However, in the latter case, there was considerable variation of the fold activation depending on the position and orientation of the enhancer. Furthermore, the HPFH-1 enhancer also exhibited a minimal activity in HeLa cells, in contrast to the HPFH-3 enhancer.

We further investigated the specificity of the HPFH-3 enhancer to activate other heterologous globin and non-globin promoters. The enhancer was capable of activating the human embryonic -globin promoter but had no effect on the adult - or -globin promoters in K562 cells. These data imply specificity of the enhancer on fetal/embryonic promoters. We have previously shown (17) that the CAT fusion gene is inactive in K562 cells, in contrast to the CAT gene. Therefore, it is conceivable that the lack of activation of -promoter by the HPFH-3 enhancer could alternatively reflect a variable enhancer strength, since a considerable activation of the CAT gene was achieved in K562 cells by the powerful and non-stage-specific enhancer element contained within the 5` hypersensitive site 2 (5` HS2) of the LCR (38) . It is interesting that the HPFH-3 enhancer down-regulated the DHFR-CAT gene by nearly 4-fold. A similar effect has been observed with the 3` A-enhancer (16) exerting strong repression on the same DHFR-CAT gene but in 293 cells, which constitutively express adenovirus E1A protein, a known repressor on some viral and cellular enhancers (39, 40, 41) . It is not clear why this repression occurred in K562 cells in our case, since these cells do not express the E1A protein and also why the and promoters when linked to the same enhancer failed to be down-regulated in the same cell line.

In view of the functional detection of the enhancer element, we pursued its detailed sequence analysis. The region contained within the F fragment has been sequenced previously by Henthorn et al. (10) . We further sequenced an additional 431 bp downstream to encompass the 700-bp region that exhibited the full enhancer activity by using as templates derivatives of fragments A and G. The enhancer lies within a complex array of multiple repeat elements including an inverted palindrome repeat and a set of seven short tandem repeats consisting of a 41-bp sequence, covering a region of 350 bp. It is noteworthy that a number of regions within the enhancer exhibit homology with other known globin or non-globin enhancers and display specific motifs for transcription factors. Thus, the enhancer exhibits six regions of homology (54-62%) with a conserved region of 24 bp, shared by both the chicken 3` -enhancer (30, 31) and the human 3` A-enhancer (16) . Twenty-seven regions within the enhancer exhibit homology (62-87%) with the SV40 enhancer (22) core sequence GTGG(A/T) (A/T) (A/T)G. Similar homologies to the chicken 3` -globin gene enhancer, the human 3` A-gene enhancer, and to the SV40 core enhancer element have also been detected within the region of the recently described HPFH-1 enhancer, located 3` of the breakpoint of the HPFH-1 deletion (8) . Furthermore, seven consensus CCAAT motifs for the ubiquitous CP1 transcription factor are found within the enhancer while at the end of the set of repeats, the enhancer exhibits the consensus motif AGATAA for the erythroid-specific transcription factor GATA-1 which binds specifically to erythroid-specific promoters and enhancers (32, 33) . A GATA-1 motif has been found also within the HPFH-1 enhancer (8) . Therefore, the HPFH-3 enhancer element shares features with other globin and non-globin enhancers, and probably its function may be mediated by the additional binding of erythroid and non-erythroid factors. The structural and functional properties of the juxtaposed 700-bp region located immediately 3` to the breakpoint of the HPFH-3 deletion documented in this study are consistent with an enhancer element that has the properties of modifying the developmental specificity of the fetal -genes and thus maintaining a high level of expression in the adult erythroid cells. The effects of the enhancer on the continued transcription of the -promoters may be mediated either via the enhancer sequences per se and/or by additional parameters that are associated with the nature of the chromatin status of the juxtaposed sequences that promotes and maintains the active configuration of the -genes (7, 42, 43) . Thus, the presence of this juxtaposed enhancer element can explain the resulting HPFH phenotype in the HPFH-3 deletion. A similar enhancer 3` to the breakpoint of HPFH-1 juxtaposed to the vicinity of the -genes has been proposed to contribute to the HPFH phenotype for both the HPFH-1 and HPFH-2 deletions (8) . It is conceivable that the HPFH-3 enhancer might be operating also for the generation of the phenotype of HPFH-4 deletion, since their 3` deletion breakpoints are differing only by about 2.0 kb (3, 10) .

The generality of the enhancer mechanism on the generation of the HPFH phenotype is further supported by two additional examples: ( a) the Kenya HPFH deletion (1) caused by an intergenic deletion between the A- and the -genes, results in a fusion A-gene and in a continued high level expression of both G- and A-genes with a pancellular distribution of fetal hemoglobin in erythrocytes (1) . Without excluding the possibility that the deletion of putative negative elements between the A- and -genes have contributed to the HPFH phenotype, the high levels of expression of both G- and A-genes cis to the deletion can be readily explained by the juxtaposition of the 3` enhancer (11, 34, 44) to the proximity of the A (2 kb) and G (6 kb) promoters; ( b) we have recently (45) identified an additional enhancer element 3` to the breakpoint of the HPFH-6 deletion, originally described as Thai (A)° thalassemia (4) . This enhancer, located 53 kb from the 3` end of the -globin gene, seems to be responsible for the generation of the HPFH phenotype, since its loss by the Chinese (A)° thalassemia, a perfectly comparable deletion to HPFH-6, results in a ()° thalassemia phenotype, associated with much lower output of fetal hemoglobin and heterocellular distribution in erythrocytes (46) . The presence of this enhancer also near the 3` breakpoints of the recently described Yunnanese (A)° thalassemia (47) can explain the efficient production of fetal hemoglobin and thus the mildness of the phenotype of a homozygote for the Yunnanese deletion ( i.e. Hb 10.7 g/dl, no splenomegaly) compared to the severe phenotype of the Chinese (A)thalassemia deletion (46) .

The data both from the present study and the other studies mentioned above support the validity of the hypothesis that DNA elements located within the 3` juxtaposed sequences of the HPFH deletion mutations can override the developmental specificity of the fetal -genes and are responsible for the resulting discrete phenotype. These data do not rule out the role of putative negative elements (48) between the A- and -genes that are removed by these HPFH deletions and which may independently contribute to the HPFH phenotype. Further studies will be needed to clarify and characterize the exact role of these enhancer elements in their natural positions within the cluster.

  
Table: Expression of HPFH-3 transgenes in embryonic blood, fetal liver, and adult blood


  
Table: Expression of transient HPFH-3 transgenes in fetal liver


  
Table: Relative CAT activity in uninduced () and hemin-induced K562 (+) cells, transfected with the various plasmids containing the individual fragments of the juxtaposed region

All transfections and assays were performed at least in three separate experiments. Relative CAT activity was determined by dividing the average percent conversion for each fragment by the average percent conversion of the control CAT plasmid. CAT values were normalized based on protein quantitation, Lac-Z assays, and confirmation of the amount of DNA used. Equimolar amounts of DNA from each plasmid were used for the transfection experiments. As a positive control for the CAT assay system, the pRSV CAT plasmid (16, 22) was used, which increased consistently CAT activity 35-40-fold in both K562 and HeLa cells. The relative CAT activity of the rest of the 12 recombinant plasmids (not shown) containing fragments H, J, I, B, C, and D, was similar to that of the control reference CAT plasmid, i.e., 1.0.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 45365 (to G. S., and N. P. A.) and by the Greek Ministry of Health and the General Secretariat of Research and Technology Grants E-138/89 and 89-ED 271 (to N. P. A.).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X81476.

§
To whom reprint requests should be addressed: University of Crete, School of Medicine, 711 10 Heraklion, Greece. Tel.: 30-81-542-077; Fax: 30-81-542-112.

The abbreviations used are: HbF, fetal hemoglobin; HPFH, hereditary persistence of fetal hemoglobin; LCR, locus control region; kb, kilobase(s); nt, nucleotide(s); CAT, chloramphenicol acetyltransferase; DHFR, dihydrofolate reductase; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. O. Smithies for the recombinant HPFH-3 bridging fragment, Drs. P. Henthorn and D. Mager for many fruitful discussions and help, Drs. D. Bodine and A. Schechter for the CAT plasmids, G. Houlaki for valuable help with the graphics and Bonnie Lenk for excellent secretarial assistance.


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