Maximal Activity of an Erythroid-specific Enhancer Requires the Presence of Specific Protein Binding Sites in Linked Promoters*

Persis J. Amrolia, Wesley Gabbard, John M. CunninghamDagger , and Stephen M. JaneDagger §

From St. Jude Children's Research Hospital, Memphis, Tennessee and the § Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Australia 3050

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

High level expression of many eukaryotic genes is achieved through the action of distal regulatory sequences or enhancers. We have utilized the interaction between the erythroid-specific enhancer in hypersensitivity site 2 (HS2) of the human beta -globin locus control region and the globin gene promoters as a model to elucidate the mechanisms governing promoter/enhancer interactions. HS2 contains a 400-base pair core element consisting of tandem AP1/NF-E2 motifs flanked by binding sites for multiple ubiquitous and erythroid-specific factors. We have compared the enhancer activity of this core element with a synthetic enhancer lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2M). In fetal/erythroid K562 cells, enhancement of a linked gamma -promoter was significantly greater with wild-type HS2 than with HS2M. In contrast, the increase in beta -promoter activity in these cells was equivalent with either enhancer fragment. Truncation of the binding site for the fetal/erythroid-specific stage selector protein in the gamma -promoter abolished the additional enhancer activity of HS2. Similarly, insertion of the stage selector protein site into the beta -promoter boosted enhancer activity observed with HS2 but not HS2M. In adult erythroid MEL cells, enhancement of a linked beta -promoter was significantly greater with HS2 than with HS2M. This effect was dependent on the binding of the adult stage-specific factor, erythroid Kruppel-like factor, to the beta -promoter. Taken together, this data suggests that the stage-specific factors binding the proximal globin promoters and the factors flanking the AP1/NF-E2 motif of HS2 act in synergy.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Transcriptional regulation of eukaryotic genes is dependent on the formation of multiprotein complexes on critical cis-acting regulatory sequences. Interactions between these proteins can provide tissue and stage-specificity, potential for regulation, and high expression levels (1, 2). Complexes assembled on adjacent DNA sites exert their influence through cooperative binding and/or transcriptional synergy (3, 4). In proximal promoter regions, activator proteins are thought to increase the rate of preinitiation complex formation through interactions with their cognate TATA-binding protein-associated factors or other components of the initiation complex (5-11). In this setting, the position and arrangement of the factor binding sites is critical for transcriptional activation. The spatial arrangement of factor binding sites within distal enhancer elements is also critical for their function. This is evident in the human interferon-beta gene enhancer and the T cell receptor-alpha enhancer in which the binding of "architectural proteins" such as LEF-1 and HMG I(Y) induce a change in DNA conformation, allowing the formation of nucleoprotein complexes or enhanceosomes (12, 13). The coordinate action of these architectural proteins and transcriptional activators such as NFkappa B or ATF-2/c-Jun is essential for optimal enhancer function.

In contrast to this, relatively little is known about the synergistic interactions observed between complexes binding to non-adjacent DNA sites, typified by promoter/enhancer interactions. One current model proposes that initially, DNA looping or bending is required to juxtapose the two elements with subsequent transcriptional activation depending on the efficient recruitment of the enhancer by its preferred promoter (14). This process may be mediated through direct interaction between an enhancer binding protein(s) and the polymerase complex or indirectly through another protein bound to the proximal promoter (15-17). Implicit to this and other models of enhancer action is the need for constraint where only the appropriate gene and not adjacent genes will be activated. This is particularly important in the context of intergenic enhancers, which are capable of activating multiple promoters but opt to activate only one at a given developmental stage. This process may be mediated by the presence of domain elements that demarcate functional units or by promoter selectivity (18-21).

The human beta -globin cluster provides an ideal model for the study of enhancer/promoter interactions in the context of a multigene locus. High level expression from the genes in this locus is dependent on a region containing four erythroid cell-specific and developmentally stable DNase I hypersensitivity sites (HS1 1-4), the locus control region (LCR) (22-24). One of these sites, HS2, contains a powerful erythroid-specific enhancer active in transient transfections, stable cell lines, and transgenic mice (25-28). The core of this enhancer consists of tandem binding sites for members of the AP1 family of proteins, including the erythroid-specific factor NF-E2, flanked by consensus sites for multiple ubiquitous and erythroid-specific proteins (29-33).

High level developmentally specific expression of the individual globin genes requires the preferential recruitment of the enhancing sequences of the LCR to their respective promoters (34). We have previously shown that the preferential interaction of HS2 with the gamma -globin gene promoter in fetal/erythroid K562 cells is mediated through the binding of a fetal and erythroid-specific protein complex, the stage selector protein (SSP). This complex is assembled on a sequence immediately adjacent to the TATA box, the stage selector element (SSE) (35, 36). A second stage-specific factor, erythroid Kruppel-like factor (EKLF) is essential for the preferential expression of the beta -globin gene in adult erythropoiesis (37). Mice lacking this protein die in utero of a beta -thalassemic-like illness (38, 39).

In the studies reported here, we explore the mechanisms governing enhancer/promoter interactions using HS2 and the gamma - and beta -globin gene promoters as a paradigm. Our findings reveal that maximal enhancer activity of HS2 in fetal/erythroid K562 cells is dependent on the presence of the SSP binding site in the proximal gamma -promoter. Similarly, EKLF appears integral for the full enhancement of beta -promoter activity by HS2. Interestingly, these effects appear to depend upon synergistic transcriptional activation between the promoter bound stage-specific proteins and factor binding sites flanking the tandem AP1/NF-E2 element of the enhancer.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

DNA Construction-- Constructs containing the beta -globin promoter (-385-+38) or a hybrid beta SSE promoter subcloned 5' to the coding sequence of the chloramphenicol acetyltransferase reporter gene (beta CAT, beta SSECAT) and the gamma -promoter (-260-+35, -53-+35, or -35-+35) subcloned 5' of the firefly luciferase gene (gamma LUC, -53gamma LUC, -35gamma LUC) have been described previously (26, 36). Constructs containing the 734-bp HindIII-BglII fragment of HS2 or a synthetic HS2 (HS2M), containing the tandem NF-E2/AP-1 core enhancer flanked by neutral DNA to ablate all previously reported co-enhancer footprints but retaining the spacing between the core enhancer and adjacent promoter have been described previously (40). Briefly, the neutral DNA was designed to be devoid of LCR consensus sequences. The 5' component of the neutral DNA consisted of a 169-bp XhoI-HindIII polymerase chain reaction fragment of pBR322 backbone and a 189-bp BglII-SalI fragment of the ampicillin resistance gene. An additional 370-bp BglII-SalI polymerase chain reaction fragment of the tetracycline resistance gene was utilized as the 3' component.

The XhoI-AatII fragments of the reporter constructs described above were subcloned 3' to either HS2 or HS2M AatII-SalI fragments. HS2 truncation mutants were made by subcloning the 374-bp HindIII-XbaI fragment of HS2 or polymerase chain reaction-generated fragments containing its 5' or 3' halves upstream of the same neutral DNA fragments used in HS2M, to once again retain identical spacing to the original 734-bp HS2. These were subcloned 5' to beta CAT and beta SSECAT using the XhoI/SalI-AatII strategy outlined above. HS2 single site mutants, which ablate DNA contacts and disrupt DNA binding of the previously described electrophoretic mobility shift complexes were made using polymerase chain reaction site-directed mutagenesis (31).

To ensure that the neutral DNA used in these constructs was inactive we performed two control experiments. In the first we replaced the original neutral DNA with totally different neutral DNA and obtained identical results in transfection studies. In the second we linked the neutral fragment to the gamma -promoter and demonstrated no enhancement of activity of this construct compared with the gamma -promoter alone (data not shown).

DNA Transfection and Transient Assays-- Plasmid DNA was prepared over double cesium chloride gradients. DNA was transfected into human (K562) or mouse (MEL) erythroleukemia cells by electroporation as described previously (36). K562 cells were then induced with 20 mM hemin and MEL cells with 2% Me2SO. After 48 h, cells were harvested and lysed with Triton X-100 for luciferase assays or repeated freeze-thaw cycles for CAT assays. Luciferase activity was assayed using the Promega system on a Monolight 2001 luminometer (Analytical Luminescence Laboratories), and linearity of the assay was confirmed with serial dilutions. Luciferase lysates had equivalent protein levels. CAT activity was determined as described previously (36) and standardized for lysate protein concentration. Constructs were only compared within the same series of experiments, and the results shown represent at least six different transfections with two independent plasmid preparations. Statistics were calculated using Student's t test; values of p < 0.05 were considered significant.

Production of Stable Cell Lines-- K562 erythroleukemia cells (2 × 107) were transfected with 10 µg of appropriate gamma -promoter vector DNA and 1 µg of pCINeo (Promega). Cells were selected in G418 (500 µg/ml) for 21 days. Multiple pools were analyzed for luciferase reporter gene expression after 48 h of incubation with hemin. To establish a K562 cell line overexpressing EKLF, these cells were transfected with the eukaryotic expression vector pCIFLAGEKLF, which contains the complete murine cDNA tagged with the FLAG epitope under the control of the viral cytomegalovirus promoter. Cells were selected in G418 (500 µg/ml), and clones with the highest level of transgene expression were identified by Western blotting utilizing a specific anti-FLAG antibody and the ECL chemiluminescence system (Amersham).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

HS2 Enhancer Activity in K562 Cells-- The erythroid-specific enhancer in HS2 has been mapped to a 46-bp element containing a tandem binding site for the AP1 family of proteins (41). Adjacent to this element (5' and 3') are multiple consensus sites for erythroid-specific and ubiquitous transcription factors (YY1, USF, SCL, GATA-1, Sp1) (Fig. 1) (30-33). We initially examined the ability of the AP1/NF-E2 binding sites alone or in the presence of the adjacent factor binding sites to enhance expression from the gamma - or beta -promoter in K562 cells, a model of the fetal erythroid environment (42). To achieve this we compared the 734-bp HindIII-BglII fragment of HS2, which contained the AP1/NF-E2 element and adjacent sites, to a synthetic HS2 (HS2M), which contained the 46-bp element flanked by neutral DNA to retain the spacing between the enhancer and adjacent promoter. Each HS2 fragment was linked to a -260gamma -promoter/luciferase or a -378beta -promoter/CAT hybrid gene and electroporated into K562 cells. Enhancerless promoter/reporter hybrids served as controls. As shown in Fig. 2, the addition of the 46-bp AP1/NF-E2 element alone to either the gamma - or beta -promoter containing construct augmented expression 6-fold (constructs 1 and 2; constructs 4 and 5). This finding is consistent with other studies which have shown that partial enhancer activity is observed in the context of 46-bp element alone (29, 30). Interestingly, linking the wild-type HS2 to the gamma -promoter construct resulted in an additional 4-fold increase in transcriptional activity to that observed with HS2M (constructs 2 and 3) (p < 0.05). In contrast, no further increase in beta -promoter activity was seen with wild-type HS2, despite the presence of the multiple transcriptional activator sites flanking the 46-bp AP1/NF-E2 element (constructs 5 and 6).


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Fig. 1.   Arrangement of factor binding sites within human HS2. A, the protein binding sites in the 734-bp HindIII-BglII fragment are illustrated. The shaded box represents the tandem NF-E2/AP-1 binding motif. The open boxes represent the binding sites flanking the AP1/NF-E2 site, with each labeled with its cognate factor. The E, CACC/SCL, and AT labels represent the erythroid-specific footprint with homology to the Friend virus LTR, the CACC motif footprinted by SCL in adult erythroblasts, and the ubiquitously footprinted AT-rich sequences, respectively. B, the HS2 fragments used were the wild-type sequence (HS2) or the tandem AP1/NF-E2 sites flanked by neutral DNA to retain equivalent spacing (HS2M).


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Fig. 2.   HS2 and HS2M activity in K562 cells. A, diagrammatic representation of the constructs used for transfection into K562 cells induced with hemin. Solid boxes represent the 734-bp HindIII-BglII fragment of HS2 (constructs 3 and 6) or the HS2M fragment (constructs 2 and 5). The open boxes represent the -260 gamma -promoter linked to the luciferase gene. The hatched boxes represent the -385 beta -promoter linked to the CAT gene. B, reporter gene activity of these constructs. Hatched bars represent CAT conversion standardized per microgram of protein; open bars represent luciferase activity (lysates had equivalent protein concentration). Values shown represent the mean of at least six separate transfections with at least two different plasmid preparations. Statistical significance was assessed using Student's t test.

Maximal Enhancer Activity of HS2 Is Dependent on Specific Sequences in the Linked Promoter-- The preferential interaction of HS2 with the gamma -promoter in K562 cells is mediated, in part, through binding of the SSP to the SSE (35, 36). We therefore postulated that the difference in maximal enhancer activity observed with HS2 and HS2M may also be dependent on the presence of a specific binding site in the promoter. To examine this, we evaluated the effect of truncation mutants of the gamma -promoter on the ability of HS2 or HS2M to enhance expression. As seen in Fig. 3A (constructs 1 and 2), the differential activity between HS2 and HS2M was retained with truncation of the gamma -promoter to nucleotide position -53 relative to the CAP site (p < 0.001). In contrast, deletion of the SSE (between -53 and -35) resulted in diminution of the enhancer activity of the wild-type HS2 to levels not statistically significantly different to those observed with HS2M (construct 3 and 4). In fact, the reporter gene expression observed with deletion of either the SSE or the sites flanking the AP1/NF-E2 element, or both, was not statistically different (compare construct 2, 3, and 4).


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Fig. 3.   Maximal enhancer activity is dependent on the SSE. Diagrammatic representation of the constructs used for transfection into K562 cells induced with hemin. Solid boxes represent the 734-bp HindIII-BglII fragment of HS2 (constructs 1 and 3) or the HS2M fragment (constructs 2 and 4). The open boxes represent either the -53 (constructs 1 and 2) or the -35 (constructs 3 and 4) gamma -promoter linked to the luciferase gene. A, reporter gene activity of these constructs in transient assays. Open bars represent luciferase activity (lysates had equivalent protein concentration). B, reporter gene activity of these constructs in stable K562 pools. Open bars represent luciferase activity (lysates had equivalent protein concentration).

To examine these findings in a genomic context, stable K562 cell lines containing these constructs were derived. As seen in Fig. 3B, the results we had observed in the transient assays were completely reproduced in the stable lines suggesting that maximal response of the gamma -promoter to the HS2 enhancer requires the presence of factors binding adjacent to the AP1/NF-E2 motifs and the SSE.

To confirm these findings, we linked HS2 or HS2M to a hybrid beta -promoter in which the -53 to -35 beta -sequence was replaced by the SSE and transfected these constructs into K562 cells (Fig. 4A). As seen in Fig. 4B (constructs 1 and 2), equivalent levels of activity were seen with HS2 and HS2M linked to the wild-type beta -promoter. Similar CAT activity was also noted with the hybrid promoter linked to HS2M (construct 4). In contrast, a significant increase in transcriptional activity was observed with the hybrid beta -promoter containing the SSE linked to HS2 (construct 3) (p < 0.01). This observation was not simply attributable to the insertion of a transcriptionally active sequence into the proximal beta -promoter, as no difference in activity was observed between HS2Mbeta CAT and HS2Mbeta SSECAT (compare constructs 2 and 4). Experiments utilizing beta -promoter and beta SSE-promoter containing constructs in stable cell lines yielded similar results (data not shown). These findings confirm that both the SSE and HS2 binding sites flanking the AP1/NF-E2 motif are necessary for maximal enhancement of globin promoters in fetal/erythroid K562 cells.


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Fig. 4.   Increased enhancement of a hybrid beta -promoter containing the SSE by HS2 in K562 cells. A, diagrammatic representation of the constructs used for transfection into K562 cells induced with hemin. Solid boxes represent the 734 bp HindIII-BglII fragment of HS2 (constructs 1 and 3) or the HS2M (constructs 2 and 4). The hatched boxes represent the wild-type beta -promoter linked to the CAT gene (constructs 1 and 2). The hatched boxes with open insert (beta SSE) represent a hybrid beta -promoter containing the -53 to -35 region of the gamma -promoter inserted into the corresponding position (constructs 2 and 4) linked to the CAT gene. B, reporter gene activity of these constructs. Hatched bars represent CAT conversion standardized per microgram of protein. Values shown represent the mean of at least six separate transfections with at least two different plasmid preparations. Statistical significance was assessed using Student's t test.

Multiple Binding Sites Flanking the AP1/NF-E2 Element Are Required for Maximal Enhancer Activity-- To determine whether our previous findings were secondary to a direct interaction between an HS2 binding protein and the SSP, we constructed a series of scanning mutants ablating each of the HS2 protein binding sites. These mutants were linked to the hybrid beta SSE-promoter and a CAT reporter gene and transfected into K562 cells. Transcriptional activation of the promoter was maintained with mutation of all sites flanking the AP1/NF-E2 element suggesting that no individual site is essential for the synergy observed with the SSE (Table I). In contrast, mutation of the AP1/NF-E2 binding site substantially reduced the activity of the hybrid promoter (Table I).

                              
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Table I
Effect of mutation of footprinted motifs in HS2 on beta SSE expression
K562 cells were electroporated with plasmids containing the hybrid beta SSE promoter linked to the CAT reporter gene 3' of a HS2 fragment in which the various footprints had been mutated individually. The expression of these altered HS2-containing constructs was assayed, and activation of the hybrid promoter was corrected with respect to wild-type expression. The asterisk indicates a significant difference from the wild-type sequence (p < 0.01).

To determine whether combinations of binding sites could be important, we linked a series of 5' and 3' truncation mutants of HS2 to either the wild-type or hybrid beta -promoter. In each case, neutral DNA was inserted to retain the original spacing between the AP1/NF-E2 element and the promoter. These constructs were transfected into K562 cells. As shown in Fig. 5, the 374-bp HindIII-XbaI fragment of HS2 (construct 1) was able to significantly enhance transcriptional activity of the beta SSE-promoter. Similarly, with the 5' half of the HindIII-XbaI fragment (which contains an erythroid-specific footprint and a Sp1 binding site) or the 3' half (which contains the binding sites for GATA-1, USF, SCL, and YY1) significant enhancer activity was observed in the presence of the SSE. These findings suggest that no individual site or combination of sites flanking the AP1/NF-E2 element is absolutely required for enhancer activity.


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Fig. 5.   Effect of site deletion in HS2 on transcriptional synergy with the SSE. Indicated fragments of HS2 were linked to either the beta CAT reporter construct or the beta SSECAT. Reporter gene activity of these constructs was measured as CAT conversion per microgram of protein. Values obtained represent the mean of at least six separate transfections with at least two different plasmid preparations. Data is presented as a ratio beta SSECAT activity to beta CAT activity. beta SSECAT activity was significantly different from beta CAT activity for experiments one to three (p < 0.05).

Maximal HS2 Enhancer-dependent Transcription of the beta -promoter Is Dependent on Binding of EKLF-- The inability of the binding sites flanking the AP1/NF-E2 element to augment beta -promoter activity in K562 cells coupled with the observation that these sites are essential for high level beta -gene expression in adult erythroid cells suggested that a developmentally specific protein may also be necessary for maximal enhancer-dependent transcription of the beta -promoter. To assess this, HS2 or HS2M was linked to the wild-type beta -promoter/CAT gene and transfected into the adult erythroid cell line, MEL. An enhancerless beta -promoter served as a control. As seen in Fig. 6A, reporter gene activity was 5-fold higher with HS2M than with the promoter alone (constructs 1 and 2). In contrast to the results in K562 cells, an approximate 5-fold enhancement was observed in the presence of the sites flanking the AP1/NF-E2 motif (constructs 2 and 3) (p < 0.01). To determine whether the adult stage-specific factor EKLF was essential for maximal enhancer activity, we examined the effect of mutating the beta -promoter CACCC box on the expression of HS2beta CAT and HS2Mbeta CAT in MEL cells. Disruption of the EKLF binding site crippled expression from both promoters to undetectable levels (data not shown). To circumvent this problem, we utilized the observation that significantly higher levels of EKLF are observed in MEL cells than K562 cells (43). We examined whether maximal HS2 enhancer-dependent transcription of a linked beta -promoter could be restored in K562 cells overexpressing EKLF (Fig. 6B). A parental K562 cell line (EKLF-) or a line overexpressing EKLF (EKLF+) were transfected with HS2beta CAT, HS2Mbeta CAT, or beta CAT alone (Fig. 6C). beta -promoter activity in the absence of the enhancer fragment was increased 2.5-fold in the EKLF+ line compared with the parental line (Fig. 6C, (1)). The addition of HS2M yielded a similar increase in beta -promoter activity in both parental and EKLF-overexpressing lines (Fig. 6C, compare (1) with (2)). In contrast, the addition of the HS2 fragment containing sites flanking the AP1/NF-E2 element demonstrated a marked differential effect between the two cell lines, with an 18-fold increase in the EKLF+ line (Fig. 6C, (3)) compared with a 4-6-fold increase in the parental line (Fig. 6C, compare (1) with (3)). Similar activation was observed in the context of an HS2 fragment in which the CACCC motif had been mutated indicating that binding of EKLF to the enhancer is not required for this effect (data not shown).


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Fig. 6.   Enhancement of beta -promoter activity by HS2 is dependent on EKLF. A, diagrammatic representation of the constructs used for transfection into MEL cells induced with Me2SO and their reporter gene activity. Solid boxes represent the 734-bp HindIII-BglII fragment of HS2 (construct 3) or the HS2M enhancer (construct 2). The hatched boxes represent the -385 beta -promoter linked to the CAT gene. In the lower panel, hatched bars represent CAT conversion standardized per microgram of protein. Values obtained represent the mean of at least six separate transfections with at least two different plasmid preparations. Statistical significance was assessed using Student's t test. B, Western analysis of K562 lines overexpressing FLAG-tagged EKLF. Nuclear extract (10 µg) from parental K562 cells (K562), K562 cells transfected with the expression vector pCI alone (pCI), or K562 cells transfected with pCIEKLF tagged with the FLAG epitope (two clones) was electrophoresed on a 10% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane and immunoblotted with a monoclonal anti-FLAG antibody. Migration position of molecular weight standards is shown on the left. C, diagrammatic representation and reporter gene activity of constructs transfected into parental (-) or EKLF-overexpressing (+) K562 cell lines. Constructs are as detailed in A. A representative example of CAT activity is shown. Values shown below are the fold activation of the constructs observed in each cell line and represent the mean of at least six separate transfections with at least two different plasmid preparations. Statistical significance was assessed using Student's t test.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The studies presented here explore the mechanisms underlying enhancer-dependent transcription of a linked promoter. Utilizing elements in the human beta -globin locus as a model, we have demonstrated that the maximal activity of HS2 is dependent on the binding of multiple erythroid and ubiquitous factors to the enhancer. It is also dependent on the binding of the erythroid-specific factors EKLF and SSP to the linked globin promoters. By virtue of the restricted expression pattern of these factors, maximal enhancer-dependent activation of the beta - and gamma -globin promoters is confined to adult or fetal erythroid cell lines, respectively. EKLF and SSP appear to act in synergy with the enhancer-binding proteins flanking the AP1/NF-E2 binding site to maximize the transcriptional activation of the globin promoters by HS2.

Human HS2 represents an ideal element for the study of enhancer/promoter interactions. Like many enhancers it requires multiple binding motifs within its core to mediate positive effects on expression of linked genes in transfected cells or transgenic mice (30-32). As seen in our studies and those of other investigators, the 46-bp element containing the AP1/NF-E2 binding site is essential for enhancer function and inducibility (26, 31). However, disruption of the sites flanking the AP1/NF-E2 element reduce enhancer function to varying degrees suggesting that HS2 functions as a multiprotein enhanceosome. Data from our studies, and others, indicate that no single site outside the 46-bp element is critical for enhancer effects as the reduction in activity seen with mutation of footprinted sites are modest (31-33). Studies in transgenic mice and MEL cells show that maximal enhancement of beta -gene expression is only observed in the presence of all binding sites (31, 44-46). Replacement of the sites in HS2 that flank the AP1/NF-E2 element with multiple AP1/NF-E2 sites results in dramatic reduction in enhancer activity, suggesting that a specific cooperativity exists between all enhancer binding proteins (31).

The importance of promoter sequences in transcriptional activation of the beta -gene by the LCR has been previously demonstrated in MEL cells. These studies identified TATA, CACCC, and CAAT boxes as sequences critical for LCR responsiveness. Although enhanced beta -gene expression was observed in the presence of the TATA box alone, maximal enhancement was dependent on the presence of all three sites (47). Our findings suggest that the binding sites for the stage-specific proteins EKLF and SSP in their respective promoters are important for maximal enhancement by HS2. The increase in transcriptional activation observed in the presence of these factors cannot solely be attributed to their binding regulatory sequences, as significant differential effects are observed in the presence or absence of the wild-type enhancer. Truncation of the SSE in the presence of the HS2M does not alter the level of promoter activity and the addition of this element to the beta -promoter has no effect in the absence of the fully active HS2. Similarly, the transcriptional activation of the beta -promoter observed with EKLF expression in K562 cells is markedly different in the presence of the wild-type enhancer. Thus maximal enhancer activity appears to depend on EKLF or SSP and the factors binding to sites adjacent to the AP1/NF-E2 motif.

Although the studies presented here utilize HS2, the studies of Morley et al. (28) indicate that this site alone is sufficient for developmentally specific expression of the human globin genes in transgenic mice. This effect may be due to functional homology between the sites mediated through the conservation of topology of protein binding sites in all human HS and in the corresponding HS in mouse, chicken, and rabbit (48-50). Our observations with HS2 may therefore be representative of the interactions between the globin gene promoters and the complete LCR.

Despite the fact that HS2 is situated immediately adjacent to the globin promoters in these and other studies, it appears that this element does not simply function as an extension of the promoter in this context (31). This is most evident in constructs in which the AP1/NF-E2 site has been mutated and transcriptional activation by the entire enhancer is lost. Similarly, replacement of sites flanking the AP1/NF-E2 motif with alternate transcriptional activator sites significantly reduces promoter enhancement (31). The effects of the sites flanking the AP1/NF-E2 motif do not appear to be secondary to the topology of the plasmids as results with supercoiled DNA were identical to those observed with linearized plasmids (data not shown). Similarly, the presence of flanking chromatin did not influence cooperative effects of the enhancer bound proteins and EKLF or SSP as stable transfectants reproduced the results we observed in the transient assays (Fig. 3B and data not shown).

The mechanisms governing the recruitment of multiprotein enhanceosomes to individual promoters remains unclear. We have recently demonstrated a protein/protein interaction between NF-E2 and hTAFII130, which allows juxtaposition of the LCR and proximal globin promoters (51). However, we have been unable to define a similar direct interaction between enhancer-bound proteins outside the AP1/NF-E2 motif and EKLF or SSP, suggesting that the stage-specific transcriptional activation observed with these factors is secondary to a separate mechanism.2 An alternate hypothesis to explain our findings is that binding of the stage-specific factors to their cognate sites increases the accessibility of the enhancer-bound factors to the transcriptional machinery assembled on the promoter. In support of this is our recent evidence that EKLF interacts with cAMP response element-binding protein, a co-activator putatively involved in chromatin remodeling.3 Interestingly, mice nullizygous for EKLF demonstrate loss of the hypersensitivity site that forms on the beta -promoter during definitive erythropoiesis. The EKLF/cAMP response element-binding protein interaction may result in displacement of core histones allowing recruitment of the basal transcriptional machinery and subsequent interaction between this complex and enhanceosome-bound proteins.

    ACKNOWLEDGEMENTS

We thank A. W. Nienhuis for continuing support and helpful comments on the manuscript. We thank Jim Bieker for the EKLF cDNA and N. Tran and A. Becher for excellent technical assistance.

    FOOTNOTES

* This work was supported by the National Health and Medical Research Council of Australia, the Wellcome Trust (to S. M. J.), National Institutes of Health Grant PO1 HL53749-03, Cancer Center Support CORE Grant P30 CA 21765, an ASH Junior Faculty Scholars Award (to J. M. C.), and the American Lebanese Associated Charities (ALSAC).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.

Dagger Joint senior authors of this work.

To whom correspondence should be addressed.

1 The abbreviations used are: HS, hypersensitivity site; LCR, locus control region; SSP, stage selector protein; SSE, stage selector element; EKLF, erythroid Kruppel-like factor; bp, base pair(s); CAT, chloramphenicol acetyltransferase.

2 J. M. Cunningham and S. M. Jane, unpublished material.

3 S. Pattinson, S. M. Jane, and J. M. Cunningham, unpublished material.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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