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Mol Cell Biol, January 1998, p. 102-109, Vol. 18, No. 1
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Activation of beta -Globin Promoter by Erythroid Krüppel-Like Factor

Haruhiko Asano and George Stamatoyannopoulos*

Division of Medical Genetics, University of Washington, Seattle, Washington

Received 6 February 1997/Returned for modification 21 March 1997/Accepted 2 October 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the beta -globin gene CACCC box and is essential for beta -globin gene expression. EKLF does not activate the gamma  gene, the CACCC sequence of which differs from that of the beta  gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the beta  gene by EKLF, the CACCC boxes of beta  and gamma  genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the beta promoter carrying a gamma  CACCC box at a level comparable to that at which it activated the wild-type beta  promoter, whereas EKLF failed to activate a gamma  promoter carrying the beta  CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the beta  promoter carrying the gamma  CACCC box, or that EKLF activated the mutated beta  promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the beta  or gamma  promoter determined EKLF specificity, the proximal beta  CACCC box sequence was created at the position of the beta  promoter (-140) which corresponds to the position of the CACCC box on the gamma  promoter. Similarly, the beta  CACCC box was created in the position of the gamma  promoter (-90) corresponding to the position of the CACCC box in the beta  promoter. EKLF retained weak activation potential on the beta -140CAC promoter, whereas EKLF failed to activate the gamma -90beta CAC promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the beta  promoter by EKLF is determined by the overall structure of the beta  promoter rather than solely by the sequence of the beta  gene CACCC box.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The programmed expression of globin genes is tissue and developmental stage specific. In humans, five beta -like globin genes (varepsilon , Agamma , Ggamma , delta , and beta ) form a cluster on the short arm of chromosome 11, and their expression is characterized by two major switches initially from embryonic (varepsilon ) to fetal (Agamma and Ggamma ) and subsequently to adult (delta  and beta ) globin gene expression (28). Although a number of cis-acting elements of globin genes and corresponding trans-acting factors have been identified (7, 19), the precise molecular mechanisms of globin gene regulation are still unclear.

The CACCC (or GT) box is a cis-acting element found in a variety of genes expressed in erythroid and nonerythroid tissues. Each beta -like globin gene (except the delta  gene) has one or two CACCC boxes among the conserved promoter sequences. The importance of the CACCC box for beta -globin gene transcription has been demonstrated by the existence of naturally occurring mutations in the proximal CACCC box which cause beta + thalassemias (13, 20, 21). The importance of the gamma  gene CACCC box is shown by the finding that gamma  gene transcription is reduced when the gamma  gene CACCC box is deleted (4, 14, 27, 33) and by in vivo footprinting studies showing significant protein binding in the gamma  CACCC sequence of gamma  gene-expressing cells (11).

Among the proteins binding to globin gene CACCC boxes, Sp1, a ubiquitous protein (12), and erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific (16) Krüppel-like zinc finger protein, are well characterized. Sp1 is known to interact with the varepsilon  (37), gamma  (9), and beta  (10) gene CACCC boxes, but its in vivo role for globin gene transcription remains unknown. EKLF binds to the proximal beta  gene CACCC element (2, 16), which enables EKLF to increase the beta  gene promoter activity in vitro (6). Disruption of the EKLF gene results in a beta -thalassemia-like phenotype characterized by lethality of the EKLF-/- mouse embryos beyond embryonic day 15 due to the deficient beta -globin production (18, 23). Similarly, EKLF-deficient mice carrying human beta -globin loci cannot express the human beta -globin gene but display no reduction in gamma  gene expression, indicating that beta  but not gamma  gene production is dependent on EKLF (22, 36). Thus, EKLF preferentially activates the beta  gene instead of the gamma  gene, despite the fact that in the mouse, EKLF is expressed in primitive erythroid cells as well as definitive erythroid cells (26).

Currently, the preferential activation of the beta  gene by EKLF is attributed to its binding affinity to the target DNA sequences. EKLF binds to an extended 9-bp CACCC box sequence (CCA CAC CCT), which can be recognized by the three zinc fingers of EKLF (2, 16). The analogous CACCC box sequence of the gamma  gene promoter is CTC CAC CCA. The CACCC box sequence of the beta  gene shows an affinity to EKLF that is eightfold higher than that of the CACCC box of the gamma  gene (6). However, there is no evidence that the binding affinity of EKLF to the gamma  CACCC box is low enough to ablate the gamma  gene activation by EKLF. beta  CACCC box sequences carrying a point mutation known to produce a beta + thalassemia show a binding affinity for EKLF that is 40- to 100-fold lower than that of the wild-type beta  CACCC box sequence (8), i.e., much lower than the binding affinity of EKLF for the 9-bp sequence of the gamma  CACCC box. Other reasons, in addition to the decreased affinity, may account for the lack of activation of the gamma  gene promoter by EKLF.

The purpose of this study was to test whether the difference in the 9-bp CACCC box sequence between the beta  and gamma  gene promoters is the sole determinant of the preferential beta  gene activation by EKLF. Our results show that the selective activation of the beta  gene by EKLF is dependent on the whole promoter context of the beta -globin gene rather than exclusively on the sequence of the beta gene CACCC box.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid constructions. pHS2gamma beta CACLuc, containing the beta  gene CACCC box in the gamma  promoter, and pHS2beta gamma CACCAT, containing the gamma  gene CACCC box in the beta  promoter, were generated by using the Altered Sites II in vitro mutagenesis system (Promega). Briefly, a KpnI-BamHI fragment of pHS2gamma Luc (a generous gift from Tim M. Townes) and a PstI-BamHI fragment of pHS2beta CAT (also a gift from Tim M. Townes) were subcloned into KpnI- and BamHI-digested and PstI- and BamHI-digested pALTER-1 vectors, respectively. gamma and beta  CACCC boxes were substituted by beta  and gamma  CACCC boxes, respectively, by using 5' phosphorylated oligonucleotides 5'-pGATTGGCCAACCCATGGGTGGAGTTCCACAGGGTGA-3' and 5'-pGTCCCTGGCTAAGCCACACCCTTGGGTTGGCCAG-3', respectively. After a mutagenesis reaction, a KpnI-BamHI fragment with a gamma  promoter containing a beta  CACCC box and a PstI-BamHI fragment with a beta  promoter containing a gamma  CACCC box were put back into KpnI- and BamHI-digested pHS2gamma Luc and PstI- and BamHI-digested pHS2beta CAT, respectively. Similarly, plasmids containing a beta  promoter with point mutations which cause beta + thalassemia (pHS2beta -88mutCAT, pHS2beta -87mutCAT, and pHS2beta -86mutCAT) were constructed from pHS2beta CAT. pHS2beta gamma CACDelta dCACCAT, in which the distal CACCC box sequence of the beta  promoter was disrupted and the proximal CACCC box sequence was substituted by the gamma  CACCC sequence, was derived from pHS2beta gamma CACCAT. pHS2beta -140CACCAT, in which the proximal beta  CACCC box sequence was moved to the gamma  CACCC box position, was prepared from gamma  CACCC sequence-disrupted pHS2beta gamma CACDelta dCACCAT (pHS2beta Delta dpCACCAT). pHS2gamma -90beta CACLuc, in which the proximal beta  CACCC box sequence was generated at the position where it is located in the beta  promoter, was prepared from gamma  CACCC sequence-deleted pHS2gamma Luc (pHS2gamma Delta CACLuc). 5' phosphorylated oligonucleotides used for these plasmid constructions are 5'-pGCC AACCCTAGGATGTGGCTCCACA-3', 5'-pGGCCAACCCTAGCGTGTGGCTCCAC-3', 5'-pTGGCCAACCCTACGGTGTGGCTCCA-3', 5'-pGTGGAGTTCCACACTAGTAGGTCTAAGTGAT-3', 5'-pATTGGCCAACCCTTCATATGAGTTCCACACTAGT-3', 5'-pCGTACCTGTCCTTGAGGGTGTGGAGCTCTTCTGGCACT-3', 5'-pGTCCCTGGCTAAATGGGTTGGCCAG-3' and 5'-pCAAACTTGACCAATACCACACCCTAGGTCTTAGAGTATCCA-3' for pHS2beta -88mutCAT, pHS2beta -87mutCAT, pHS2beta -86mutCAT, pHS2beta gamma CACDelta dCAC CAT, pHS2beta Delta dpCACCAT, pHS2beta -140CACCAT, pHS2gamma Delta CACLuc, and pHS2gamma -90beta CACLuc, respectively. Plasmids with mutations were verified by DNA sequencing by using a kit (Cyclist; Stratagene).

Transactivation analysis. CV-1 cells and K562 cells were cultured in Eagle's minimal essential medium and RPMI 1640, respectively, supplemented with 10% fetal calf serum. Transient transfections of CV-1 cells were performed by the calcium phosphate coprecipitation method (1). Briefly, 1.8 × 105 CV-1 cells were plated in a 6-cm-diameter culture dish 24 h prior to transfection; 3.6 ml of fresh complete medium was added 2 to 4 h before transfection. A DNA mixture containing 3.6 µg (except where indicated otherwise) of each activator, reporter, and pSVbeta -Gal control vector (Promega) was ethanol precipitated, rinsed with 80% ethanol, air dried, dissolved in 180 µl of distilled H2O, and mixed with 20 µl of 2.5 M CaCl2. The DNA solution was mixed with 200 µl of 2× HEPES-buffered saline and added to the culture medium. After overnight incubation, cells were glycerol shocked. The cells were completely washed, further incubated for 24 h in the complete medium, and then lysed in 325 µl of reporter lysis buffer (Promega).

Transient transfection of K562 cells was performed with reporter, expression (10 times greater molar amount of the reporter plasmid), and pSG5 vectors (to total 40 µg) and 10 µg of pSVbeta -Gal. Log-phase cells (3 × 107 to 4 × 107) in RPMI 1640 medium were electroporated at 960 µF and 320 mV (Bio-Rad Gene Pulser). After standing at room temperature for 10 min, the cells were plated in 10 ml of the complete medium, incubated at 37°C for 24 h, and then harvested. The cell extracts were prepared in 400 µl of reporter lysis buffer. Aliquots (100 µl) of the extracts, which had been diluted 1:10 in the EKLF samples, were heat inactivated and assayed for chloramphenicol acetyltransferase (CAT) activities by the phase extraction method (25). For luciferase assays, the cell extracts were diluted 1:10, and 100-µl aliquots were analyzed by using the (Promega) luciferase assay system.

All transfection assays were performed multiple times and with different preparations of the same plasmid. CAT and luciferase activities obtained were corrected for transfection efficiencies by beta -galactosidase (beta -Gal) A405.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

EKLF activates a beta -globin gene promoter which contains the gamma  CACCC box sequence. EKLF interacts with the beta -globin gene CACCC box by recognizing the 9-bp sequence CCA CAC CCT (2, 16). The sequence of the CACCC box of the gamma -globin promoter is CTC CAC CCA. To test whether the 9-bp CACCC box sequence difference between beta - and gamma -globin gene promoters is the sole determinant of the selective function of EKLF on the beta  gene promoter, we substituted the original beta  gene CACCC box sequence of pHS2beta CAT with the gamma  gene CACCC box sequence. The resultant construct was designated pHS2beta gamma CACCAT (Fig. 1).


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  		FIG. 1.   (A) Structure of pHS2beta gamma CACCAT, containing a beta  gene promoter with a gamma  CACCC box. The CAT gene is driven by a 1.5-kb KpnI-BglII fragment of HS2 and the beta  gene promoter (a fragment extending from bp -265 to +48 relative to the cap site) carrying a gamma  CACCC box. Uppercase letters denote the 9-bp gamma  CACCC sequence analogous to the beta  CACCC sequence recognized by EKLF. Numbers above the gamma  CACCC box sequence show base pair distances from the cap site. (B) Transactivation of a beta  promoter containing a gamma  CACCC box by EKLF. CAT activities in CV-1 cells were normalized to beta -Gal activity and expressed as relative percentages of CAT activity of pHS2beta CAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are expressed as mean (columns) ± SD (error bars) derived from four independent transfections using two different plasmid sets. Notice that EKLF transactivates the beta  gene promoter despite the fact that the promoter contains the gamma  CACCC box.

The reporter constructs pHS2beta CAT and pHS2beta gamma CACCAT and the activator plasmid pSG5/EKLF were transiently cotransfected into CV-1 cells with plasmid pSVbeta -Gal as an internal control of transfection efficiency. CV-1 is an established cell line derived from monkey kidney and has been previously used by Bieker and Southwood (3) to evaluate the activity of EKLF on globin gene promoters. As shown in Fig. 1, EKLF increased the activity of the beta  gene promoter of pHS2beta CAT (i.e., the construct containing the normal CACCC box) by roughly 800% of the control value. Notice that EKLF activated the beta gamma CAC gene promoter as effectively as the wild-type beta  promoter, even though the beta gamma CAC gene promoter contains the gamma  CACCC box instead of the beta  CACCC box. The average CAT activities driven by the beta  promoter carrying the gamma  CACCC box sequence were 97% (without EKLF) and 960% (with EKLF) relative to that driven by pHS2beta CAT in the absence of EKLF stimulation (taken as 100%).

These findings suggested that EKLF functions in the context of the whole beta  gene promoter rather than exclusively through its affinity to the 9-bp sequence of the beta  CACCC box.

EKLF fails to activate a gamma -globin gene promoter which contains the beta  CACCC box sequence. If the 9-bp beta  CACCC box sequence has a critical role for EKLF function, we would expect EKLF to activate a gamma -globin gene promoter containing the beta  CACCC box sequence instead of the gamma  CACCC box sequence. To test this possibility, we substituted the gamma  gene CACCC box sequence of pHS2gamma Luc with the beta  gene CACCC box sequence. The resultant construct was designated pHS2gamma beta CACLuc (Fig. 2). The reporter constructs pHS2gamma Luc and pHS2gamma beta CACLuc, plus pSG5/EKLF and pSVbeta -Gal, were transfected into CV-1 cells.


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  		FIG. 2.   (A) Structure of the construct pHS2gamma beta CACLuc containing a gamma  gene promoter with a beta  CACCC box. The luciferase gene is driven by the 1.5-kb fragment of HS2 and the gamma  gene promoter (bp -299 to +37 relative to the cap site) carrying a beta  CACCC box. Uppercase letters denote the 9-bp beta  sequence recognized by EKLF. Numbers above the beta  CACCC box sequence show base pair distances from the cap site. (B) Transactivation of a gamma  promoter containing a beta  CACCC box by EKLF. Luciferase activities in CV-1 cells were normalized to beta -Gal activity and expressed as relative percentages of luciferase activity of pHS2gamma Luc in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that EKLF cannot transactivate the gamma  gene promoter despite the fact that the promoter contains the beta  CACCC box.

As shown in Fig. 2, in the presence of EKLF, the average luciferase activity derived from pHS2gamma Luc was 120% of the activity obtained in the absence of EKLF (100%). Thus, EKLF did not activate the gamma  gene promoter. The average luciferase activities from pHS2gamma beta CACLuc were 58% (without EKLF) and 92% (with EKLF) relative to the activity obtained from pHS2gamma Luc without EKLF (100%). Therefore, EKLF failed to activate the gamma beta CAC gene promoter, although the gamma beta CAC gene promoter carried the beta  CACCC box sequence, an optimal EKLF binding site. These results provided further evidence that the 9-bp beta  CACCC box sequence is not enough to mediate beta -globin gene-specific EKLF function.

The effects of EKLF on the beta gamma CAC and gamma beta CAC promoters are reproduced in the erythroid environment. The results described above were obtained in transactivation assays using a nonerythroid line, CV-1. It was possible that these results reflected the lack of other transcriptional factors which are present in erythroid cells. For these reasons, we repeated the transient transfection assays with K562 cells, a human erythroleukemia line which exhibits an embryonic/fetal globin phenotype. There is no endogenous beta  gene expression in K562 cells, but beta  gene constructs linked to locus control region cassettes or to DNase I-hypersensitive site 2 (HS2) display efficient beta  gene transcription (34, 38). The reporter constructs pHS2beta CAT, pHS2gamma Luc, pHS2beta gamma CACCAT, and pHS2gamma beta CACLuc, plus pSG5/EKLF and pSVbeta -Gal, were transiently transfected into the K562 cells.

As shown in Fig. 3, EKLF activated pHS2beta CAT and pHS2beta gamma CACCAT to similar degrees. The average CAT activities driven by beta  and beta gamma CAC in the presence of EKLF were 1,062 and 936%, respectively, relative to that of pHS2beta CAT in the absence of EKLF (100%). As in the experiments with CV-1 cells, substitution of the CACCC box of the gamma  promoter by the beta  CACCC box did not increase the effect of EKLF on the gamma  promoter. The average luciferase activities stimulated by EKLF were 292% (pHS2gamma Luc) and 235% (pHS2gamma beta CACLuc); thus, the beta  CACCC box-containing gamma  promoter was not activated by EKLF more than the wild-type gamma  gene promoter. These results provide further evidence that CACCC box recognition is not the sole determinant of EKLF activity.


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  		FIG. 3.   Studies using K562 cells. Results of transactivation by EKLF of a beta  promoter carrying a gamma  CACCC box and a gamma  promoter carrying a beta  CACCC box are depicted. CAT and luciferase activities were normalized to beta -Gal activity and expressed as relative percentages of CAT and luciferase activities of pHS2beta CAT and pHS2gamma Luc in K562 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that the CACCC box substitutions do not influence the level of activation of the beta  or gamma  gene promoter by EKLF.

The pattern of promoter activation by EKLF in K562 cells (Fig. 3) is very similar to that in CV-1 cells (Fig. 1 and 2), indicating that the results observed in CV-1 cells are not an artifact caused by the nonerythroid environment. Hence, we used CV-1 cells in all subsequent experiments.

Activation of the beta gamma CAC promoter by EKLF cannot be attributed to EKLF overexpression. Since the transactivator gene used in a transient expression system is generally overexpressed for full activation of the reporter gene, the results described above, especially those for beta gamma CAC gene promoter activation by EKLF, could be attributed to overexpression of EKLF. We examined the relationship between the transfected amount of pSG5/EKLF and the CAT activity from pHS2beta CAT to (i) determine whether EKLF is overexpressed under the experimental condition that we used and if so (ii) find transfection conditions which are not associated with pSG5/EKLF overexpression.

Amounts of pSG5/EKLF ranging from 0 to 3.6 µg were cotransfected with pHS2beta CAT and pSVbeta -Gal into CV-1 cells. Results are shown in Fig. 4. The highest CAT activity obtained from cells transfected with 3.6 µg of pSG5/EKLF was taken as 100%, and CAT activities obtained with the lower concentrations of pSG5/EKLF were expressed as percentages of this highest activity. CAT activity exhibited a plateau between 0.7 and 3.6 µg of pSG5/EKLF (Fig. 4), indicating that EKLF was overexpressed in our previous CV-1 transfection studies in which 3.6 µg of pSG5/EKLF was used. Between 0 and 0.7 µg, CAT activity increased toward a plateau level along with the increase in the amount of pSG5/EKLF. Thus, transfection using less than 0.7 µg of pSG5/EKLF does not produce full activation of the beta  gene promoter and is considered to give rise to unsaturated EKLF expression in CV-1 cells.


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  		FIG. 4.   Relationship between amounts of transfected activator plasmid and degree of activation of the reporter gene in CV-1 cells. The reporter construct, pHS2beta CAT, was cotransfected with various amounts of pSG5/EKLF. CAT activities were normalized to beta -Gal activity and expressed as relative percentages of CAT activity of pHS2beta CAT transfected with 3.6 µg of pSG5/EKLF (100%). Average values ± SD (error bars) were derived from three independent transfections using two different plasmid sets. Notice that CAT activities show two phases, ascending (0 to 0.7 µg of EKLF plasmid) and plateau (0.7 to 3.6 µg of EKLF plasmid).

To test whether pSG5/EKLF overexpression enabled EKLF to activate the beta  gene promoter carrying the gamma  CACCC box instead of the beta  CACCC box, we repeated the transient transfection experiments with CV-1 cells, with 0.5 µg of pSG5/EKLF as a transactivator. The amount of the EKLF plasmid used should create unsaturated EKLF expression in the cells.

As shown in Fig. 5, transfection of 0.5 µg of pSG5/EKLF activated the beta  and beta gamma CAC gene promoters to similar degrees. The average CAT activities of pHS2beta CAT and pHS2beta gamma CACCAT stimulated by EKLF were 639 and 601%, respectively, relative to that of pHS2beta CAT lacking EKLF stimulation (100%). Thus, the activation by EKLF of a beta  promoter carrying the gamma  CACCC box sequence was consistently comparable to that of the beta  gene promoter containing the beta  CACCC box. These data suggest that pSG5/EKLF overexpression is not the cause of activation of the beta gamma CAC promoter by EKLF.


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  		FIG. 5.   Transactivation of the beta  promoter containing a gamma  CACCC box by a small amount of pSG5/EKLF (0.5 µg per transfection). CAT activities in CV-1 cells were normalized to beta -Gal activity and expressed as relative percentages of CAT activity of pHS2beta CAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from three independent transfections using two different plasmid sets. Notice that EKLF transactivates the beta  gene promoter containing the gamma . CACCC box, a result which is similar to the results of assays using a standard amount (3.6 µg) of pSG5/EKLF (Fig. 1).

The distal CACCC box of the beta -globin promoter is not the cause of activation of the beta  promoter containing a gamma  CACCC box sequence. The beta  gene promoter has two CACCC boxes, one proximal and one distal, at bp -90 and -105 relative to the cap site; EKLF recognizes the proximal (-90) CACCC box sequence (16). A possible interpretation of our findings that EKLF can activate a beta  promoter carrying a gamma  CACCC box is that in the absence of the wild-type beta  gene CACCC box at -90, EKLF interacts with the distal CACCC box, resulting in beta  promoter activation. This interpretation is unlikely because naturally occurring mutations of the proximal CACCC box of the beta  gene cause beta + thalassemia (35), and one of these promoter mutations has been shown by Donze et al. (6) to significantly decrease beta  gene promoter activation by EKLF in transient expression assays; therefore there is evidence that the distal CACCC box contributes minimally, if at all, to beta  gene activation.

To test whether there is functional interaction between EKLF and the distal CACCC box under the conditions of our experiments, we generated three types of point mutations in the proximal beta gene CACCC box: -88 (relative to the cap site) Cright-arrowT (pHS2beta -88mutCAT), -87 Cright-arrowG (pHS2beta -87mutCAT), and -86 Cright-arrowG (pHS2beta -86mutCAT). Reporter plasmids carrying these mutations, 3.6 µg of EKLF plasmid, and pSVbeta -Gal were transiently cotransfected into CV-1 cells. Absolute promoter activities were decreased to similar degrees in all three mutated beta  promoters. The CAT activities with and without EKLF were 151% ± 23% (mean ± standard deviation [SD]) and 20% ± 7%, respectively, for pHS2beta -88mutCAT, 109% ± 15% and 25% ± 5% for pHS2beta -87mutCAT, and 148% ± 10% and 18% ± 7% for pHS2beta -86mutCAT. In the same experiments, EKLF activated the wild-type beta  promoter of pHS2beta CAT to 634% ± 81% of the control without EKLF (100%). These results indicated that the lack of functional interaction between EKLF and the distal CACCC box is also observed under the experimental condition that we have used.

To test the role of the distal CACCC element more directly, we disrupted the distal CACCC box of the pHS2beta gamma CACCAT construct by producing the nucleotide substitutions CCT CAC CCTright-arrowCCT ACT AGT; the resulting construct was designated as pHS2beta gamma CACDelta dCACCAT (Fig. 6A). When they are introduced in the comparable residues in the proximal CACCC box, the point mutations denoted by the underlined three C residues cause thalassemias and reduce the interaction of the CACCC box with EKLF. The reporter constructs pHS2beta gamma CACCAT and pHS2beta gamma CACDelta dCACCAT, plus pSG5/EKLF and pSVbeta -Gal, were transfected into CV-1 cells. The transfected amount of the EKLF plasmid was 0.5 µg in this experiment. CAT activity was increased about sixfold by the addition of EKLF in both pHS2beta gamma CACCAT and pHS2beta gamma CACDelta dCACCAT compared to that of pHS2beta gamma CACCAT without EKLF (100%), although the variation was relatively large (Fig. 6B). Therefore, EKLF activated the beta gamma CAC promoter with a disrupted distal CACCC box, even though this promoter had no original beta  CACCC box sequences. This finding provides direct evidence that the presence of an intact distal CACCC box is not the cause of activation of the gamma  CACCC box-containing beta promoter by EKLF.


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  		FIG. 6.   (A) Disruption of the distal CACCC box sequence (beta gamma CACDelta dCAC). The distal CACCC box sequence (CCT CAC CCT) of the beta  promoter carrying a gamma  CACCC box sequence (CTC CAC CCA) at the proximal CACCC site is altered to CCT ACT AGT. Numbers above the promoter sequences show base pair distances from the cap site. (B) Transactivation of a distal CACCC box-disrupted beta gamma CAC promoter by EKLF. A small amount of EKLF plasmid (0.5 µg) was used for transfection. CAT activities in CV-1 cells were normalized to beta -Gal activity and expressed as relative percentages of CAT activity of pHS2beta gamma CACCAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from three independent transfections using two different plasmid sets. Notice that in the presence of EKLF, similar levels of CAT activities were obtained from beta gamma CAC and beta gamma CACDelta dCAC.

The position of the CACCC box is not critical for activation of the beta  gene promoter by EKLF. The results described above demonstrate that the difference in the 9-bp CACCC box sequence between beta  and gamma  gene promoters is not a critical determinant of the specificity of the beta  gene activation by EKLF. As shown in Fig. 7, there are two major differences between the beta  and gamma  CACCC boxes: one is the position of the CACCC box relative to the cap site, and the other is the configuration of the surrounding cis elements. To test whether the position of the CACCC box confers the beta  gene specificity on EKLF, we generated a gamma  promoter which contained a beta  CACCC box sequence placed in the position in the beta  promoter (i.e., 90 bp upstream from the cap site). We also generated a beta  promoter which contained a beta CACCC box in the position where the gamma  CACCC box is normally located in the gamma  promoter (i.e., 140 bp upstream from the cap site). The normal CACCC box sequence of each promoter was deleted or disrupted.


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  		FIG. 7.   Comparison of the locations of cis elements of the beta  and gamma  gene promoters. The positions of the functional CACCC box are shown by solid rectangles.

The construct pHS2gamma -90beta CACLuc, in which the original gamma  CACCC box was deleted and the proximal beta  CACCC box was created at exactly the same position as in the beta  promoter, is shown in Fig. 8A. The reporter constructs pHS2gamma Luc and pHS2gamma -90beta CACLuc, plus pSG5/EKLF and pSVbeta -Gal, were transiently transfected into CV-1 cells. If the position of the CACCC box sequence is an important determinant for selective activation of the beta  gene promoter by EKLF, we would expect activation of the gamma -90beta CAC gene promoter by EKLF. As shown in Fig. 8B, the average luciferase activity of pHS2gamma -90beta CACLuc was less than 10% of that of pHS2gamma Luc without EKLF stimulation (100%), and the addition of EKLF did not alter the low activity. Thus, EKLF failed to activate the gamma  gene promoter even though its optimal CACCC box sequence is placed at a distance from the transcription start site which is optimal for functioning in the beta  gene promoter. These results suggest that the beta -globin gene specificity of EKLF is not determined solely by the position of the CACCC box.


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  		FIG. 8.   Effect of the position of the CACCC box on activation of the gamma  gene promoter by EKLF. (A) Generation of a gamma  promoter containing a beta  CACCC box at bp -90 (gamma -90beta CAC). The original gamma  CACCC box sequence (CTC CAC CCA) was deleted, and the proximal beta  CACCC box sequence was inserted into position -90, i.e., the position of the beta  CACCC box in the beta  promoter. Numbers above the promoter sequences are base pair distances from the cap site. (B) Transactivation of a gamma  promoter containing a beta  CACCC box at position -90 (beta -90beta CAC) by EKLF. Luciferase activities in CV-1 cells were normalized to beta -Gal activity and expressed as relative percentages of luciferase activity of pHS2gamma Luc in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that the promoter activity is almost ablated by the CACCC box movement. In addition, EKLF cannot activate the mutant promoter even though this promoter contains an optimal binding sequence at its optimal site.

Figure 9A shows the reporter construct pHS2beta -140CACCAT, in which both proximal and distal CACCC boxes were disrupted and the proximal beta  CACCC box was created at exactly the same position as that where the gamma  CACCC box is located in the gamma  promoter. This CACCC box relocation decreased the basal CAT activity without EKLF stimulation to about 20% of that of pHS2beta CAT (100%). In contrast to the relocation of the CACCC box in the gamma  gene promoter, EKLF activated the beta -140CAC promoter to about 165% relative to the original beta  promoter without EKLF (100%) (Fig. 9B). Thus, the beta  promoter containing a relocated CACCC box (beta -140CAC) retained mild reactivity to stimulation by EKLF.


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  		FIG. 9.   Effect of the position of the CACCC box on the activation of the beta -globin gene promoter by EKLF. (A) Generation of a beta  promoter containing a CACCC box at bp -140 (beta -140CAC), i.e., the position of the CACCC box in the gamma  promoter. The proximal CACCC box sequence (CCA CAC CCT) and the distal CACCC box sequence (CCT CAC CCT) were disrupted by base substitutions (shown in underlined italics). Subsequently, the proximal CACCC box sequence was inserted into the gamma  CACCC position in the gamma  promoter. Numbers above the promoter sequences correspond to base pair distances from the cap site. (B) Transactivation of a beta  promoter containing a CACCC box at position -140 (beta -140CAC) by EKLF. CAT activities in CV-1 cells were normalized to beta -Gal activity and expressed as relative percentages of CAT activity of pHS2beta CAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that the promoter activity is remarkably decreased by the relocation of the CACCC box to a position equivalent to that of the CACCC box of the gamma  gene and that EKLF is still capable of weakly activating this mutant promoter.

These results suggest that the position of the CACCC box in the beta  or gamma  gene promoter is not the critical determinant of EKLF function. The relationship of the CACCC box with the surrounding cis elements and the interactions of EKLF with some other protein(s) binding to these elements, i.e., the overall context of the promoter, may determine the specificity of the activation of the beta -globin gene promoter by EKLF.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The purpose of this study was to investigate whether the 9-bp CACCC box sequence underlies the specificity of activation of the beta -globin gene by EKLF. To address this issue, we swapped the CACCC boxes between the beta  and gamma  gene promoters and analyzed the activities of EKLF on these mutated promoters by transient transfection assays. The results indicate that the CACCC box sequence of the beta  gene promoter is not the only determinant of the specific activation of the beta  gene by EKLF. We have further shown that factors such as the lack of an erythroid environment in the initial transactivation studies, the overexpression of EKLF in the transactivation assays, or the activation of the gamma  CACCC box-containing beta  gene promoter through its intact distal CACCC box cannot account for our results. Thus, the selective transcriptional activation of the beta -globin gene (compared to that of the gamma -globin gene) by EKLF (22, 36) is not due exclusively to the higher affinity of EKLF to the beta gene CACCC box sequence. Rather, the specificity of the activation of the beta -globin gene by EKLF is dependent on the whole promoter context of the beta -globin gene. Thus, just the protein-DNA interaction between the DNA-binding domain of EKLF and the beta  CACCC box is not sufficient to activate the beta -globin gene. Most likely, protein-protein interactions between the EKLF transactivator domain and the transcriptional complex are necessary to bring about the specific activation of the beta  gene promoter by EKLF.

Insights on the nature of the promoter context-dependent beta  gene activation by EKLF were provided by the experiments using beta  and gamma  gene promoters in which the positions of the CACCC boxes were interchanged. It is reasonable to assume that EKLF, like other transcriptional activators (32), interacts directly or indirectly with the basal transcriptional machinery. Our findings can be explained by assuming that when EKLF is tethered onto the beta  gene promoter, it interacts with the basal transcriptional machinery formed on the TATA box and flanking regions of the beta  gene, whereas when it is tethered onto the gamma  gene promoter by the beta  CACCC box, it cannot interact with the basal transcriptional machinery on the gamma  gene. If the beta  and the gamma  genes use the same basal transcriptional machinery, a simple explanation of why EKLF, although bound on the beta  CACCC box of the gamma beta CAC promoter, cannot activate gamma  gene transcription is that the position of the beta  CACCC box in the beta gamma CAC promoter does not allow EKLF to interact with the basal transcriptional machinery. However, our results of assays using a gamma  promoter carrying a beta  CACCC box at -90 and a beta  promoter carrying a beta  CACCC box at -140 (Fig. 8 and 9) do not agree with this hypothesis. Instead, our results argue that the location of the CACCC box relative to the transcription start site is not the critical determinant of the specificity of beta -globin gene activation by EKLF.

An alternative explanation of our findings is based on the recruitment model of action of transcriptional activators (24, 29). This model proposes that a transcriptional activator functions by recruiting the transcriptional machinery to the DNA (the regulatory motifs of the promoter). EKLF may act similarly and recruit a subcomplex of the basal transcriptional machinery to the beta -globin gene as illustrated in Fig. 10A. If this is so, the putative subcomplex recruited by EKLF must be unique and critical for the assembly of the basal transcriptional machinery on the beta  gene because disruption of the EKLF gene totally ablates beta  gene transcription but not gamma  gene transcription (22, 36). The nonresponsiveness of the gamma  gene to EKLF can be explained by assuming that the assembly of the basal transcriptional machinery of the gamma  gene does not utilize the putative subcomplex which is essential for the basal transcriptional machinery on the beta  gene (Fig. 10B). Instead, the basal transcriptional machinery on the gamma  gene requires a different subcomplex which can be recruited to the gamma  gene by a factor that interacts with the gamma  CACCC box (Fig. 10C).


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  		FIG. 10.   Proposed mechanism of beta -globin gene activation by EKLF. (A) EKLF bound to the beta  gene recruits a subcomplex of the transcriptional machinery and enables formation of a transcription initiation complex (IC) of the beta  gene together with TFIID containing TATA box-binding protein (TBP), RNA polymerase II (pol II) holoenzyme, and probably other subcomplexes recruited by other transcriptional activators. This initiation complex gives rise to high-level beta  gene transcription. (B) EKLF tethered to the gamma  gene by a beta  CACCC box also recruits the same subcomplex as described above. However, the EKLF-bound subcomplex is different from the subcomplex normally interacting with the gamma  gene transcriptional machinery and fails to assemble with other components, resulting in failure of initiation of gamma  gene transcription. (C) Putative gamma  CACCC box-binding factor recruits an appropriate subcomplex of the transcriptional machinery of the gamma  gene. The appropriate assembly of the initiation complex on the gamma  gene gives rise to high-level gamma  gene transcription.

The hypothesis that the beta  gene specificity of EKLF is dependent on two factors, protein-protein interaction(s) mediated by the transactivation domain and protein-DNA interaction mediated by the DNA-binding domain, was previously proposed by Bieker and Southwood (3). Although DNA-binding specificity is considered to be the main determinant of promoter specificity of transcriptional activators (17), there is also evidence, from studies of a limited number of transcription factors, that the transactivation domain may also critically influence the promoter specificity of a transcriptional activator (15, 31). Since the transactivator domain of EKLF is composed of multifunctional subdomains (5), it is possible that the DNA binding of EKLF is augmented by the activator domain as is the case of the Oct-2 POU DNA-binding domain (30). In that case, the activator domain of EKLF may play a dual role, i.e., increase the binding of EKLF on the CACCC box of the beta  promoter and recruit a component of the transcriptional machinery of the beta -globin gene.

    ACKNOWLEDGMENTS

This study was supported by NIH grants HL20899 and DK45365.

    FOOTNOTES

* Corresponding author. Mailing address: University of Washington, Division of Medical Genetics, Box 357720, Seattle, WA 98195. Phone: (206) 543-3526. Fax: (206) 543-3050. E-mail: gstam{at}u.washington.edu.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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