©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Roles of in Vivo Footprinted DNA Motifs within an -Globin Enhancer
ERYTHROID LINEAGE AND DEVELOPMENTAL STAGE SPECIFICITIES(*)

Qingyi Zhang , Irene Rombel , G. Narender Reddy , Jong-Back Gang , C.-K. James Shen(§)

From the (1) Section of Molecular and Cellular Biology, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Transcriptional regulation of the human -like globin genes, embryonic 2 and adult , during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5`NF-E2/AP1, GT II, and GATA-1(c), positively regulate the 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult -globin promoter activity in adult erythroid MEL cells. On the other hand, the 3`NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human -like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo.


INTRODUCTION

The human -like globin gene family is clustered on chromosome 16. The transcriptionally active members of the gene cluster are arranged in the order of their expression during erythroid development: 5`-2 (embryonic)-2 (fetal/adult)-1 (fetal/adult)-1 (fetal/adult, minor)-3` (Higgs et al., 1989). Of these members, 2 is transcribed predominantly in yolk sac, the embryonic erythroid tissue. As development proceeds, the expression of 2 is shut off, and the two -globin genes, 2 and 1, are turned on in fetal liver. Finally, in adult humans, the two -globin genes are expressed at high levels in nucleated erythroblasts originated from appropriate stem cells in the bone marrow. In the adult and fetal erythroblasts, however, low levels of 2 and 1 messages could still be detected (Albitar et al., 1989; Chui et al., 1989). The expression of these -like globin genes, similar to the -like globin family members, is regulated predominantly at the level of transcription (Collins and Weissman, 1984; Karlsson and Nienhuis, 1985).

Both genetic and molecular data (Higgs et al., 1990) have indicated that the appropriate erythroid lineage- and developmental stage-specific expression of the human -like globin gene family is, like the -like globin family (Orkin, 1990; Stamatoyannopoulos, 1991), modulated by the so-called locus control region (LCR).() The -LCR is located at approximately 40 kilobase pairs upstream of the 2-globin gene, and its functional domain has been mapped, by transgenic mice and DNA transfection studies, to a 300-bp sequence containing an erythroid-specific DNase I-hypersensitive site (HS-40). Similar to the regulation of the -like globin genes by -LCR, the -LCR or HS-40 confers high levels of erythroid-specific expression of linked human 2- or -globin genes in transgenic mice or stably integrated cell lines (Jarman et al., 1991; Sharpe et al., 1992; Vyas et al., 1992; Sharpe et al., 1993; Gourdon et al., 1994). It also behaves as a classical enhancer for the promoter activity of 2- or -globin genes in transiently transfected erythroid cell culture (Pondel et al., 1992; Ren et al., 1993; Zhang et al., 1993).

The molecular mechanisms of how the -LCR controls 2- or -globin gene transcription are not well understood. By analogy to what have been hypothesized for the -LCR function (Grosveld et al., 1987; Tuan et al., 1989; Evans et al., 1990; Forrester et al., 1990; Engel, 1993), the -LCR could play a dominant role in the formation of an active chromatin structure of the entire -like globin locus domain. It could also, like other eukaryotic gene enhancers, facilitate the assembly of active transcriptional initiation complexes at the 2- or -globin upstream promoters. All of these regulatory processes are likely to be modulated by diffusible nuclear factors, as suggested by previous chromosome transfer and cell fusion experiments (Deisseroth and Hendrick, 1979; Baron and Maniatis, 1986).

A number of nuclear factors bind in vitro to several different sequence motifs of the -LCR (Jarman et al., 1991; Strauss et al., 1992). Subsequent genomic footprinting work has demonstrated the formation in vivo of specific nuclear factor-DNA complexes at a subset of these sequence motifs in erythroid cells (Strauss et al., 1992; Zhang et al., 1993). Interestingly, a single set of nuclear factor binding motifs is found in the -LCR as well as in the -LCR (Fig. 1 A). These motifs include GATA-1, which binds a factor specifically expressed in erythroid, megakaryotic, and mast cell lineages (Martin et al., 1990; Romeo et al., 1990); the NF-E2/AP1 motif that can bind either NF-E2, which has a similar cell type specificity of expression as GATA-1, or the widely expressed AP1 factor (Andrews et al., 1993a, 1993b; Chan et al., 1993; Chang et al., 1993; Ney et al., 1993; Caterina et al., 1994; Igarashi et al., 1994); and the GT motif, which binds Sp1, USF, or TEF (Jarman et al., 1991; Ellis et al., 1993).

The functional contributions of these nuclear factor binding motifs to the enhancement of human embryonic 2-globin promoter activity by HS-40 have been partially analyzed before (Zhang et al., 1993). The results suggested that one of the four GATA-1 motifs, GATA-1(c) (Fig. 1), and both NF-E2/AP1 motifs played positive regulatory roles (Zhang et al., 1993). However, the mutant HS-40 sequences used in the above study were generated mainly by Bal31 deletion, which could remove more than one functional motif during successive deletion steps. This would have prohibited the definitive assignment of the positive/negative role of a particular motif. Furthermore, the previous analysis was carried out only with transfected K562 cells, a human erythroid cell line of embryonic/fetal origins (Lozzio and Lozzio, 1975). Thus, it did not allow us to assess the functional significance of the developmental stage-specific difference of nuclear factor binding patterns within HS-40, as seen in previous genomic footprinting analysis of HS-40 in adult versus embryonic/fetal erythroid cells (Strauss et al., 1992; Zhang et al., 1993).


Figure 1:A, nuclear factor binding motifs of HS-40. The motifs that bind nuclear factors in vitro are indicated by the blank boxes on the left map. Those motifs that bind nuclear factors in vivo in K562 cells ( K) and human adult erythroblasts ( E) are indicated by the filled or hatched boxes. Genomic footprints are similar for motifs with filled boxes, while those of the hatched motifs are different between the two types of erythroid cells. The three GATA-1 motifs shown are GATA-1(b), GATA-1(c), and GATA-1(d), respectively, as arranged on HS-40 from left to right. The two NF-E2/AP1 motifs are 5`NF-E2/AP1 and 3`NF-E2/AP1, also arranged from left to right. The single GT motif is termed GT II throughout the text. The maps are constructed from the data of references (Jarman et al., 1991; Strauss et al., 1992; Zhang et al., 1993). B, mutants of HS-40 motifs. The mutations of the nuclear factor binding motifs of HS-40 were generated by site-directed mutagenesis. For each motif and its flanking DNA, the wild type sequence is shown in full, with the central binding site of nuclear factor(s) boxed. The mutated base or bases are indicated by downward arrows. Note that two different mutants of the 3`NF-E2/AP1 motif, I and II, have been generated and analyzed (see text for details).



To gain further insights into the functions of the multiple nuclear factor-DNA complexes of HS-40, we have generated various HS-40 mutants by site-directed mutagenesis. The activity of either the embryonic 2- or the adult -globin gene promoter cis-linked to these HS-40 mutants was then assayed after transient expression in K562 cells and in MEL, a mouse adult erythroid cell line (Friend et al., 1971). As shown below, the present study has clearly defined the positive/negative roles of different HS-40 DNA motifs, including a dual role of the 3` NF-E2/AP1 motif. The functionality of these motifs also correlates remarkably well with their erythroid lineage- and developmental stage-specific occupancies by nuclear factors in vivo.


EXPERIMENTAL PROCEDURES

Plasmid Constructs and Site-directed Mutagenesis

We first constructed pBlue-HS-40 by blunt end ligation of a 373-bp EcoRI- BamHI fragment containing the HS-40 enhancer (Higgs et al., 1990) into the HindIII and EcoRI sites of pBluescript II KS(-). Site-directed mutagenesis was conducted using a commercially available kit, Mutatorsite-directed mutagenesis kit (Stratagene). Different oligonucleotides were designed and synthesized so that they were homologous to specific target regions in the wild type enhancer but with one or more base mismatches in the core binding sites for different nuclear factors. Many of them also contained specific restriction enzyme sites in order to facilitate the identification of the mutants. HS-40 mutations were first introduced into the plasmid pBlue-HS-40. The wild type and different HS-40 mutant-containing fragments were then isolated from agarose gels after XbaI digestion of pBlue-HS-40 or the above pBlue-HS-40-based plasmids and cloned into the XbaI site of p-597GH (Zhang et al., 1993), which contains 597 bp of the 2-globin promoter region (-559 to +38) linked to the human growth hormone reporter gene in p0GH (Nichols Institute). Each of the enhancer fragments was also cloned, by blunt end ligation, into the HindIII site of pB-590GH, which contains a 590-bp HindIII- DdeI fragment of the human -globin promoter (-574 to +21) located immediately upstream of the human growth hormone gene of p0GH. Only plasmids containing HS-40 in the genomic orientation relative to the globin promoters were used in the transfection assay. All mutant plasmids were identified by restriction enzyme cutting and dideoxynucleotide sequencing. Corresponding to the lower strand of HS-40, the mutant oligonucleotides used for site-directed mutagenesis of the HS-40 enhancer are: GATA-1(b), 5`-GTTGGCCCAGaatTCTGCTCCCTCAAGT-3`; 5`NF-E2/AP1, 5`-CAGAAGCACTGAtcgATGGTTGGCCCAG-3`; 3`(NF-E2/AP1)-I, 5`-CCCCCACAGGATcga-TCAGCAGTCCTGT-3`; 3`(NF-E2/AP1)-II, 5`-CCCCCACAGGATGACTCAGaAGTCCTGT-3`; GT II, 5`-CCCACCTCCACgtgCACAGGATGACTC-3`; GATA-1(c), 5`-GCACCAGAGGTTGaatTCAGCAGTACCA-3`; GATA-1(d), 5`-CTTCCCTCTCAGgTtAACAGGAGGGGGA-3`.

The plasmids containing different site-directed mutants are named according to the following examples. The plasmids containing the wild type HS-40 are pHS-40-597GH and pHS-40-B-590GH; the plasmids containing a mutation in the 5`NF-E2/AP1 motif of HS-40 are pHS-40 (5`NF-E2/AP1)-597GH and pB-HS-40 (5`NF-E2/AP1)-590GH, etc.

Transient Transfection Analysis

The maintenance of the erythroid K562 and MEL cells in culture is as described (Zhang et al., 1993; Reddy and Shen, 1993). DNA transfection was accomplished by electroporation (Potter et al., 1984). Procedures for transfection of K562 cells are similar to those previously described (Zhang et al., 1993) except that the cells were harvested at the densities of 5-8 10cells/ml. It was observed that the lower densities of cells at harvesting prior to the electroporation gave more reproducible data of transient expression.

For transient expression in dimethyl sulfoxide (MeSO)-induced MEL, the cells growing in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin were harvested at the densities of 1-5 10cells/ml. A total of 10cells was collected by centrifugation and resuspended in 0.4 ml of medium containing 10 µg of the test plasmid(s), 1 µg of pSV2CAT (Gorman et al., 1982), and 50 µg of sheared salmon sperm DNA. After electroporation, the cells were grown in media with 2.5% fetal bovine serum and 2% MeSO for 5 days, with 1:1 dilution on the 3rd day.

The relatively low levels of globin promoter activities from enhancerless plasmids were measured by the sensitive human growth hormone (GH) assay (Selden et al., 1986). The level of HS-40-directed 2-globin promoter activities in K562 cells and HS-40-directed -globin promoter activities in MeSO-induced MEL cells were measured by the GH assay and/or RNA primer extension (Sambrook et al., 1989), which gave comparable results.

Data from either GH assay or RNA primer extension analysis were calibrated against the chloramphenicol acetyltransferase activities expressed from co-transfected pSV2CAT plasmids (Gorman et al., 1982), although the chloramphenicol acetyltransferase activities usually were constant within the range of ±10%. The quantities of the endogenous 2 mRNAs were also used to standardize the expression levels of transfected plasmids. The average expression level for each plasmid was derived from three or more independent DNA transfection experiments. The details of the above GH assay, chloramphenicol acetyltransferase assay, and RNA primer extension analysis are as described previously (Sambrook et al., 1989; Zhang et al., 1993).


RESULTS

The HS-40 enhancer consists of a total of four GATA-1 motifs, two NF-E2/AP1 motifs, and one GT motif, as defined by nuclear factor binding studies in vitro (Jarman et al., 1991; Strauss et al., 1992) (Fig. 1 A). Except for GATA-1(a), which exhibited no genomic footprint and was apparently non-functional in previous Bal31 deletion analysis (Zhang et al., 1993), we have introduced 2- or 3-bp substitutions into each of the above mentioned motifs (Fig. 1 B). The enhancer effects of these HS-40 mutants on the 2-globin promoter activity in K562 cells and on the -globin promoter activity in MeSO-induced MEL cells were then measured by transient expression assay. Under our experimental conditions, the wild type HS-40 enhances the activity of cis-linked embryonic 2-globin promoter in K562 cells by 160 ± 15-fold and that of cis-linked adult -globin promoter in MeSO-induced MEL cells by 13 ± 2-fold.

As exemplified by the primer extension analysis in Fig. 2 and summarized in Fig. 3the enhancer function of HS-40 on 2-globin promoter activity in K562 cells was not affected by mutations either in the GATA-1(b) motif (Fig. 2 A, lane 1), which is consistent with our previous result (Zhang et al., 1993), or in the GATA-1(d) motif (Fig. 2 A, lane 6). However, the HS-40 enhancer function dropped by 40% when the GATA-1(c) motif was mutated (Fig. 2 A, lane 5).


Figure 3: Summary of functional roles of individual nuclear factor binding motifs of HS-40 enhancer. The motifs in the 355-bp enhancer are labeled as described in Fig. 1 A except for GATA-1(d), which is half-filled to indicate its occupancy by nuclear factors, presumably GATA-1, in adult erythroid cells but not in embryonic/fetal erythroid cells. The small x in the lines below the top diagram indicates the individual motifs changed by site-directed mutagenesis. The enhancer effects of wild type and mutant HS-40 sequences on the 2-globin promoter activity in K562 cells and on the -globin promoter in MeSO-induced MEL cells were analyzed by transient expression assay. The relative enhancer strengths, with the wild type ( WT) HS-40 being 100%, were calculated from RNA primer extension and GH assay data as described in the text and are listed. The standard deviations, as derived from two to four independent transfection experiments, are listed in the parentheses.




Figure 2: A, example of primer extension assay of RNAs from cells transfected with plasmids containing 2-GH hybrid gene cis-linked with wild type HS-40 ( lane 7) or with HS-40 enhancer mutated at GATA-1(b) ( lane 1); 5`NF-E2/AP1 ( lane 2); 3`NF-E2/AP1, the 1-bp mutation ( lane 3); GT II ( lane 4); GATA-1(c) ( lane 5); GATA-1(d) ( lane 6); lane 8 is from enhancerless plasmid p-597GH. B, primer extension assay of RNAs from K562 cells transfected with plasmids: lane 1, wild type p-HS-40-597GH; lane 2, p-HS-40(3`NF-E2/AP1-II)-597GH; lane 3, p-597GH; lane 4, p-HS-40(3`NF-E2/AP1-I)-597GH. The endogenous 2-globin mRNAs gave primer extension products 93 nucleotides ( nt) long, while the major primer extension product from 2-GH hybrid RNAs of transfected plasmids is 64 nucleotides long.



Mutation of either 5`NF-E2/AP1 or GT II motif essentially abolished the enhancer function of HS-40 in K562 cells (Fig. 2 A, lanes 2 and 4). Surprisingly, mutant HS-40 with the 1-bp mutation in the 3`NF-E2/AP1 motif, 3`(NF-E2/AP1)-II (Fig. 1 B), exhibited a 2-3-fold higher level of enhancer activity than the wild type (Fig. 2 A, lane 3 and Fig. 2 B, lane 2). This is in contrast to the previous observation of similar base mutation in a dimeric NF-E2/AP1 motif, which greatly repressed the enhancer function of 5`HS2 or -LCR (see ``Discussion''). On the other hand, the 3-bp mutation of 3`NF-E2/AP1 motif, 3`(NF-E2/AP1)-I (Fig. 1 B), greatly decreased the HS-40 enhancer function by 75% (Fig. 2 B, lane 4).

As summarized in Fig. 3, all mutations introduced into HS-40 greatly decreased its enhancer effect on the -globin promoter activity in MeSO-induced MEL cells. Interestingly, the increase of HS-40 enhancer function in K562 cells due to the 3`NF-E2/AP1-II mutation was not observed in MEL cells. Also, mutation of the GATA-1(d) motif, which is genomic footprinted only in the adult erythroid environment (Strauss et al., 1992; Zhang et al., 1993), decreased the HS-40 enhancer function in MEL cells by 60%.


DISCUSSION

For the first time, site-directed mutagenesis has been used to systematically analyze the functional roles of different nuclear factor-DNA complexes of HS-40 in the transcriptional activation of human -like globin upstream promoters by the -LCR in erythroid cells at different developmental stages. Since only transient expression assay has been used, our data most likely reflect the structure-function relationship during the final stage of transcriptional activation when the globin promoters are presumably physically associated with the HS-40 enhancer. Our primary targets for site-directed mutagenesis of HS-40 are those sequence motifs that bind specific nuclear factors in vitro and/or in vivo (Fig. 1). Despite the fact that genomic footprints most likely reflect only relatively stable binding of proteins to DNA helix and that nuclear factor(s) functioning at an earlier stage of human -like globin gene regulation may remain bound to its cognate DNA sequence(s), our data have demonstrated a remarkable correlation between the erythroid-specific occupancy of specific nuclear factor binding motifs at the HS-40 enhancer and their regulatory roles in the transcriptional activation of the human -like globin promoters in transiently transfected erythroid cell cultures.

Positive Regulatory and Neutral Roles of Different GATA-1 Motifs

Several previous studies have analyzed the function of the single GATA-1 motif in the 5`HS-2 enhancer of -LCR. Mutation in the GATA-1 motif of a synthetic core 5`HS-2 fragment showed no effect on the level of expression of a linked human globin gene in multicopy transgenic mice (Ellis et al., 1993). The same construct exhibited a 40% reduction of -globin gene expression in stably integrated MEL cells (Ellis et al., 1993). Mutation of the same GATA-1 motif in a native 5`HS-2 environment, however, did not affect the expression of a cis-linked -globin promoter when stably integrated in K562 cells as a single copy (Miller et al., 1993). Finally, GATA-1 mutation showed no effect on the 5`HS-2 enhancer function on -globin promoter in transiently transfected K562 cells (Gong and Dean, 1993). It was suggested by these workers that GATA-1 motif may function by conferring position-independent expression of linked genes, instead of acting as a classical enhancer element. However, interpretation of the above data was complicated by 5`HS-2 being only a part of the -LCR.

Our data indicate that at least GATA-1(c) is involved in the final stage of transcriptional activation of the -like globin promoters by HS-40 in embryonic/fetal as well as in adult erythroid cells, although the effect of GATA-1(c) mutation on HS-40 enhancer-directed activation of the 2-globin promoter in K562 cells is not as great as the GT II or NF-E2/AP1 motifs (Fig. 3; see below). Interestingly, mutation of GATA-1(d) motif, which is bound with nuclear factor(s) in the adult erythroid environment but not in K562 cells (Strauss et al., 1992; Zhang et al., 1993), indeed has no effect on HS-40 enhancer function in K562 cells. It is involved, however, in the transcriptional activation of the human -globin promoter by HS-40 in transiently transfected MEL cells (Fig. 3). Since the above three GATA-1 mutations would prohibit binding of the GATA-1 factor (Ko and Engel, 1993), our data indicate that nuclear factor-DNA complexes formed at the GATA-1(c) and GATA-1(d) motifs play active roles in the erythroid cell-specific and developmental stage-specific enhancer function of the HS-40 or -LCR. On the other hand, mutation in the GATA-1(b) motif, which is occupied by nuclear factors in both K562 cells and adult erythroblasts (Zhang et al., 1993), showed no effect in transfected K562 (Figs. 2 and 3). We suspect that in erythroid cells of embryonic/fetal lineage such as K562, this GATA-1 motif is involved at an earlier regulatory step of HS-40 function such as the assembly of an active chromatin structure.

The GT II Motif Is Essential for HS-40 Enhancer Function

The function of the GT motif of 5`HS-2 of the -LCR has been analyzed previously. Its mutation has only a minimal effect on the enhancer function of either a synthetic 5`HS-2 fragment in stably integrated cells (Ellis et al., 1993) or a native 5`HS-2 in transgenic mice (Caterina et al., 1991). The drastic decrease in the enhancer effect of HS-40 carrying a mutation in its GT motif, GT II (Fig. 3), strongly suggests that the nuclear factor-DNA complex(es) at the GT motif is an essential and integral part of the multiple protein-DNA complexes formed in HS-40 during its final step of activating the -like globin promoters in erythroid cells of different developmental stages. Sp1 is the major protein in K562 nuclear extract that binds the wild type HS-40 GT motif. However, it cannot bind to the mutant GT motif.() The close proximity of GT II to the 3`NF-E2/AP1 motif also implies that nuclear factor-DNA complexes formed at these two motifs may directly interact with each other.

Positive and Negative Regulatory Roles of the NF-E2/AP1 Motifs

The function of the tandemly arranged, dimeric NF-E2/AP1 motifs of 5`HS-2 of the -LCR has been extensively studied previously by the analysis of mutated 5`HS-2 or -LCR in transiently transfected cells, in stably integrated cell lines, or in transgenic mice. These results have demonstrated that intact dimeric NF-E2/AP1 motifs are required for 5`HS-2 to function as a potent erythroid-specific enhancer. However, the dimer motifs do not play any role in -LCR or 5`HS-2-mediated, integration site-independent expression of different human -like globin promoters (Moi and Kan, 1990; Ney et al., 1990a, 1990b; Caterina et al., 1991; Talbot and Grosveld, 1991; Miller et al., 1993).

The NF-E2/AP1 binding domain of HS-40 consists of two motifs separated by 6 bp, 5`-ATGACTCAGTG-3` and 5`-TGCTGAGTCAT-3`, both of which can bind NF-E2 as well as AP1 (Jarman et al., 1991; Strauss et al., 1992; Andrews et al., 1993a). As we have discovered, contrasting with 5`HS-2, the NF-E2/AP1 motifs of the -LCR, or HS-40, can function as either a positive or negative regulatory element. Introduction of a 3-bp mutation into the central core of either the 5` or 3` NF-E2/AP1 motif (Fig. 1 B) significantly lowered the HS-40 enhancer function to 5-25% of the wild type level (Fig. 3). However, when a GC base pair at one boundary of the 3`NF-E2/AP1 binding site is changed to TA (3`(NF-E2/AP1)-II, Fig. 1 B), the activity of the HS-40 enhancer, when placed in the genomic orientation relative to the 2-globin promoter in the plasmid, is increased by a factor of 2-3-fold in K562 cells. This is interesting since extensive studies have shown that the same 1-bp mutation abolishes the binding of NF-E2, but not AP1, to the sequence (5`-GCTGAGTCAT-3`/3`-CGACTCAGTA-5`), which is identical in the 5`NF-E2/AP1 motif of the 5`HS-2 enhancer, 3`NF-E2/AP1 motif of HS-40, and the NF-E2/AP1 site in the porphobilinogen deaminase promoter (Mignotte et al., 1989; Moi and Kan, 1990; Ney et al., 1990b; Talbot and Grosveld, 1991; Andrews et al., 1993a; Miller et al., 1993). On the other hand, the 3-bp mutation would greatly affect the binding of both NF-E2 (3) and AP1 (Smeal et al., 1989; Abate et al., 1991).

The opposite effects of the 3- and 1-bp mutations of the 3`NF-E2/AP1 motif thus strongly suggest that binding of NF-E2 factor to the 3`NF-E2/AP1 motif, and possibly the 5`NF-E2/AP1 motif as well, represses the HS-40-mediated 2-globin promoter activity, while the binding of other AP1 factor(s) to the same motif(s) positively regulates the HS-40 function on 2 promoter expression in K562 cells. Given that K562 is an erythroid cell line trapped during the embryonic to fetal transition (Lozzio and Lozzio, 1975) and the relative concentration of NF-E2 to AP1 in erythroid cells appears to increase as development proceeds (Andrews et al., 1993a), it is likely that transcriptional activation and silencing of the human 2-globin gene during embryonic to fetal erythroid development is partly modulated by the competitive binding of positive regulatory AP1 and negative regulatory NF-E2 factors to the -LCR. Of course, we cannot rule out the possible involvement of other nuclear factors capable of binding to the NF-E2/AP1 motif(s) in the positive and negative regulation of the HS-40 enhancer effect (Chan et al., 1993; Chang et al., 1993; Caterina et al., 1994; Igarashi et al., 1994; Kataoka et al., 1994; Kerppola and Curran, 1994).

The apparent negative regulatory effect of NF-E2 binding on the HS-40 enhancer-directed expression of 2-globin promoter activity is reminiscent of the negative regulation of several non-erythroid eukaryotic gene systems mediated through binding of the c-Fos-c-Jun complex at AP1 binding sites (Nicholson et al., 1990; Lopez et al., 1993; Hsu et al., 1994). It is also known that specific nuclear factor binding DNA motif(s) can function as either a positive or negative regulatory element, depending on the cellular environment (Miner and Yamamoto, 1991). Furthermore, an enhancer could sometimes function as a silencer (Jiang et al., 1993).


FOOTNOTES

*
This research was supported by National Institutes of Health Grant DK-29800. 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.

§
To whom correspondence should be addressed. Tel.: 916-752-8860; Fax: 916-752-3085.

The abbreviations used are: LCR, locus control region; bp, base pair(s); MeSO, dimethyl sulfoxide; GH, growth hormone.

G. N. Reddy, unpublished data.


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