©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Flanking and Intragenic Sequences Regulating the Expression of the Rabbit -Globin Gene (*)

(Received for publication, September 22, 1994; and in revised form, November 22, 1994)

Magdalena James-Pederson Susan Yost Brian Shewchuk Timothy Zeigler Randall Miller Ross Hardison (§)

From the Department of Biochemistry and Molecular Biology, The Center for Gene Regulation, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Despite their descent from a common ancestral gene and the requirement for coordinated, tissue-specific regulation, the alpha- and beta-globin genes in many mammals are regulated in distinctly different ways. Unlike the beta-globin gene, the rabbit alpha-globin gene is transiently expressed at a high level without an added enhancer in transfected erythroid and non-erythroid cells. By examining a series of alpha/beta fusion genes, we show that internal sequences of the rabbit alpha-globin gene (within the first two exons and introns) are required along with the 5` flank for this enhancer-independent expression. Furthermore, deletion of the introns of the alpha-globin gene, or replacement by introns of the beta-globin gene, results in severely decreased expression of the transfecting genes. Hybrid constructs between segments of the alpha-globin gene and a luciferase gene confirm that internal alpha-globin sequences are needed for high level production of RNA in transfected cells. The flanking and internal sequences implicated in regulation of the rabbit alpha-globin gene coincide with a prominent CpG-rich island and may comprise an extended promoter (including both flanking and intragenic sequences) that is active in transfected cells without an enhancer.


INTRODUCTION

The expression of alpha- and beta-globin genes must be coordinated and balanced to produce the functional alpha(2)beta(2) hemoglobin in erythrocytes, but the mechanisms leading to this coordination are surprisingly complex. In particular, the promoters of the alpha- and beta-globin genes are regulated differently. The human alpha-globin gene itself, with no added enhancer, is expressed after transient transfection of non-erythroid cells (Mellon et al., 1981; Humphries et al., 1982) and constitutively at a high level after stable integration in transformed murine erythroleukemia (MEL) cells (Charnay et al., 1984). In contrast, the human and rabbit beta-globin genes require the presence of a viral enhancer in cis for transient expression in non-erythroid cells (Banerji et al., 1981; Treisman et al., 1983) and are inducible in stably transformed MEL cells (Chao et al., 1983; Wright et al., 1983). Although the alpha-globin gene appears to be deregulated when introduced as a DNA fragment in transfected cells, both the human alpha- and beta-globin genes are appropriately inducible when carried on an entire chromosome in hybrid human times MEL cells (Deisseroth and Hendrick, 1978; Willing et al., 1979; Pyati et al., 1980). This indicates that the genes are regulated at least in part by distal DNA sequences, and, in fact, linkage to a locus control region (Grosveld et al., 1987) or a major control region (Higgs et al., 1990) allows the beta- and alpha-globin genes to be expressed at a high level in a position-independent, erythroid-specific manner in transgenic mice.

The differences in regulation of the human alpha- and beta-globin genes correlate closely with the striking differences in their DNA sequences and genomic context (reviewed in Hardison et al.(1991) and Hardison and Miller(1993)). Both human and rabbit alpha-globin genes are largely contained within CpG islands embedded in a long stretch of G + C-rich DNA that constitutes a very dense isochore, whereas the beta-like globin gene cluster is contained within an A + T-rich isochore characteristic of the bulk of mammalian genomic DNA (Bernardi et al., 1985). The human alpha-globin CpG island is not methylated in any tissue or stage of development examined (Bird et al., 1987), whereas critical sequences around the beta-like globin genes (Shen and Maniatis, 1980; van der Ploeg and Flavell, 1980) are methylated in nonexpressing tissues. The presence of CpG islands encompassing the alpha-globin gene may be a general requirement for its enhancer-independent expression. Indeed, the mouse alpha1-globin gene is not in a CpG island, and it requires an enhancer for expression in transfected cells (Whitelaw et al., 1989).

However, the particular sequences within this CpG island that account for the enhancer independence of the human alpha-globin gene have not been identified precisely (Charnay et al., 1984; Whitelaw et al., 1989; Brickner et al., 1991), nor is it clear whether this effect is derived from specific activating proteins or is a more general effect of the genomic DNA context (e.g. being in a CpG island). Further information can be gleaned from analysis of a similar mammalian alpha-globin gene that is related to the human gene but differs in some potentially important internal and flanking sequences. Like the human gene, the rabbit alpha-globin gene is part of a CpG island (Hardison et al., 1991), and it is transcribed when transfected into HeLa cells (Cheng et al., 1988). The present study examining the expression of various hybrids of the rabbit alpha-globin gene with either a beta-globin gene or a luciferase gene suggests a model of an extended promoter (encompassing both 5`-flanking and internal sequences of the alpha-globin gene) within the CpG island with multiple, positive regulatory elements.


MATERIALS AND METHODS

Recombinant Plasmids

The structures of plasmids containing the alpha-globin, beta-globin, or hybrid genes are summarized in Fig. 1. All the constructs except CAJO were cloned into the polylinker region of the plasmid vector Bluescript I KS(+) from Stratagene, which will be referred to here as pBS. The rabbit alpha-globin gene is on a SacII to BglII fragment that extends from -226 to +942 (200 bp (^1)past the polyadenylation site) (Cheng et al., 1988), corresponding to nucleotides 6517 to 7684 in the sequence of the rabbit alpha-globin gene cluster (GenBank accession number M35026, Hardison et al.(1991)). A HindIII site was subsequently inserted at the former BglII site (pBSalpha in Fig. 1). The parental fragment containing the rabbit beta-globin gene is a SacI to KpnI fragment that extends from -1221 to +3325 (pBSbeta4.5 in Fig. 1), corresponding to nucleotides 29696 to 34241 in the sequence of the rabbit beta-like globin gene cluster (EMBL accession number X07786, Margot et al.(1989)). Shorter fragments were truncated in the 5` flank at the sites for PstI (-100, pBSbeta3.4) or PvuII (-12, pBSbeta3.3) or truncated in the 3` flank at the HpaI site (+1766, pBSbeta3.0), which is 477 nucleotides past the polyadenylation site. The 196-bp SV40 enhancer fragment present in pBSbeta.en, CAJO, and pBSalpha.en includes the two 72-base pair repeats (Banerji et al., 1981). This enhancer was inserted at the BglII site at -424 in the beta-globin 5` flank (pBSbeta.en, Fig. 1) and in the polylinker of pBS 5` to the alpha-globin gene (pBSalpha.en, Fig. 1).


Figure 1: alpha-Globin and beta-globin gene constructs used in transfection assays. The rabbit alpha-globin gene is shown with dotted lines for flanking sequences, dark dotted boxes for exons, and light dotted boxes for introns. The rabbit beta-globin gene is shown with black lines for flanking sequences, black boxes for exons, and white boxes for introns. The restriction endonuclease cleavage sites used in the construction of each recombinant are shown. The box labeled enh is the SV40 enhancer, which includes the two 72-bp repeats.



An alpha-globin gene fragment from NcoI (+35, the ATG initiation codon) to PvuII (+796, or 84 nucleotides past the polyadenylation site) was inserted into pBSbeta3.0 at the HpaI site to generate pBSbetaalpha and pBSbetaalpha.in (opposite orientation, Fig. 1).

Fusions between alpha- and beta-globin genes were made at the alpha-globin gene NcoI site (+35) and the beta-globin gene PvuII site(-12) for the exon 1 fusions, at the AccI sites in exon 2 of the alpha-globin gene (+356) and beta-globin gene (+283), at the BalI sites in exon 3 of the alpha-globin gene (+525) and beta-globin gene (+1165), and at the EcoRI sites in exon 3 of the alpha-globin gene (+545) and the beta-globin gene (+1116). Single sites were used for the alpha/beta and beta/alpha fusion gene constructs, and combinations of sites were used for the alpha(beta) and beta(alpha) replacement constructs (Fig. 1).

Fusions between alpha-globin and luciferase genes are shown in Fig. 2. The alpha-Luc construct consists of the rabbit alpha-globin gene from the PstI site at -1096 to the PstI site at +494 fused in-frame to the luciferase coding segment (nucleotides 1757 to 45 from plasmid pGEM-luc from Promega). The fusion is at the 3` end of intron 2 of alpha-globin, maintaining the splice junction. This results in a hybrid protein encoded by exons 1 and 2 of alpha-globin and the luciferase cDNA. Nucleotides +544 to +941 of rabbit alpha-globin, containing the 3` half of exon 3 and the polyadenylation site, are fused to the 3` end of the luciferase coding region. In alpha(inverted)-Luc, the 5` rabbit alpha-globin fragment (-1096 to +494) is inserted in the opposite orientation with respect to alpha-Luc. The construct alpha(Deltae12)-Luc has a 206-bp deletion in the 5` alpha-globin fragment from nucleotides +105 to +309 (inclusive). In the alphap-Luc construct, rabbit alpha-globin 5`-flanking region (nucleotides -241 to +34 relative to the cap site) was inserted upstream of the luciferase coding region in the plasmid pGL2Basic (Promega). The alpha-globin start codon was deleted, such that the luciferase start codon was utilized.


Figure 2: alpha-Luciferase fusion constructs used in transfection assays. The rabbit alpha-globin gene is shown with black boxes for exons, wide white boxes for introns, and thin white boxes for flanking sequences. The luciferase coding region is shown as a dotted box. SV40 untranslated sequences are shown with diagonal stripes, with the large T antigen intron indicated by a light stipple pattern.



Transfections of Cells with DNA

K562 cells grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum, 2% penicillin/streptomycin, and 0.5 µg/ml amphotericin B, were transfected by electroporation (Neumann et al., 1982; Potter et al., 1984) at 450 V, 500 µF for 500 ms (Promega electroporator). For transfections with the alpha/beta fusion constructs, 1 ml of cell suspension (10^7 cells/ml) was mixed with 100 µg of the test DNA, electroporated, and harvested after 48 h of growth. In transfections for transient expression of luciferase, 5 times 10^6 K562 cells were mixed with 4 µg of test DNA, 10 µg of pRSVlacZ, and 36 µg of carrier DNA in a total volume of 700 µl, electroporated, and allowed to grow as above. To generate pools of cells stably transfected with the luciferase constructs, 10^7 cells in a 1-ml suspension were mixed with 90 µg of linearized test DNA and 10 µg of linearized pM5neo (Laker et al., 1987), which contains the gene for neomycin resistance driven by the promoter-enhancer of myeloproliferative virus M5. 24 h after electroporation, G418 was added to each culture to a final concentration of 1.2 mg/ml. Cultures were maintained until a pool of 10^7 G418-resistant cells per transfection culture was obtained, at which point the cells were harvested.

HeLa cells grown in Eagle's minimal essential medium with Earle's salts and L-glutamine (MEM), supplemented with 10% fetal calf serum and 1% penicillin/streptomycin, were transfected by the calcium phosphate procedure (Wigler et al., 1978). The HeLa cells (5 times 10^5 cells/ml, 10 ml per 10-cm^2 Petri dish) were transfected with a calcium phosphate precipitate containing 50 µg of test DNA. The media were replaced after 24 h, and the RNA was harvested after 48 h.

RNA Analysis

After transfection, RNA was isolated from both HeLa and K562 cells by the guanidine thiocyanate-acid phenol procedure (Chomczynski and Sacchi, 1987). Several different probes were used in the S1 nuclease protection assays (Favaloro et al., 1980). To analyze the 3` end of alpha-globin RNA, a 402-bp EcoRI-HindIII fragment from pBSalpha was 3` end-labeled at the EcoRI site in exon 3 with [alpha-P]dATP and Klenow polymerase. Other probes were uniformly labeled (Bentley, 1984) by copying the inserts in the single-stranded M13 clones alphaAs (NcoI to PstI, detecting exon 1 and exon 2 of the alpha-globin gene), betaA2s (PstI to BamHI, detecting exon 1 and exon 2 of the beta-globin gene), and betaCs (BglII to BglII, detecting exon 3 of the beta-globin gene) (Rohrbaugh et al., 1985; Vandenbergh et al., 1991). For each RNA analysis from a set of transfection assays, 0.1, 1.0, or 10 ng of rabbit reticulocyte poly(A) RNA (Life Technologies, Inc.) was mixed with 100 µg of Escherichia coli tRNA to serve as a positive control and quantitation standard. Hybridizations were in a solution of 80% formamide, 40 mM Hepes, pH 7.5, 400 mM NaCl, 1 mM EDTA, at temperatures of 51 °C for the alpha probes, 42 °C for the 5` beta probe, and 37 °C for the 3` beta probe. RNA-DNA hybrids were digested with 400 units of S1 nuclease and resolved on denaturing polyacrylamide gels.

Luciferase-encoding RNAs were detected by the RNase protection assay of Melton et al.(1984) as described by Ausubel et al.(1993), using a 176-nucleotide probe generated against the 3` end of the luciferase region by transcribing pGEMluc (Promega) digested with HpaII with T7 RNA polymerase in the presence of 30 µCi of [alpha-P]UTP. The fragments protected from RNase digestion were 125 nucleotides long when annealed to alpha-Luc and alpha(Deltae12)-Luc RNA and 115 nucleotides long when annealed to alphap-Luc RNA.

Quantification of the Relative Levels of Rabbit Globin RNA

Transfections followed by S1 analysis of RNA were performed multiple times for each construct, and the resulting autoradiographs were quantified by densitometry. The signal for each construct was normalized to the pBSbeta.en or pBSalpha signal from the same autoradiograph, and the values from different experiments were averaged. For transfections into K562 cells, the amount of alpha-globin RNA from pBSalpha was consistently about 10 times that of beta-globin RNA from pBSbeta.en (by reference to the rabbit reticulocyte RNAs used as controls), so all values could be reported relative to the alpha-globin RNA from pBSalpha. However, in HeLa cells, the amount of RNA from pBSbeta.en relative to pBSalpha varied widely in different experiments, so the values for HeLa cell transfections are reported separately as relative to alpha-globin RNA from pBSalpha or beta-globin RNA from pBSbeta.en (depending on the probe used in the analysis). The results of the RNase protection assays of luciferase-containing constructs were quantified on a beta-scope (Betagen).

Chloramphenicol Acetyltransferase Assay

A series of restriction fragments from the flanking and internal regions of the rabbit alpha-globin gene (Yost et al., 1993) were inserted into the enhancer trap plasmid pCATpromoter (Promega), which contains the gene for chloramphenicol acetyltransferase (CAT) driven by the SV40 early promoter, but no enhancer. K562 cells were transfected with 100 µg of the pCAT promoter as above, and freeze-thaw extracts of transfected cells were assayed for CAT activity (Gorman et al., 1982), which is reported relative to the activity generated by pSV2CAT. The amount of CAT activity for pSV2CAT in 5 independent transfections ranged from 30 to 77 pmol of chloramphenicol acetylated per min per mg of protein in the extract (corresponding to 24 to 64% acetylation of the chloramphenicol in 60 min by 25 µl of extract).

Luciferase Assay

Luciferase assays were performed on transfected cells as described in the Promega Luciferase Assay System technical bulletin. The same extracts prepared by this procedure were used to assay for beta-galactosidase activity as described in Sambrook et al.(1989). Luciferase activities in relative light units per second (RLUbullets) as detected by a Berthold luminometer were divided by the beta-galactosidase activities (expressed as A) to correct for transfection efficiency.


RESULTS

Transcription of the Rabbit alpha-Globin Gene in Transfected Cells Does Not Require an Enhancer

The ability of the rabbit alpha-globin gene to be transiently expressed was tested in both erythroid (human K562 cells, which express endogenous alpha-, -, -, and -globin genes, but not the beta-globin gene) and non-erythroid (HeLa) cells. Cells transfected with plasmids containing the alpha-globin gene (clone pBSalpha, Fig. 1) produced about 2 ng of RNA in K562 cells (Fig. 3A, lanes 2, 3, and 13) and 6 ng in HeLa cells (Fig. 3B, lanes 1 and 2). Inclusion of the SV40 enhancer (pBSalpha.en, Fig. 1) caused only a modest increase in the amount of RNA (Fig. 3A, lanes 6, 7, and 14; Fig. 3B, lanes 5 and 6). The results from several determinations show the SV40 enhancer caused at most a 2-fold increase in alpha-globin gene expression in either K562 cells or HeLa cells (summarized in Fig. 5).


Figure 3: S1 nuclease protection assays on RNA from K562 and HeLa cells transfected with alpha-globin gene constructs. Autoradiographs of the gels resolving fragments protected from nuclease S1 digestion are shown. Abbreviated names of the DNA constructs are given at the top of each lane: M, mock-transfected cells; pr, input probe; rR, rabbit reticulocyte poly(A) RNA; ex1, ex2, ex3, protected fragments from exons 1, 2, or 3, respectively. A, RNA from transfected K562 cells was hybridized to a uniformly labeled probe extending from the NcoI site in exon 1 to the PstI site in intron 2 of the rabbit alpha-globin gene, and the portions of the probe protected by RNA from digestion by S1 nuclease are shown. The multiple bands probably result from S1 nibbling into the ends of the duplex. B, RNA from transfected HeLa cells was hybridized with a 3` end-labeled probe extending from the EcoRI site in exon 3 to an artificial HindIII site inserted 234 bp 3` to the polyadenylation site of the alpha-globin gene. Lanes 2-9 in A and 1-6 in B show the results of duplicate transfections (separate plates of cells transfected with the same DNA).




Figure 5: Summary of the relative levels of rabbit globin RNA in transfected K562 and HeLa cells. The constructs used in the transfection assays are shown in a simplified form and are not drawn to scale; the conventions in the drawing are the same as in Fig. 1. Multiple determinations of the amount of RNA produced from individual constructs were normalized to the pBSalpha or pBSbeta.en signals. The averages ± S. D. (or half the range for n = 2) are reported for n determinations. Based on Student's t test, the p values for pBSalpha/beta.2 and pBSalpha/beta.3 versus pBSbeta4.5 are <0.001, except for pBSalpha/beta.2 versus pBSbeta4.5 in K562 cells (p < 0.01).



As expected from earlier work (Banerji et al., 1981), this contrasts sharply with the requirement of an enhancer for expression of the rabbit beta-globin gene in HeLa cells. The S1 analysis in Fig. 4B (lanes 3, 4, and 8) shows that the rabbit beta-globin gene (clones pBSbeta3.0 and pBSbeta4.5, Fig. 1) directs the synthesis of barely detectable RNA in HeLa cells, but introduction of the SV40 enhancer (pBSbeta.en, Fig. 1) causes a large increase in the amount of RNA produced (13- to 25-fold, Fig. 5). Like the endogenous homolog, the rabbit beta-globin gene is also not actively expressed in K562 cells (Fig. 4A, lane 5), but even addition of the SV40 enhancer (CAJO, Fig. 1) does not rescue its expression (Fig. 4A, lane 9).


Figure 4: S1 nuclease protection assays on RNA from K562 and HeLa cells transfected with alpha/beta hybrid genes and beta-globin genes. A, RNA from transfected K562 cells was hybridized with uniformly labeled probes extending from the BglII site in exon 3 to a BglII site located 350 bp 3` to the polyadenylation site of the beta-globin gene (lanes 1-9) or from a PstI site located 100 bp 5` to the cap site to the BamHI site in exon 2 of the beta-globin gene (lanes 10-18). Lanes 10-15 show the results of duplicate transfections with the same DNA. Lanes 10-18 are from a longer exposure than lanes 1-9, so that 1.0 ng of rabbit reticulocyte RNA in lane 17 gives a signal comparable to 10 ng of RNA in lane 3. Bands resulting from cross-hybridization between the endogenous human -globin RNA and the rabbit beta-globin probe are labeled (). B, RNA from HeLa cells was hybridized with the uniformly labeled probe for the 3` end of beta-globin RNA described for A. M(r), size markers of pBR322 digested with HinfI.



A Promoterless alpha-Globin Gene Does Not Serve as an Enhancer of beta-Globin Gene Expression

Addition of the rabbit alpha-globin gene (minus its promoter) to the beta-globin gene in either orientation (clones pBSbetaalpha and pBSbetaalpha.in, Fig. 1) does not cause an increase in the amount of beta-globin RNA produced in transfected cells. As shown in Fig. 4B (lanes 9 and 10), no RNA is seen when the constructs are transfected into HeLa cells. Fig. 4A (lanes 10-13) shows a small amount of beta-globin RNA (about 0.1 ng) produced in K562 cells transfected with these constructs, equal to that obtained from the beta-globin gene with or without the SV40 enhancer in this same experiment (pBSbeta3.0, pBSbeta.en; data not shown). The summary in Fig. 5confirms this absence of enhancement in multiple determinations.

Internal Sequences of the alpha-Globin Gene Are Required for Transcription

A series of alpha/beta fusion genes was tested in the transient expression assay. The 5` portion of the alpha-globin gene was joined to the 3` portion of the beta-globin gene in either exon 1, exon 2, or exon 3 (clones pBSalpha/beta.1, pBSalpha/beta.2, and pBSalpha/beta.3, respectively; Fig. 1). Fusion of the 5` flank of the alpha-globin gene onto the body of the beta-globin gene (pBSalpha/beta.1) does not confer active expression in either K562 cells (Fig. 4A, lane 6) or HeLa cells (Fig. 4B, lane 5). However, inclusion of internal alpha-globin sequences up to exon 2 or exon 3 does generate a substantial amount of hybrid RNA both in transfected K562 cells (Fig. 4A, lanes 7 and 8) and HeLa cells (Fig. 4B, lanes 6 and 7), detected with a 3` end-labeled beta-globin probe. Analysis of the hybrid RNA with a uniformly labeled probe from the 5` portion of the alpha-globin gene confirms that the alpha/beta genes fused at exons 2 or 3 are transcriptionally active (Fig. 3A, lanes 8, 9, 15, and 16), producing about as much RNA as the parental alpha-globin gene. The summary of several experiments (Fig. 5) shows that a construct with both the 5` flank and internal alpha-globin gene sequences (pBSalpha/beta.3) produces about 15 times as much RNA as the beta-globin gene construct pBSbeta4.5 in K562 cells and about 10 times as much in HeLa cells, whereas fusion of the alpha-globin gene 5` flank onto the beta-globin gene has little effect.

Deletion or Replacement of Internal alpha-Globin Gene Sequences Reduces RNA Production

The introns of the alpha-globin gene were removed by replacing the internal sequences with an alpha-globin cDNA (pBScalpha, Fig. 1). Transfection of either K562 or HeLa cells with this construct produced very little alpha-globin RNA (Fig. 3A, lanes 4 and 5; Fig. 3B, lanes 3 and 4). This decrease in production of RNA could result from the loss of positive cis-acting sequences in the alpha-globin gene introns or from the absence of splicing, or a combination of both. Thus, the internal regions of the alpha-globin gene were replaced with analogous regions of the beta-globin gene, leaving the splice junctions intact (clones pBSalpha(beta).12, pBSalpha(beta).23, and pBSalpha(beta).13; Fig. 1). Transfection of either HeLa cells (Fig. 6, lanes 9-11) or K562 cells (Fig. 7, lanes 11-13) with these plasmids produced very small amounts of hybrid RNA. The replacement of the internal alpha-globin gene sequences with beta-globin sequences caused about a 5- to 7-fold decrease in the amount of RNA produced, compared to the parental alpha-globin gene (Fig. 5).


Figure 6: S1 nuclease protection assay on RNA from transfected HeLa cells. The transfecting DNAs included beta-globin genes with and without an enhancer, alpha-globin genes with internal regions replaced by beta-globin gene sequences (the pBSalpha(beta) series), a beta/alpha hybrid gene (pBSbeta/alpha.2) and a beta-globin gene with internal sequences replaced with alpha-globin gene sequences (pBSbeta(alpha).23). The RNA was hybridized with the uniformly labeled probe specific for exons 1 and 2 of the beta-globin gene (Fig. 4).




Figure 7: S1 nuclease protection assays on RNA from transfected K562 cells. The transfecting DNAs include the pBSalpha(beta) replacement constructs and alpha/beta as well as beta/alpha fusion genes. The RNA was hybridized with the uniformly labeled probe for exons 1 and 2 of the beta-globin gene (Fig. 4).



Fusion of Internal alpha-Globin Gene Sequences into a beta-Globin Gene Does Not Activate Its Transcription

The beta-globin gene promoter could not be activated by internal alpha-globin gene sequences in reciprocal beta/alpha hybrid gene constructs (pBSbeta/alpha.2 and pBSbeta/alpha.3; Fig. 1). Transfections with these plasmids, as well as a plasmid with the second intron of the beta-globin gene replaced by that of the alpha-globin gene (pBSbeta(alpha).23; Fig. 1), produced only very small amounts of RNA in both HeLa (Fig. 6, lanes 12-14) and K562 cells (Fig. 7, lanes 14-16). Thus, the positive cis effects of these alpha-globin gene internal sequences do not act on the beta-globin gene promoter, whether the internal sequences are in the normal position (as in the hybrid genes in pBSbeta/alpha.2 or pBSbeta(alpha).23) or inserted at a distant site, as with the promoterless alpha-globin genes added to plasmids with the beta-globin gene (pBSbetaalpha and pBSbetaalpha.in; Fig. 5).

Internal and Flanking Sequences of the alpha-Globin Gene Can Increase Expression from the SV40 Promoter

A complementary study showed that several nuclear proteins, including an Sp1-like protein, will bind specifically in both the 5` flank and internal regions of the alpha-globin gene (Yost et al., 1993). This suggests that these DNA sequences could perhaps act as enhancers of promoters containing Sp1-binding sites, despite the fact that the alpha-globin gene sequences were not effective as enhancers of the beta-globin gene promoter. To test this hypothesis, four fragments of the alpha-globin gene (Fig. 8A) were inserted 3` to the coding sequences of the chloramphenicol acetyltransferase gene in the expression vector pCATpromoter, which is driven by the SV40 promoter (but not the enhancer). After transfection into K562 cells, all four fragments cause a clear but moderate increase in CAT activity relative to the parental pCATpromoter (6- to 18-fold, Fig. 8B). This is a specific effect of adding these fragments since an insertion of a fragment containing hypersensitive site 3 of the human beta-globin locus control region (Philipsen et al., 1990) did not increase the CAT activity (only 0.4% relative to pSV2CAT, data not shown); this DNA fragment is not an enhancer of the SV40 promoter (Tuan et al., 1987). These data indicate that several internal and flanking regions of the rabbit alpha-globin gene are able to increase expression from the SV40 promoter, but individually they are not as effective as the SV40 enhancer.



Figure 8: Test of the ability of alpha-globin gene fragments to enhance expression of the CAT gene from the SV40 promoter. A, four fragments of the rabbit alpha-globin gene were placed 3` to the CAT gene driven by an SV40 promoter (pCAT promoter), transfected into K562 cells and CAT activity was measured. Proteins implicated in binding to the DNA motifs are indicated. CP1 is a CCAAT-box binding protein, alphaIRP is the alpha-globin inverted repeat binding protein (a relative of Sp1), TBP is the TATA-box binding protein, YY1 is involved in both positive and negative regulation of various genes, Sp1 is a transcriptional activator, CACBP refers to any protein binding to a CACC motif, and CnBP refers to the protein binding to a string of Cs in the 3`-untranslated region. B, an autoradiograph of the thin layer chromatogram separating chloramphenicol (Cm) from its acetylated products (1-Ac-Cm and 3-Ac-Cm) is shown for one set of duplicate transfections with each construct. CAT activity was calculated as nanomoles of chloramphenicol acetylated min mg of protein and is reported relative to the activity of pSV2CAT. The first column of numbers gives the activities (± half the range) determined for the experiment shown in the autoradiograph. The second column gives the results (±S.D.) for several independent experiments. All values for the constructs containing alpha-globin gene fragments are significantly greater than those for pCATpromoter (p < 0.001 by Student's t test).



Internal Sequences of the Rabbit alpha-Globin Gene Are Required for Production of High Levels of RNA from Luciferase Reporter Gene Fusion Construct

Since the beta-globin gene is not expressed in K562 cells, interpretation of the results from the alpha/beta fusions is complicated by the possible presence of negative regulatory elements in the beta-globin gene sequences. In order to address this concern, segments of the alpha-globin gene were fused with a different reporter gene that should not contain any negative elements. The luciferase reporter gene was chosen because of the relative ease with which enzyme activity could be measured in transfected cells. However, transfection of cells with the constructs shown in Fig. 2showed the fusion of rabbit alpha-globin coding sequences to the luciferase coding region to generate a hybrid enzyme caused a decrease in luciferase activity (alpha-Luc versus alphap-Luc, Table 1), which was contrary to the result expected based on previous data with the alpha/beta fusion constructs. Subsequent observations on the hybrid enzyme produced in stably transfected K562 cells and in bacteria indicate that the alpha-globin amino acid sequence has a deleterious effect on the activity of the hybrid luciferase protein, making enzymatic activity an unreliable and unpredictable measure of expression for the alpha-luciferase fusions. This likely explains the increase in luciferase activity seen for the deletion construct, alpha(Deltae12)-Luc (Table 1), which has less alpha-globin coding sequence in the hybrid gene.



Because measurement of enzymatic activity was not a reliable indicator of expression, the production of RNA from these alpha-luciferase fusion constructs was measured directly using an RNase protection assay. RNA from pools of K562 cells stably transfected with alpha-Luc (containing the internal sequences, Fig. 2) yielded a clear, luciferase-specific, protected fragment of 125 nucleotides, whereas transfection with a construct with the alpha-globin sequences in the reverse orientation, alpha(inverted)-Luc, produced no detectable RNA (Fig. 9). In contrast, no 115-nucleotide protected fragment was detected above background in RNA from cells transfected with alphap-Luc, which does not contain the internal alpha-globin gene sequences. These data using the luciferase reporter constructs are congruent with the results from the alpha/beta fusion gene experiments; in both cases, the alpha-globin internal sequences are required for production of high levels of RNA. The construct using only 5`-flanking sequences as a promoter is expressed, as shown by the enzymatic activity in Table 1, but from an amount of RNA that is not detectable relative to that from a construct containing internal sequences (alpha-Luc, Fig. 9).


Figure 9: RNase protection assays on RNA from K562 cells transfected with alpha-luciferase fusion constructs. RNA from pools of stably transfected K562 cells was hybridized to a uniformly labeled RNA probe for the luciferase portion of the hybrid message. An autoradiogram on the gel resolving the protected fragments is shown. The positions of the 176-nucleotide probe and the expected protected fragments (125 nucleotides for alpha-Luc, 115 nucleotides for alphap-Luc) are indicated by arrows. The positive control in the second lane is a clone (D8) of K562 cells stably transformed with the alpha-Luc construct.



Deletion of a 206-bp Fragment from the alpha-Globin Structural Sequence That Contains a YY1-binding Site and Two Sp1-binding Sites Has No Effect on RNA Production

Previous binding studies with the rabbit alpha-globin gene identified a YY1-binding site near the 3` end of exon 1 and two tandem Sp1-binding sites within intron 1 (Yost et al., 1993). To test the contribution of these sites to the requirement for internal sequences for high level alpha-globin expression, a 206-bp fragment was deleted from the construct alpha-Luc by oligonucleotide-directed mutagenesis, generating the construct alpha(Deltae12)-Luc (Fig. 2). After stable transfection into K562 cells, the amount of RNA produced is about the same as that from the parental construct alpha-Luc (Fig. 9), showing that these three sites are not required for the enhancer-independent expression of the alpha-globin gene.


DISCUSSION

Like its homolog in humans, the alpha-globin gene from rabbits does not require an added enhancer for expression in transfected erythroid and non-erythroid cells. The role of internal alpha-globin gene sequences in expression has been controversial. Studies with human alpha/beta hybrid genes showed that sequences internal or 3` to the alpha-globin gene are required for its constitutive expression in stably transfected MEL cells (Charnay et al., 1984). However, Whitelaw et al.(1989) argued that the human alpha-globin gene is expressed without an added SV40 enhancer only when replicating in HeLa cells, and no erythroid-specific enhancers were found in or around the gene. Our studies show that internal regions of the rabbit alpha-globin gene are required for efficient expression. Inclusion of these internal sequences in alpha/beta hybrid genes allows expression without an enhancer, whereas their replacement with internal segments from the beta-globin gene causes a loss of expression. Furthermore, inclusion of internal sequences caused a large increase in RNA production from alpha-luciferase hybrid genes. The regulatory regions implicated for the rabbit gene are similar to those recently mapped for the human alpha-globin gene by Brickner et al.(1991), who found that a DNA segment extending from the 5` flank through exon 1 and intron 1 efficiently drove expression of a CAT reporter gene.

Internal regulatory sequences have been discovered in a growing number of genes, often within the introns. In some cases, these operate independently of position or orientation, forming enhancers in introns (e.g. Banerji et al.(1983)). In other cases, the internal regulatory sequences are active only in their native position, as in the genes for c-myc (Yang et al., 1986) and ribosomal protein L32 (Atchison et al., 1989; Chung and Perry, 1989). The analogous internal sequences in the rabbit alpha-globin gene have only a modest enhancing effect on the SV40 promoter (Fig. 7), and this effect may not be sufficient to explain the enhancer-independent phenotype of the intact gene. The internal regulatory sequences of the rabbit alpha-globin gene may work best within their natural context, perhaps constituting part of the promoter. In fact, the internal regulatory segments of the human alpha-globin gene are not effective when placed 3` to the gene or in the distal 5` flank (Brickner et al., 1991). Thus, the alpha-globin gene may not contain a classic internal enhancer, but rather the promoter of the gene may be unusually long, extending into the first two exons and introns.

Consistent with the absence of a strong internal enhancer, the intragenic sequences that confer enhancer-independent expression do not localize to one precise region. Although a segment extending into exon 2 will produce a significant amount of RNA in alpha/beta hybrid genes, inclusion of DNA up to the 5` end of exon 3 will produce more RNA (Fig. 4). Also, replacement of the region surrounding either intron 1 or intron 2 with equivalent sequences from the rabbit beta-globin gene will decrease the amount of RNA produced. All the internal and flanking regions tested increased CAT expression from the SV40 promoter to a comparable extent. Surprisingly, deletion of an internal region containing YY1- and Sp1-binding sites has no effect on RNA production. It is possible that multiple, redundant positive elements may be involved in establishing enhancer-independent expression.

The DNA segments required for enhancer-independent expression of the rabbit and human alpha-globin genes correspond to the CpG islands encompassing the genes (Bird et al., 1987; Hardison et al., 1991). The CpG island in rabbits begins about 500 bp 5` to the alpha-globin cap site and extends to the third exon, and it contains all the sequences shown here to be important in transient expression. The CpG islands are not found around the mammalian beta-like globin genes or around the mouse alpha1-globin gene, all of which require enhancers in transient expression assays. Thus, the CpG island may be critical for generating a widely expressed, enhancer-independent promoter, either by the binding of ubiquitous trans-activating proteins (Whitelaw et al., 1989; Yost et al., 1993) or by establishing a unique chromatin structure that is readily transcribed (Charnay et al., 1984). Chromatin from CpG islands differs dramatically from that of bulk chromatin, with much less histone H1 and an elevated level of acetylation of histone H4 (Tazi and Bird, 1990), which may facilitate access of transcription factors to the promoter. These two hypotheses are not mutually exclusive, and the combination of specific binding by transcriptional activators within a type of chromatin that is permissive for transcription could account for the ability of the rabbit and human alpha-globin genes to be expressed after introduction into a wide variety of cells.

Thus, the CpG island with appropriate binding sites for transcription factors may constitute an extended promoter, including both 5`-flanking and internal sequences, that operates independently of an enhancer. Understanding the mechanisms that allow this deregulated promoter to be expressed in a wide range of transfected cell types should provide the basis for determining how, during normal development, it is turned off in non-erythroid cells and activated in erythroid cells to produce tight, tissue-specific regulation. Enhanced expression in erythroid cells is dependent on the major control region located 40 kilobases 5` to the gene cluster (Higgs et al., 1990). Although the proximal 5`-flanking region of the human alpha-globin gene is sufficient to respond to this enhancer (Pondel et al., 1992; Ren et al., 1993), (^2)inclusion of the internal gene region leads to an even greater level of expression (Ren et al., 1993). In addition, given the capacity of the alpha-globin gene to express after transfection into a wide variety of cells, one could propose that a negative regulator is required to prevent expression in non-erythroid cells. Further experiments are required to test this possibility.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants DK-27635 and HL-44491, a United States Public Health Service Research Career Development Award DK-01589 (to R. C. H.), and by a Sigma Xi grant-in-aid (to S. E. Y.). 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 and reprint requests should be addressed: 206 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802. Tel.: 814-863-0113; Fax: 814-863-7024.

(^1)
The abbreviation used is: bp, base pair(s).

(^2)
B. Shewchuk and R. Hardison, unpublished data.


ACKNOWLEDGEMENTS

We thank W. Schaffner and E. Schreiber for gifts of the SV40 enhancer and the CAJO construct and Steve Pullen, Hania Petrykowska, and Martin Sigg for help with the experiments.


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