©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Analysis of DNase-I Hypersensitive Sites at the Mouse Porphobilinogen Deaminase Gene Locus
DIFFERENT REQUIREMENTS FOR POSITION-INDEPENDENT EXPRESSION FROM ITS TWO PROMOTERS (*)

(Received for publication, March 21, 1995; and in revised form, May 23, 1995)

Catherine Porcher (1), Christiane Picat (1), Dominique Daegelen (2), Carole Beaumont (1), Bernard Grandchamp (1)(§)

From the  (1)From INSERM U409, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris and (2)INSERM U129, Hôpital Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Porphobilinogen deaminase (EC 4.3.1.8; PBG-D) is the third enzyme of the heme biosynthetic pathway. In both human and mouse, the gene encoding PBG-D posseses two promoters, lying in close proximity. We have previously reported the mapping of six nuclear DNase-I hypersensitive sites at the PBG-D locus which could contribute to the regulation of the gene. In the present study, and in order to define all the elements necessary for a high level of expression and an integration site independence, we studied the pattern and the level of expression of a cloned PBG-D gene following integration into a host genome.The longest construct that we tested (12.5 kilobases) contained sufficient regulatory elements to promote expression levels similar to that of the endogenous gene, both in transgenic mice and in transfected cells. The overall contribution of individual DNase-I hypersensitive sites to the expression of the gene was then studied using a series of mutants that were stably transfected into mouse erythroleukemia cells. Two regions seem to play a critical role in the erythroid-specific expression of the PBG-D gene: the proximal promoter and a region situated at -1000 relative to the initiation site. Study of individual clones of mouse erythroleukemia cells revealed that the erythroid-specific expression of the gene was submitted to position effects in the absence of the upstream region, although the housekeeping transcription is not sensitive to such effects. The tandem arrangement of the housekeeping and tissue-specific promoters of the PBG-D gene raises some questions about the functioning of these two overlapping transcriptional units in erythroid cells. Previous data have suggested that in erythroid cells most of the transcripts initiated at the upstream promoter stop downstream of the first ubiquitous exon, between the two promoters. Here, we show that the deletion of a constitutive DNase-I hypersensitive site that is located in the region of the elongation block results in opposite effects on the steady state levels of housekeeping and tissue-specific RNA. This finding is consistent with the hypothesis that this region promotes premature termination of the housekeeping transcripts therefore preventing promoter interference.


INTRODUCTION

Enzymes involved in the heme biosynthetic pathway are expressed in all cell types, though in variable amounts, and their activities are coordinately induced during erythroid differentiation, leading to an increase in heme synthesis. Porphobilinogen deaminase (EC 4. 3. 1. 8; PBG-D)()is the third enzyme of the heme biosynthetic pathway. In both human and mice, the gene encoding PBG-D posseses two promoters, lying in close proximity(1, 2) . Differential splicing of transcripts initiated at each individual promoter yields two distinct mRNA species which give rise to two isoforms of the protein. One isoform is ubiquitous, whereas the other is erythroid-specific. We have previously analyzed some of the regulatory elements that contribute to the tissue-specific promoter utilization of the mouse PBG-D gene. Six DNase-I hypersensitive (DHS) sites were identified in DNA from erythroid and nonerythroid cells(3) . The three most upstream sites (DHS A, B, and C) are present in all tissue types, while the three downstream sites are either exclusively detected (DHS E) or more pronounced (DHS D and F) in erythroid cells (see Fig. 1for position of the DHS). DHS B corresponds to the ubiquitous promoter region which does not possess a TATA box or CAAT box, but two SP1-binding sites(1) , an usual feature for gene promoters expressed in all cell types in a constitutive manner(4) . DHS E is located near the erythroid start site. Two cis-acting sequences were found in the erythroid promoter, namely a GATA-1-binding site situated downstream of a duplicated CACC motif. GATA-1 is a major element of erythroid differentiation, as experiments involving the disruption of the GATA-1 gene in mouse embryonic stem cells have shown(5, 6) . The CACC motif binds ubiquitous factors, such as TEF-2 or SP1(7) , as well as the erythroid-specific Krüppel-like factor(8, 9) . DHS C, which maps to the first exon-intron boundary, is situated within the region where a block to elongation of ubiquitous transcripts has been characterized(1) . Finally, DHS D, which is localized 1 kb upstream of the erythroid transcription start site, consists of two inverted repeats of the GATA-1-binding site. Functional analysis of the erythroid PBG-D gene activity, using a reporter gene in stable transfection experiments, showed that combination of at least one of the two CACC elements and of the GATA-1 site was sufficient to promote a basal and tissue-specific expression of the PBG-D gene(3) . However, these studies did not permit comparison of the expression level of the reporter gene with that of the endogenous PBG-D gene, and variability in the expression levels of some of the constructs suggested that the integration site in the host cells may have lead to position effects. In the present study, and in order to define all the elements necessary for a high level of expression and integration site independence, we examined the pattern and the level of expression initiated at the two promoters following integration of a whole PBG-D gene into a host genome. A DNA fragment containing the entire gene with 2.5 kb of 5`- and 2.5 kb of 3`-flanking regions was used to produce transgenic mice and the same sequences were stably transfected into mouse erythroleukemia (MEL) cells, a cell line in which both promoters of the gene are active. To accurately assess the transgene copy number and the relative level of expression from each individual promoter, we used a quantitative PCR assay as described previously(10) . The longest construct contained sufficient regulatory elements to confer a pattern of expression similar to that of the endogenous gene in both transgenic mice and in transfected cells. A series of deletional mutant constructs was then used to characterize the contribution of the previously identified DHS (B-E, Fig. 1) to the regulation of the PBG-D gene expression. The two overlapping transcriptional units whose function depends on the presence of two separate promoters lying within 2.5 kb of each other displayed marked differences in sensitivity to position effects.


Figure 1: Schematic representation of the mouse PBG-D gene and position of the DHS. Positions of the transcriptional start sites from the housekeeping (HK) and the erythroid-specific (E) promoters are indicated (horizontal arrows). Exons are numbered from 1 to 15. Exon 1 is specific for the ubiquitous transcripts, exon 2 for the erythroid transcripts. The location of the DNase-I hypersensitive sites (DHS) is shown by vertical arrows (B-F). Thin arrows indicate DHS observed in all tissue types, and thick arrows indicate DHS either specifically observed (E) or more pronounced (D and F) in erythropoietic cells. A partial restriction map (distances are calculated relative to the erythroid start site) and position of the oligonucleotides used in this study (horizontal arrows in the lower part of the figure) are shown. The same representation of GATA-1 binding sites () and of CACC motifs () is used throughout the figures.




EXPERIMENTAL PROCEDURES

Plasmid Constructs

The organization of the mouse PBG-D gene, together with a partial restriction map, and the location of all oligonucleotides used in this study are presented in Fig. 1.

A 12.5-kb fragment of mouse genomic DNA, isolated from a previously described cosmid(1) , was subcloned into pGEM 4Z (Promega). This fragment consisted of the entire PBG-D gene (7.5 kb) flanked by 2.5 kb of 5` and 2.5 kb of 3` sequences. This initial construct contained the DNA regions corresponding to DHS B to F and was named pBCDEF. Two modifications were subsequently introduced into this construct that modified either exon 2 by insertion of a 5-mer oligonucleotide (GATCC) into the unique BamHI restriction site, or exon 3 by addition of six nucleotides (AAGCTT). The latter modification was introduced as follows: two partially overlapping fragments were synthesized by PCR using primers E2S/E3HdAS and E3HdS/E4HS and the initial construct as template. After purification, these two products were mixed, denatured, and reamplified using primers E2S/E4HS (see Fig. 1for position of the oligonucleotides). The final product was then digested with BamHI and SpeI and ligated to the corresponding sites in the initial construct.

Different constructs were derived from plasmid pBCDEF, labeled in exon 3, as follows (positions are numbered relative to the erythroid start site): p5`BCDEF, the 5` end of the initial construct was deleted leaving only 50 bp upstream of the ubiquitous start site; pBDEF, an internal SmaI(-1579)/XhoI (-1450) fragment was deleted; pBCEF, plasmid pBCDEF was digested by HindIII(-1038), then incubated with 2 units of Bal 31 nuclease (Life Technologies, Inc.) for 2-25 min. After incubation with the DNA polymerase I Klenow fragment, plasmids were religated, transformed, then sequenced by the chain termination procedure (11) in order to determine the extent of the deletions. One of the resulting plasmids had a 59 bp deletion that removed the two GATA-1 motifs. This plamid was named pBCEF and retained for further analysis; pCDEF, DNA sequences situated upstream of the SmaI site(-1579) were deleted; pBCDE, DNA sequences downstream of the HincII site (+6000) situated downstream of the polyadenylation signal were deleted.

All the following constructs are derived from pBCDE: pBCE, a XhoI(-1450)/BamHI (+98) fragment (lacking the two GATA-1 motifs) was isolated from plasmid pBCEF and subcloned into the corresponding sites of plasmid pBCDE; pBCD, the GATA-1 motif in the erythroid-specific promoter (-40 from the erythroid start site) was modified by site-directed mutagenesis following the same procedure as that described for introducing the modification into exon 3. The mutation changed the CTTATC motif into CTCACT. The first pair of primers was I1S/GatamAS and GatamS/EIIIK and reamplification was performed using primers I1S/EIIIK. The final product was then digested by HindIII and BamHI and ligated into the corresponding sites of plasmid pBCDE; pE, 5` sequences upstream of the PstI site(-494) in pBCDE were removed.

All modifications introduced by PCR were verified by sequencing.

Sequences of the oligonucleotides used in the PCR reactions are as follows (F = fluorescein): pSE1 sense, ACACCAGGGGACCGCAGCGGACT; I1S sense, CTAGTAGAGACACACCTGAA; E2S sense, F-AGTGTCCTGTTGCTGCTGCC; EIIIK antisense, ACGGGTACCCACTCGAATCA; E4HS antisense, F-GCCAGGGTACAAGGCTTTCA; GATAmS sense, GATGGGCCTCATCATTTT; GatamAS, AAAATGATGAGGCCCATC; E3HdS sense, CAAAGATGAAGCTTAGGGTGATTCGAGTGGGCAC; E3HdAS antisense, CACCCTAAGCTTCATCTTTGAGCCGTTTTCTTCC.

Transgenic Mice

DNA to be microinjected was derived from plasmid pBCDEF modified in exon 2. After enzymatic digestion, a 12.5-kb EcoRI fragment was separated from the plasmid sequences by electrophoresis and purified by GeneClean (Bio 101). A few hundred copies of the DNA fragment were injected into one of the pronuclei of fertilized eggs from mating B6D2F mice. Surviving eggs were transferred to pseudopregnant females(12) . Transgenic mice were identified by PCR analysis of genomic DNA from blood (see below). Lines were established, and expression of the transgene was analyzed on F or F generations.

Cell Culture and Transfections

Mouse erythroleukemia cells (clone 745) were grown in suspension in a minimal essential medium (MEM) supplemented with 2 mML-glutamine and 10% fetal calf serum (Life Technologies, Inc.). Cells were transfected by electroporation. Briefly, 2 10 mid-log growth phase cells were washed, resuspended in 0.8 ml of MEM and mixed to the linearized PBG-D and pMC1neo plasmids (Stratagene), present in a 5:1 molar ratio, respectively. Electroporation was performed by a single pulse of 240V at 960 µF with the Gene Pulser Apparatus (Bio-Rad). Cells were then divided into 4 independent pools and grown for 36 h in complete medium before addition of neomycin (G418) at 0.8 mg/ml. In some cases, individual clones were selected by growing the cells in methyl cellulose in the presence of G418. Cells were then harvested for DNA and RNA analyses.

PCR Quantifications

Copy number and level of expression of the transgene, in mice or in the transfected cells, were directly compared with that of the endogenous gene by quantitative PCR, using fluorescent primers(10) . The additional five bases present in exon 2 or six bases in exon 3 of the transfected genes led to the amplification of PCR products that differed in size from those derived from the endogenous gene (see Fig. 3for the size of the expected PCR fragments).


Figure 3: Housekeeping and erythroid-specific expression of the PBG-D gene in individual clones of stably transfected MEL cells. A, schematic representation of the PBG-D gene and of the modification introduced into the transfected gene. The black bar within exon 3 represents the six bases inserted for the labeling. Below are shown the PCR products resulting from the amplification of DNA or RNA of transfected cells and their size in base pairs. E, fragment deriving from the endogenous gene. T, fragment deriving from the transfected gene. Horizontal arrows indicate the localization of the primers and the stars the fluorescent primers. B, quantification of the housekeeping and erythroid mRNA in individual clones of MEL cells transfected with pBCDEF. Cells were transfected by plasmid pBCDEF modified in exon 3. After selection of stably transfected cells, DNA and RNA were prepared from 16 individual clones. Copy number and mRNA amount were quantified by PCR. Levels of expression of the transfected gene were plotted against the copy number and relative to that of the endogenous gene. Closed squares, erythroid-specific expression; open triangles, housekeeping expression. E = endogenous gene. T = transfected gene. The mean expression per copy of the transfected gene (± S.D.) and the correlation coefficient of the linear regression (r) are given.



Determination of Copy Numbers

DNA was prepared either from transfected cells (10) or from 50 to 200 µl of mouse blood. Cells were lysed in a non-ionic detergent containing buffer in the presence of proteinase K(13) . 5 µl of DNA was then amplified, using primers E2S-F and EIIIK.

Quantification of mRNA

RNA was prepared either from 5 10 transfected cells or from the organs of transgenic mice using the RNAzol B as recommended by the manufacturer (Bioprobe). 2.5 µg of RNA was then reverse transcribed in a final volume of 25 µl, using 100 ng of an oligo(dT) primer and 200 units of the Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). 2.5 µl of cDNA was then amplified by PCR, using either the E2S-F/EIIIK primers in order to specifically amplify the erythroid transcript sequences, or the pSE1/E4HS-F primers in order to specifically amplify the ubiquitous transcript sequences.

PCR products were then loaded onto a denaturing gel in an Automated Laser Fluorescent DNA sequencer (Pharmacia). At the end of the electrophoresis, the area of the fluorescence peaks was calculated using the Fragment Manager integration software (Pharmacia). Calculation of the ratio of fluorescence emitted by both kinds of molecules gives either the relative copy number of the transfected genes or the relative transcript number initiated at the erythroid or ubiquitous promoters.


RESULTS

A 12.5-kb Fragment Containing a Marked PBGD Gene Contains all Cis-regulatory Elements Sufficient for a Tissue-specific, Integration Site Independent Expression

A 12.5-kb fragment consisting of the entire gene flanked by 2.5 kb of sequences upstream of the ubiquitous promoter and 2.5 kb of 3` sequences (Fig. 1) was injected into fertilized mouse oocytes. The transgene was modified by inserting five nucleotides into a BamHI restriction site located in exon 2, in order to compare the copy number or the level of expression of the transgene with that of the endogenous gene using a quantitative PCR technique.

Four founder transgenic mice were obtained from which lines were established. No rearrangement of the construct in these four lines was detected by Southern blot analysis (data not shown). Transgene expression was analyzed in one to six animals of the F1 or F2 offspring of each founder animal, at the adult stage. Mice were perfused in order to remove any contaminating blood from tissues, and RNAs were prepared from five different tissues (bone marrow, spleen, heart, liver, and brain). After reverse transcription, the corresponding cDNAs were amplified by PCR. The oligonucleotides used in the reaction hybridized to exon 2 and 4 allowing quantification of transcripts initiated at the downstream promoter. The levels of erythroid-specific PBG-D mRNA in bone marrow and spleen from animals of each line varied between 25 and 100% of that of the endogenous gene, after correcting for the copy number (Fig. 2). Transcripts driven by the erythroid promoter were correctly initiated as determined by RNase protection experiments using RNA extracted from the spleen of transgenic mice (data not shown). No PCR product from the erythroid cDNA could be detected in non-erythroid tissues from either the endogenous PBG-D gene or the transgene.


Figure 2: Expression from the erythroid promoter in transgenic mice. A PBG-D transgene containing DHS B to F (a schematic representation is shown on the upper part of the figure, with the same symbols as in Fig. 1) modified by insertion of five nucleotides in exon 2 (represented by a black bar) was used to produce transgenic mice. RNAs were prepared from bone marrow and spleen of adult transgenic animals. Quantification was performed by PCR. The level of transcripts initiated at the tissue-specific promoter of the transgene is expressed relative to that of the endogenous gene from the analysis of 1-6 mice (mean ± standard deviation).



These data suggest that the construct used to produce transgenic mice contained all the regulatory cis-elements necessary to determine tissue specificity of the erythroid-specific promoter and a level of expression comparable to that of the endogenous gene in transgenic mice.

In order to determine the contribution of individual cis-acting elements to the regulated expression of the gene and to study the interactions between the two promoters, we performed stable transfection experiments into MEL cells. To quantify transcripts initiated at each promoter separately, the transfected gene was marked by inserting six bases into exon 3 which is present in the two mRNA species. By using two different sets of primers, it was possible to individually amplify the mRNA transcribed from the upstream housekeeping promoter and the mRNA transcribed from the downstream erythroid-specific promoter (Fig. 3A). The six-base insertion into the transfected gene enabled each type of mRNA to be recognized as either endogenous or transfected. In order to verify that this modification had no influence on the expression of the marked gene, expression levels of the erythroid mRNA were analyzed in MEL cells stably transfected with the gene marked either in exon 2 or in exon 3. In both cases, the level of expression of the erythroid promoter from the transfected gene was similar to that of the endogenous gene (not shown), in agreement with the results obtained in transgenic mice.

To assess the effects of the integration site and of copy number on the level of expression initiated at both PBG-D promoters, we analyzed 16 individual clones of transfected MEL cells. The ratio of transfected versus endogenous mRNA levels measured by RT-PCR was plotted against the number of copies of the transfected gene in each individual clone. The results in Fig. 3B show that there is a good correlation between the level of expression and the number of copies for both the ubiquitous (r = 0.98) and the erythroid (r = 0.88) mRNA. The mean level of expression per copy of the transfected gene was 0.90 and 0.61 relative to the expression of the endogenous gene for ubiquitous and erythroid mRNA, respectively.

Expression Initiated at the Erythroid PBG-D Promoter in MEL Cells Transfected with Various Deletional Mutants

In order to study the relative contribution of individual DHS to the erythroid-specific expression of the PBG-D gene, various constructs were derived from the initial plasmid pBCDEF labeled in exon 3, to remove the regions of interest. These deletion mutants were cotransfected into MEL cells with a plasmid containing a neomycin resistance gene, and analyses were carried out from populations of selected MEL cells. For construct pBCDEF, the mean expression per copy number of the transfected gene was identical to that of the endogenous gene (Fig. 4), in agreement with the results obtained on individual clones. Deletion of a 2.45-kb fragment located at the 5` end of the initial construct produced a moderate decrease in the level of expression (p5`BCDEF). Additional deletion of the region encompassing the housekeeping promoter and exon 1 had no further effect (pCDEF). When DHS C was removed by internal deletion of a SmaI-XhoI fragment (pBDEF), the level of expression fell to 47% of that of the endogenous gene, although this DHS is not erythroid-specific. Deletion of the 3`-flanking region, including DHS F, did not seem to have any detectable effect (pBCDE). In contrast, a point mutation abrogating the binding activity of the GATA-1 site at -40 bp relative to the erythroid transcriptional start site dramatically reduced the erythroid expression of the gene (pBCD). A critical role for this GATA motif has been shown in the homologous region of the human PBG-D gene (14) . However, a construct containing only 494 bp of the erythroid promoter, thus including the proximal GATA-1-binding site and a duplicated CACC motif, but no other DHS (pE) showed very weak expression. These results indicate that the proximal promoter is necessary for the tissue-specific expression but does not lead by itself to full promoter activity and suggests that upstream sequences may cooperate with the erythroid promoter.


Figure 4: Erythroid-specific PBG-D gene expression in stably transfected MEL cell populations. Upper part, schematic representation of the PBG-D gene (for symbols, see Fig. 1). Details for obtaining each construct derived from pBCDEF modified in exon 3 are described under ``Experimental Procedures.'' Analysis of the erythroid-specific expression was performed after selection of transfected MEL cell populations. Levels of expression of the tissue-specific promoter were determined by quantitative PCR. They are expressed as a percentage of that of the endogenous gene, after correction for the copy number. Results are given as the mean (± S.D.) of three to six independent experiments. Mean number of integrated copies ranged from 1 to 15. , mutagenized GATA-1-binding site.



DHS D Is Necessary for Obtaining a High and Integration Site Independent Level of Expression at the Erythroid Promoter

When compared to the full-length pBCDEF construct, deletion of DHS D alone resulted in a reduced accumulation of the erythroid mRNA, but the level of expression remained proportional to the copy number (Fig. 5, construct pBCEF). Deletion of DHS F (construct pBCDE) did not dramatically modify the erythroid expression. In contrast, when both DHS D and F were deleted from the construct (pBCE), many clones did not express the transfected gene and a poor correlation was observed between the expression level and the copy number (r = 0.337). It therefore appears that with this construct the erythroid expression is highly sensitive to position effects. However, all the individual clones obtained with this construct expressed the housekeeping mRNA at a level similar to that of the endogenous gene when corrected for copy number (data not shown).


Figure 5: Quantification of erythroid-specific mRNA from deletional mutants of the PBG-D gene in individual clones of transfected MEL cells. A schematic representation of each construct is shown at the top of each graph (for symbols, see Fig. 1). After transfection into MEL cells, 12-25 independent clones were selected for each construct. Levels of expression are plotted versus the copy number, relative to that of the endogenous gene, after PCR quantification. The correlation coefficient of the linear regression (r) and mean expression per copy number (± S.D.) are also given. E = endogenous gene. T = transfected gene.



To determine whether or not DHS D is able to prevent position effects when it is present in one single copy, we selected single copy clones of MEL cells transfected with either the initial pBCDEF construct or with a construct where only DHS D has been deleted (pBCEF). Indeed, previous studies of HS function have shown that, when multiple copy integrants are analyzed, cooperative interaction between tandemly integrated genes may potentiate their activity(15) . Quantification of both erythroid and housekeeping mRNAs from single copy transfectants of the full-length construct showed that both promoters are active at the same level as in the endogenous gene (Table 1), whereas many single copy transfectants with the pBCEF construct (where site D has been deleted) did not express the erythroid PBG-D mRNA at all. Interestingly, the same clones expressed the housekeeping mRNA from the construct, although at a variable level (m = 0.61).



Deletion of DHS C Leads to an Increased Level of Housekeeping mRNA and Decreased Level of Erythroid-specific mRNA

DHS C, which maps downstream of the first exon has been observed in both erythroid and non-erythroid tissues(3) , and run-on experiments have suggested that this region may correspond to a block in the elongation of transcripts initiated at the upstream promoter(1) . Internal deletion of this region in a construct where both promoters were conserved led to a 2-fold drop in the accumulation of erythroid transcripts in the total population of transfected MEL cells (Fig. 4). When individual clones were analyzed (Fig. 6), production of both types of mRNAs remained proportional to the copy number. Furthermore, the level of housekeeping mRNA from the transfected gene was significantly increased and when corrected for copy number, it was 1.5-fold more abundant than that from the endogenous PBG-D gene (pBDEF, Fig. 6). By contrast, the level of erythroid-specific transcripts was decreased to about 20% of the endogenous level. It therefore appears that DNA sequences at DHS C have opposite effects on the level of mRNAs initiated by the two promoters of the gene.


Figure 6: Analysis of the ubiquitous and erythroid-specific PBG-D gene expression in clones transfected with a construct lacking DHS C. The pBDEF construct modified in exon 3 was stably transfected into MEL cells. 12 independent clones were isolated. Levels of expression initiated at both promoters are represented versus the copy number, relative to the endogenous products, after PCR quantification. Correlation coefficient of the linear regression (r) and mean expression per copy number (± S.D.) are also given. Closed squares, erythroid-specific expression; open triangles: housekeeping expression.




DISCUSSION

Expression of the PBG-D gene is driven by a housekeeping promoter active in all cells and a tissue-specific promoter active only in erythroid cells. One consequence of this gene organization, which also occurs in some other genes(16, 17) , is that the erythroid promoter is embedded in a ubiquitously transcriptionally active locus. Indeed it is located downstream of the housekeeping PBG-D promoter and also downstream of the HA-X histone gene. This gene, which is located in the 3`-flanking region of the PBG-D gene, is transcribed in the reverse orientation(18) . The aim of the present study was to define the cis-acting elements controlling the expression of the mouse PBG-D gene initiated at its two promoters. In this paper, we tested the ability of a 12.5-kb fragment containing the PBG-D gene and five of six DHS (B-F, Fig. 1) to promote a high level of erythroid-specific expression in transgenic mice. In order to permit a precise comparison between the level of expression of the transgene and that of the endogenous gene, we developed a precise PCR-based assay to determine the copy number and expression level of the transgene relative to that of the endogenous gene which was used as an internal standard in this assay. Of four transgenic breeding lines tested, all expressed the mRNA initiated at the downstream promoter of the transgene and the expression was restricted to erythropoietic tissues (i.e. bone marrow and spleen). The level of expression was comparable to that of the endogenous gene when corrected for the copy number even in two lines with a single copy transgene. The same fragment, when stably transfected in to MEL cells exhibited a position-independent, copy number-dependent expression.

The overall contribution of individual DHS to the erythroid expression of the gene was then studied using a series of deletion mutants that were stably transfected into MEL cells.

The ubiquitous DHS F, which is located 3` of the PBG-D gene, probably corresponds to the promoter region of the transcriptionally active HA-X gene, and the results of transfection experiments show that deletion of this region does not bring about any reduction in PBG-D expression.

Two regions seem to play a critical role in the erythroid-specific expression of the PBG-D gene: the erythroid proximal promoter and a region situated at -1000 (DHS D) relative to the initiation site. These two regions possess a chromatin structure that is modified during erythroid differentiation as demonstrated by the appearance of DNase-I hypersensitive sites specific for erythroid cells(3) . Study of individual clones of MEL cells revealed that the erythroid expression of the gene is subject to position effects in the absence of the upstream region, although the housekeeping transcription is not sensitive to such effects. In MEL clones that fail to express the erythroid mRNA from the transfected construct, either a negative position effect or the absence of a positive effect can theoretically be postulated. However, in the absence of a significant influence of the host chromatin on the transcription initiated at the housekeeping promoter, a global repression extending from the neighboring chromatin at the insertion locus is very unlikely to occur. In clones that express a construct lacking DHS D, DNA regions at integration sites upstream or downstream of the PBG-D gene may influence the erythroid promoter activity in a positive manner. Since these sequences are separated from the erythroid promoter by transcriptional complexes at the housekeeping promoter of the PBG-D gene and at the promoter of the HA-X gene, this may be achieved through DNA looping mediated by protein-protein interactions. DHS D therefore appears to function like an enhancer element which cooperates with the erythroid promoter to ensure maximal transcriptional activity, in an integration site-independent manner. Interestingly, both regions have GATA-1-binding sites in close proximity to CACC/GT motifs, an association of binding sites which is a common feature of many erythroid-specific regulatory elements(19, 20, 21, 22, 23) . In addition, DHS D shares some similarities with the hypersensitive site 3 from the -globin locus control region(24, 25) . The locus control region shows strong activator as well as insulator functions that are specific for erythroid tissues(26) , and it also contains several GATA-1 and CACC/GT motifs. Furthermore, hypersensitive site 3 has been shown to confer integration site independence to the linked gene(27) .

The tandem arrangement of the housekeeping and tissue-specific promoters of the PBG-D gene raises some questions about the functioning of these two overlapping transcriptional units in erythroid cells. Transcriptional interferences have been observed when transcription from an upstream promoter down-regulates a closely linked downstream promoter in the same orientation(16) . In the case of the PBG-D gene, previous data have suggested that no switch mechanism between the two promoters was involved during activation of the erythroid transcription unit(1) . In MEL cells, both types of mRNA are present. Run-on experiments using isolated nuclei from these cells and from mouse spleen erythroblasts suggested that in erythroid cells the initiation of transcription is also enhanced at the housekeeping promoter but that most of the transcripts initiated at the upstream promoter stop downstream of the first ubiquitous exon, between the two promoters(1) . The region corresponding to a constitutive DHS (site C, Fig. 1) maps near this transcriptional elongation block. Our present observation that the deletion of this region results in opposite effects on the steady state levels of housekeeping and tissue-specific RNA is consistent with the hypothesis that this region promotes premature termination of the housekeeping transcripts therefore preventing promoter interference.


FOOTNOTES

*
This work was supported by grants from INSERM, AFM, Association Claude Bernard, and University Paris 7. 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: INSERM U409, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris Cedex 18, France. Tel.: 33-1-44856384; Fax: 33-1-42264624.

The abbreviations used are: PBG-D, porphobilinogen deaminase; DHS, DNase-I hypersensitive; kb, kilobase(s); MEL, mouse erythroleukemia; PCR, polymerase chain reaction; bp, base pair(s).


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