(Received for publication, March 21, 1995; and in revised form, May 23, 1995)
From the
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.
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)
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 (
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): p
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.
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.
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.
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).
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.
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.
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.
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.
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 H
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 H
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
H
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.
(
)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.
) and of CACC motifs
(
) is used throughout the figures.
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.
5`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.
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).
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.
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.
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.
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.
, 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).
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.
A-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.
A-X gene, and the results of
transfection experiments show that deletion of this region does not
bring about any reduction in PBG-D expression.
A-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) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.