(Received for publication, September 22, 1994; and in revised form, November 22, 1994)
From the
Despite their descent from a common ancestral gene and the
requirement for coordinated, tissue-specific regulation, the - and
-globin genes in many mammals are regulated in distinctly
different ways. Unlike the
-globin gene, the rabbit
-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
/
fusion genes, we show that internal sequences of the
rabbit
-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
-globin
gene, or replacement by introns of the
-globin gene, results in
severely decreased expression of the transfecting genes. Hybrid
constructs between segments of the
-globin gene and a luciferase
gene confirm that internal
-globin sequences are needed for high
level production of RNA in transfected cells. The flanking and internal
sequences implicated in regulation of the rabbit
-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.
The expression of - and
-globin genes must be
coordinated and balanced to produce the functional
hemoglobin in erythrocytes, but the
mechanisms leading to this coordination are surprisingly complex. In
particular, the promoters of the
- and
-globin genes are
regulated differently. The human
-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
-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
-globin gene appears to be
deregulated when introduced as a DNA fragment in transfected cells,
both the human
- and
-globin genes are appropriately
inducible when carried on an entire chromosome in hybrid human
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
-
and
-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 - and
-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
-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
-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
-globin CpG island is not methylated in any tissue or stage of
development examined (Bird et al., 1987), whereas critical
sequences around the
-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
-globin gene
may be a general requirement for its enhancer-independent expression.
Indeed, the mouse
1-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
-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
-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
-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
-globin gene with
either a
-globin gene or a luciferase gene suggests a model of an
extended promoter (encompassing both 5`-flanking and internal sequences
of the
-globin gene) within the CpG island with multiple, positive
regulatory elements.
Figure 1:
-Globin and
-globin gene constructs used in transfection assays. The rabbit
-globin gene is shown with dotted lines for flanking
sequences, dark dotted boxes for exons, and light dotted
boxes for introns. The rabbit
-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
-globin gene fragment from NcoI (+35, the ATG
initiation codon) to PvuII (+796, or 84 nucleotides past
the polyadenylation site) was inserted into pBS
3.0 at the HpaI site to generate pBS
and pBS
.in
(opposite orientation, Fig. 1).
Fusions between - and
-globin genes were made at the
-globin gene NcoI
site (+35) and the
-globin gene PvuII
site(-12) for the exon 1 fusions, at the AccI sites in
exon 2 of the
-globin gene (+356) and
-globin gene
(+283), at the BalI sites in exon 3 of the
-globin
gene (+525) and
-globin gene (+1165), and at the EcoRI sites in exon 3 of the
-globin gene (+545) and
the
-globin gene (+1116). Single sites were used for the
/
and
/
fusion gene constructs, and combinations of
sites were used for the
(
) and
(
) replacement
constructs (Fig. 1).
Fusions between -globin and
luciferase genes are shown in Fig. 2. The
-Luc construct
consists of the rabbit
-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
-globin, maintaining the splice junction. This results in a
hybrid protein encoded by exons 1 and 2 of
-globin and the
luciferase cDNA. Nucleotides +544 to +941 of rabbit
-globin, containing the 3` half of exon 3 and the polyadenylation
site, are fused to the 3` end of the luciferase coding region. In
(inverted)-Luc, the 5` rabbit
-globin fragment (-1096
to +494) is inserted in the opposite orientation with respect to
-Luc. The construct
(
e12)-Luc has a 206-bp deletion in
the 5`
-globin fragment from nucleotides +105 to +309
(inclusive). In the
p-Luc construct, rabbit
-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
-globin start codon was deleted,
such that the luciferase start codon was utilized.
Figure 2:
-Luciferase fusion constructs used in
transfection assays. The rabbit
-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.
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
10
cells/ml, 10 ml per 10-cm
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.
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
[-
P]UTP. The fragments protected from RNase
digestion were 125 nucleotides long when annealed to
-Luc and
(
e12)-Luc RNA and 115 nucleotides long when annealed to
p-Luc RNA.
Figure 3:
S1 nuclease protection assays on RNA from
K562 and HeLa cells transfected with -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
-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
-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 pBS or
pBS
.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
pBS
/
.2 and pBS
/
.3 versus pBS
4.5 are
<0.001, except for pBS
/
.2 versus pBS
4.5 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 -globin gene in HeLa cells.
The S1 analysis in Fig. 4B (lanes 3, 4, and 8) shows that the rabbit
-globin gene
(clones pBS
3.0 and pBS
4.5, Fig. 1) directs the
synthesis of barely detectable RNA in HeLa cells, but introduction of
the SV40 enhancer (pBS
.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
-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 /
hybrid genes and
-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
-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
-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
-globin probe are labeled (
). B, RNA from HeLa cells was hybridized with the uniformly
labeled probe for the 3` end of
-globin RNA described for A. M
, size markers of pBR322 digested
with HinfI.
Figure 6:
S1 nuclease protection assay on RNA from
transfected HeLa cells. The transfecting DNAs included -globin
genes with and without an enhancer,
-globin genes with internal
regions replaced by
-globin gene sequences (the pBS
(
)
series), a
/
hybrid gene (pBS
/
.2) and a
-globin gene with internal sequences replaced with
-globin
gene sequences (pBS
(
).23). The RNA was hybridized with the
uniformly labeled probe specific for exons 1 and 2 of the
-globin
gene (Fig. 4).
Figure 7:
S1 nuclease protection assays on RNA from
transfected K562 cells. The transfecting DNAs include the
pBS(
) replacement constructs and
/
as well as
/
fusion genes. The RNA was hybridized with the uniformly
labeled probe for exons 1 and 2 of the
-globin gene (Fig. 4).
Figure 8:
Test of the ability of -globin gene
fragments to enhance expression of the CAT gene from the SV40 promoter. A, four fragments of the rabbit
-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,
IRP is the
-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
-globin gene fragments are significantly greater than those for
pCATpromoter (p < 0.001 by Student's t test).
Because measurement
of enzymatic activity was not a reliable indicator of expression, the
production of RNA from these -luciferase fusion constructs was
measured directly using an RNase protection assay. RNA from pools of
K562 cells stably transfected with
-Luc (containing the internal
sequences, Fig. 2) yielded a clear, luciferase-specific,
protected fragment of 125 nucleotides, whereas transfection with a
construct with the
-globin sequences in the reverse orientation,
(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
p-Luc, which does
not contain the internal
-globin gene sequences. These data using
the luciferase reporter constructs are congruent with the results from
the
/
fusion gene experiments; in both cases, the
-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
(
-Luc, Fig. 9).
Figure 9:
RNase protection assays on RNA from K562
cells transfected with -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
-Luc, 115 nucleotides for
p-Luc) are indicated by arrows. The positive control in
the second lane is a clone (D8) of K562 cells stably
transformed with the
-Luc construct.
Like its homolog in humans, the -globin gene from
rabbits does not require an added enhancer for expression in
transfected erythroid and non-erythroid cells. The role of internal
-globin gene sequences in expression has been controversial.
Studies with human
/
hybrid genes showed that sequences
internal or 3` to the
-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
-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
-globin gene are required for
efficient expression. Inclusion of these internal sequences in
/
hybrid genes allows expression without an enhancer, whereas
their replacement with internal segments from the
-globin gene
causes a loss of expression. Furthermore, inclusion of internal
sequences caused a large increase in RNA production from
-luciferase hybrid genes. The regulatory regions implicated for
the rabbit gene are similar to those recently mapped for the human
-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 -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
-globin gene may work best
within their natural context, perhaps constituting part of the
promoter. In fact, the internal regulatory segments of the human
-globin gene are not effective when placed 3` to the gene or in
the distal 5` flank (Brickner et al., 1991). Thus, the
-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 /
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
-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
-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
-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
-like globin genes or around the mouse
1-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
-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 -globin gene is sufficient to respond to this enhancer
(Pondel et al., 1992; Ren et al., 1993), (
)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
-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.