From the Divisions of Medical Genetics and
§ Hematology, University of Washington, Seattle, Washington
98195 and ¶ Bristol-Myers Squibb Pharmaceutical Research
Institutes, Seattle, Washington 98121
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ABSTRACT |
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To delineate the regulation of the human
-globin gene, we investigated
-gene expression during the
development of transgenic mice carrying constructs with
-promoter
truncations linked to a micro-locus control region (µLCR). Expression
levels were compared with those of µLCR
mice carrying a 2 kilobase
-promoter and
YAC controls.
mRNA in the embryonic cells
of µLCR (
179)
mice were as high as in µLCR
mice suggesting
that the proximal
-promoter contains most elements required for
-gene activation.
mRNA in adult µLCR (
179)
mice was
significantly lower than in the embryonic cells indicating that
elements involved in
-gene silencing are contained in the proximal
-promoter. Extension of the promoter sequence to
463
decreased
-gene expression in the definitive erythroid cells, supporting
previous evidence that the
179 to
463
region contains an
-gene silencer. However, the
-gene of the µLCR(
463)
mice
was not silenced in the definitive cells of fetal and adult
erythropoiesis indicating that additional silencing elements are
located upstream of position
463
. These results provide in
vivo evidence that multiple elements of the distal as well as the
proximal promoter contribute to
-gene silencing.
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INTRODUCTION |
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All animal species have different hemoglobins in the embryonic and
definitive stages of development. These hemoglobins are synthesized
under the control of globin genes whose expression is restricted to
either the definitive or the primitive stages of erythropoiesis. In the
mouse there are two embryonic genes, and
h1, and two adult genes
major and
minor; expression of embryonic genes is totally
restricted to the yolk sac stage of erythropoiesis, whereas adult gene
expression starts only after the onset of definitive hematopoiesis in
the liver of the 11-day-old mouse fetus. In humans, the first gene of
the
globin locus to be expressed is the embryonic (
) followed by
the two fetal (
) genes and the adult
and
genes (1).
-globin synthesis occurs predominately in primitive yolk sac origin
erythroblasts, where it accounts for over 80% of
-like globins at 5 weeks of gestation, falling to 15% by week 7 (2-5). In humans, the
-gene is totally and permanently silenced after the 7th week of
gestation. The silencing of the
-gene is controlled at the
transcriptional level as shown by the total absence of a DNase I
hypersensitive sites in the
-gene promoter of erythroid cells of
54-day-old human embryos (6).
The absolute nature of -globin gene silencing is remarkable in view
of the relative proximity of the
-gene to the locus control region
(LCR),1 residing 6-22 kb
upstream. The LCR, characterized physically by five DNase I
hypersensitive sites (HS), influences chromatin structure over the
entire
globin domain (7), acts as a powerful erythroid-specific
enhancer (8, 9), and protects linked globin genes from the effects of
surrounding chromatin (9, 10). Expression of the
-gene in transgenic
mice requires the presence of the LCR (11, 12); in mice bearing a
2.5-kb micro LCR cassette linked to the
-globin gene,
-globin
expression is restricted to the primitive, yolk sac origin
erythroblasts (11). Transgenic mice bearing a fragment containing the
LCR hypersensitive sites 1 and 2 and contiguous
5'-flanking
sequence fused to the coding region of the
globin gene display an
embryonic pattern of transgene regulation (13), suggesting that the
cis elements necessary for proper developmental control of
-globin gene are contained within its promoter and 5'-flanking
sequence (12-14). Several negative regulatory elements have been
identified upstream of the
-globin gene (14-18). Transient
expression assays have localized a silencer between 392 and 177 base
pairs upstream of the
-globin cap site (15, 16). Transgenic mice
carrying a 2-kb
-gene promoter from which the
177 to
392
silencer has been deleted express the
-globin gene in the adult
stage of development (14).
Transgenic mice provide an excellent model for delineation of the
sequences necessary for -gene silencing, because of the absolute
restriction of
-globin gene expression in the yolk sac cells of the
mouse. If such sequences exist, their deletion or mutation is expected
to be associated with loss of
-gene silencing resulting in
continuation of
-gene expression in the adult stage of development.
In the experiments described in this report developmental studies were
performed using transgenic mice containing an LCR cassette linked to an
-globin gene containing only 179 or 463 bp of promoter sequence. We
found that although a significant level of developmental control is
retained in the proximal promoter, the bulk of
-gene silencing
resides with sequences located upstream of
179
. These sequences
include but are not limited to the
177 to
392
-silencer. These
results provide in vivo evidence that elements located both
in the proximal as well as in the distal
-gene promoter are involved
in
-gene silencing.
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EXPERIMENTAL PROCEDURES |
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DNA Constructs (Fig. 1)--
pµLCR(463)
was produced from
a modified version of pµLCR
, which contains 2 kb of sequence
upstream of the
-globin gene transcription start site. The µLCR
cassette of pµLCR
was modified to provide an additional 0.6 kb of
sequence 5' to HS4 of the original µLCR, producing 3.1-kb pµLCR
(thereafter, the term µLCR used in the text represents this new LCR
cassette except where otherwise indicated). pµLCR
was linearized
using SmaI, partially digested using BfrI, and
the digestion products were blunted using Klenow enzyme. An 8.3-kb
product was gel purified and religated to generate pµLCR(
463)
.
To generate pµLCR(
179)
the construct pµLCR
was linearized
using SmaI, partially digested using BamHI, and
the digestion products were blunted using Klenow enzyme. A 7.9-kb product was gel purified and religated to produce
pµLCR(
179)
.
Transgenic Mice--
Transgenic mice carrying the
pµLCR(179)
and pµLCR(
463)
constructs were produced as
described previously (19). Founder animals were identified by Southern
blotting with an
-gene sequence probe. F1 progeny were obtained by
breeding founder animals with nontransgenic mice and were screened for
correct integration and to exclude the presence of mosaicism in the
founders. To study the developmental pattern of human
-gene
expression, staged pregnancies were interrupted on days 9, 12, and 16 of development. Samples from blood and yolk sac were collected on day 9 embryos; blood, yolk sac and liver were collected on day 12 fetuses;
and blood and liver were collected on day 16 fetuses.
mRNA Quantitation--
Total RNA was isolated from
transgenic tissues by the method of Chomczynski and Sacchi (20). The
mRNA level was measured by the quantitative RNase protection
assay described previously (21). Briefly, riboprobe for
mRNA
was labeled by transcribing the linearized plasmid pT7
(188) using T7
RNA polymerase (22). The
probe protects a 188-bp fragment in exon 2 of
mRNA. The mouse
and
riboprobes were used in RNase
protection assays as internal globin mRNA controls. mRNA levels
were determined in all transgenic siblings of each litter. RNA samples
from different tissues were analyzed at least twice to reduce
experimental error in mRNA quantitations. Human
and mouse
and
signals were quantitated with a PhosphorImager. Levels of human
mRNA per transgene copy were expressed as percentages of mouse
-like mRNA levels per copy, taking into account that the mouse
possesses four copies of the
-globin gene and two copies of the
-globin gene. In the adult stages of development when
mRNA
is absent, murine
mRNA per copy was calculated by dividing the
levels of murine
mRNA by four.
Copy Number Determination--
Copy number determination was
accomplished by the multiply redundant protocol described previously
(21) to reduce experimental errors. Multiple DNA samples were obtained
from each of at least three animals from each transgenic line. These
samples were digested with restriction enzyme EcoRI and
resolved by electrophoresis over 1% agarose gel. Southern blots were
hybridized with a radiation-labeled probe by using a 0.6-kb
BamHI fragment of the
-globin gene as template. The
signals were quantitated on a PhosphorImager. Copy numbers were
calculated by determining the relative intensity of signals from a
given transgenic line compared with the signals obtained from diploid
human genomic DNA.
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RESULTS |
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Constructs (Fig. 1)--
The
construct µLCR(179)
consists of a 1.9-kb
-gene fragment that
contains the
-globin gene, 179 bp of sequences of the
-gene
promoter and 280 bp of 3'-nontranslated sequences. The
463
construct contains
genomic sequences identical to those of the
179
construct, but the promoter is extended to a BfrI site at position
463. This construct therefore contains the
177 to
392 sequence previously shown in transient assays (15) and transgenic
mouse studies (14) to behave like an
-gene silencer.
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Analysis of -Globin Gene Expression in Cells of Embryonic and
Definitive Murine Erythropoiesis--
For developmental studies, timed
pregnancies were interrupted at 9, 12, and 16 days, and yolk sac,
blood, and fetal liver samples were collected for measurement of human
and murine
and
mRNA by RNase protection (Fig.
2). More than one tissue was analyzed in
each gestational day to increase the accuracy of
mRNA
measurements. Multiple members from each litter were used for
measurements of globin mRNA. Data from each line and each day are
presented in Tables I and
II as means ± S.D.
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-Gene Expression in the Embryonic Cells of µLCR(
179)
Transgenic Mice and Controls--
The µLCR(
179)
construct
contains all the transcriptional motifs of the proximal
-gene
promoter, i.e. the TATA box at
30, the CAAT box at
84,
the CACC box at
113, and a GATA-1 site in position
165 shown before
to be required for up-regulation of
-gene expression (29). Although
the proximal promoters of the
-like globin genes share a basic
organization, several differences also exist between promoters.
Differences between the
-and
gene promoters include the presence
of a duplicated CAAT box in the
-promoter and divergence of
sequences surrounding the TATA, CAAT, and CACC boxes. Presumably these
structural differences contribute to the difference in the
developmental regulation of
-and
genes in humans.
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-Gene Expression in the Embryonic Erythropoiesis of
463
-Transgenic Mice--
The µLCR(
463)
construct contains the
sequence
177 to
392
previously shown to harbor an
-gene
silencer (14, 15). This silencer contains sites that bind
erythroid-specific as well as constitutive transcriptional factors
(16-18, 28, 30), a GATA-1 binding site at
208, two GATA-1 binding
sites that overlap with a YY1 site at
269; and a CCACC site at
379
that binds Sp-1 or a related factor.
mRNA Levels in Cells of Definitive Erythropoiesis of the
µLCR
and
YAC Mice--
As shown in Table II, the 12-day fetal
liver samples of the
YAC mice contain from 2 to 3.7%
mRNA
deriving from contaminating embryonic erythroblasts. There is no
mRNA in the 16-day blood and liver samples or in adult blood of the
YAC transgenic mice. In the 3.1 µLCR
and 2.5 µLCR
mice,
traces of
mRNA are detected in the 16-day liver (0.11%) and
adult blood (0.18-0.67%). Therefore, compared with the
-locus
YACs, the two µLCR
constructs are "leaky" and allow synthesis
of residual levels of
mRNA in the cells of definitive
erythropoiesis.
mRNA Levels in Definitive Erythroid Cells of
179
Mice--
Fig. 4 shows that
-gene
expression in the 12-day fetal liver of the six µLCR(
179)
lines
is significantly higher than in the 2.5 µLCR
and 3.1 µLCR
controls. The difference is even more striking in the 16-day fetal
liver in which the levels of
mRNA in µLCR(
179)
mice are
30-50-fold higher than in the 3.1 µLCR
control (Table II). The
µLCR(
179)
construct, however, has not totally lost the ability
to down-regulate
-gene expression, because, as shown in Fig.
5A, there is a significant
decline in
mRNA levels in the adult µLCR(
179)
-transgenic
mice. This is in contrast to a µLCRA
gene containing only the
proximal
-promoter (µLCR(
201)A
), which is not down-regulated
in the adult and it directs similar levels of
mRNA in the adult
and in the embryonic cells of transgenic mice (19).
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mRNA Level in Definitive Cells of the µLCR(
463)
Mice--
Despite the presence of the
177 to
392 silencer in the
µLCR(
463)
construct, the 12-day definitive erythroid cells of
these mice had
mRNA levels that were about 2-fold higher than
the levels of the 3.1 µLCR
or
YAC controls (Table II and Fig.
4). Levels of
mRNA in the 16-day fetal tissues of
µLCR(
463)
mice are 20-40-fold higher than in the µLCR
control (Fig. 4). Therefore the definitive erythroblasts of the
µLCR(
463)
mice synthesize significant amounts of
mRNA.
The levels of
mRNA in adult µLCR (
463)
mice, are
significantly lower than in adult µLCR(
179)
mice (Fig. 5) most
likely reflecting the presence of the
179 to
392
-silencer.
-Expression in the adult µLCR(
463)
mice is 10-20-fold higher
than in the adult 3.1 µLCR
control (Table II), most likely
reflecting the presence of silencing elements upstream of
463
.
Expression of µLCR (179)
and µLCR (
463)
Transgenes Is
Independent of Position of Integration--
The results in Tables I
and II show consistency of expression of the
-gene in the mouse
lines carrying the µLCR(
179)
or the µLCR(
463)
constructs.
The mean
-gene expression in the µLCR(
179)
mice is 18.9 ± 5.5 in the day 12 embryonic blood and 5.3 ± 2.2 in adult
blood. The coefficients of variation in per copy expression between the
µLCR (
179)
lines are 0.29 for the fetal day-12 embryos and 0.41 for the adult stage, respectively. Small coefficients of variation
(less than 0.5) indicate that the expression of a transgene is
independent of the position of integration (21). Since
-gene
expression is not influenced by the position of integration of the
transgenes, the developmental profiles shown in Tables I and II must
reflect an inherent property of the µLCR(
179)
construct. It is
noteworthy that copy number-dependent expression was also
previously observed in the µLCR(-201) A
transgenic
mice containing only the proximal
gene promoter (19). Copy
number dependence of
expression is also characteristic of the
construct µLCR(
463)
. Thus, the coefficient of variation is 0.38 in the day-12 embryonic blood and 0.39 in adult blood of the
µLCR(
463)
-transgenic mice, indicating that the expression of
this transgene is also independent of position of integration.
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DISCUSSION |
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Several studies have shown that sequences in the -gene promoter
as well as in the LCR participate in the developmental regulation of
the
-globin gene. Experiments in transgenic mice have shown that, in
the absence of the LCR, the human
-genes remain silent in the
embryonic cells of transgenic mice, indicating that the LCR is
necessary for in vivo
-globin gene transcription (11, 12). The contribution of LCR sequences in the developmental control of
-gene has been clearly shown in studies of
YAC transgenic mice
containing deletions of DNase I hypersensitive site 3; mice carrying a
2.5-kb deletion removing HS3 and the surrounding flanking sequences
display decreased
-globin expression in the embryonic erythropoiesis
(26), whereas mice carrying deletions of the core element of HS3
display total absence of
-expression in day-9 embryonic cells and
severe reduction of
-expression in day-12 embryonic cells (31). Such
data suggest that sequences of the core element of HS3 are necessary
for activation of
-gene transcription. The contribution of elements
of the
-gene promoter have been investigated in vitro,
with transient expression assays and in vivo, in transgenic
mice. Studies in transiently transfected cell lines have revealed
sequences that either positively (17, 29, 30, 32-34) or negatively
(15-17, 35) influence transcription from a linked reporter gene.
Several of these sequences have been evolutionarily conserved (36).
Studies in transgenic mice have shown that all the cis
elements required for
-gene silencing are located in a 2.0-kb
fragment that contains the
-gene promoter (11, 28).
In this study we wished to examine to what degree the proximal and
distal -gene promoter are involved in the developmental regulation
of the
-globin gene in vivo. We used control constructs containing the whole
-locus or a 2-kb
-promoter and constructs with an
-promoter containing only the essential transcriptional motifs or these essential motifs as well as a previously identified upstream silencer. We found high levels of
mRNA in the adult cells of transgenic mice carrying only the proximal
-promoter indicating that structural elements located in the distal
-gene promoter are critical for
-gene silencing. The level of
mRNA in the adult cells decreased when sequences containing the previously described silencer were added, providing further evidence for the
in vivo function of this element. However, presence of this silencer did not totally suppress
-gene expression in definitive cells suggesting that additional elements located in the distal
-promoter are involved in
-gene silencing. Although mice carrying only the proximal
-promoter have high levels of
-gene expression in the adult stage of development, the level of
mRNA in the adult cells is strikingly lower than in embryonic cells, suggesting that sequences located in the proximal
-promoter participate in
-gene silencing. Overall, our results provide in vivo
evidence that multiple elements located in the distal as well as in the proximal
-gene promoter are involved in
-gene silencing.
Liu et al. (37) have recently reported that a sequence
located between position 179 to
304 of the
-gene promoter
contains a positive regulatory element, the deletion of which in the
context of the
-locus YAC results in catastrophic reduction of
-globin (as well as on
-globin) gene expression. This finding
contrasts to the results of the present study, which shows that the
levels of
mRNA in the embryonic cells of
µLCR(
179)
-transgenic mice are as high as in transgenic mice
carrying an
promoter extending 2-kb upstream of the
-gene cap
site. Such findings imply that most of the cis elements
necessary to interact with the LCR are located in the proximal
-gene
promoter. The results of Liu et al. also contrast to the
results of a previous study in which a
179 to
463
sequence was
deleted from a 2-kb
-promoter. Transgenic mice carrying this
construct have normal
mRNA levels in embryonic erythroblasts
(14), although they should have lacked
expression if a positive
element with the characteristics described by Liu et al.
(37) was located in the deleted sequence.
There are several explanations for these discrepant findings. First, it
is known that YACs have a considerable tendency for rearrangements and
YAC transgenic lines may carry several YAC integrants, each showing
different structural rearrangements (24). The finding of Liu et
al. (37) could be explained if such rearranged YACs were contained
in the transgenic lines they studied, a possibility that has not been
totally excluded with the structural analyses done. Second, as previous
studies have shown, globin genes behave differently when they are
individually linked to the LCR in short constructs or when they are
present in constructs containing the whole -locus. Thus, when the
genes are linked to a 2.5-kb µLCR (38) or a 20-kb LCR (39), they
are expressed in embryonic as well as adult cells, whereas they are
totally silenced in embryonic cells when they are located in
cosmids (38, 39) or in
YACs (27, 40). Similar behavior has been
documented with
-globin genes (19, 38, 39). Such results do not
invalidate the use of short constructs but they point to the different
insights obtained when short globin gene-LCR constructs or constructs
containing the whole
-locus are used in transgenic mouse
experiments. The short constructs are useful for delineating the
functional role of specific sequences flanking the globin genes or
elements contained in the HSs of the LCR. Such constructs have allowed
the delineation of specific cis elements of the
- (41) or
- (19) gene promoters or the HSs of the LCR (41-45). Since, in the
intact
-locus, the competition between globin gene promoters to
interact with the LCR becomes the dominant determinant of gene
expression, "whole locus" constructs are most useful for addressing
questions on the control of globin gene switching. It is likely that
certain regulatory elements have different functions in the different stages of development, and these functions could be identified by the
use of the two types of constructs. The discrepancies between our
results and those of Liu et al. (37) perhaps reveal that the
179 to
392 sequence has a dual function. In the presence of the
transcriptional environment of definitive erythropoiesis this element
may act as an
-gene silencer, and this function was depicted when
the LCR
-gene constructs were used in transgenic mice. In the
presence of an embryonic transcriptional environment and an intact
-locus, this sequence may behave as an "anchor" that facilitates
the interaction of the LCR with the
-and
genes of embryonic
cells; this function was perhaps depicted when in the studies of Liu
et al. (37) a
-locus YAC with a deleted
179 to
392
sequence was used.
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ACKNOWLEDGEMENTS |
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We thank Harold Haugen, Sara Shaw, and Heimei Han for expert technical help and Sherri Brenner for assistance in the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by Grants DK45365, HL20899, and HL53750 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Medical
Genetics, Box 357720, University of Washington, Seattle, WA 98195. Tel.: 206-543-3526; Fax: 206-543-3050; E-mail:
gstam{at}u.washington.edu.
1 The abbreviations used are: LCR, locus control region; kb, kilobase(s); HS, hypersensitive sites; bp, base pair(s).
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REFERENCES |
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