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In Vivo Search for Butyrate Responsive Sequences Using Transgenic Mice
Carrying
Gene Promoter Mutants
By Betty S. Pace, Qiliang Li, and George Stamatoyannopoulos
We describe an in vivo approach, in transgenic mice, aimed
to identify promotar elements responsible for the induction
of y globin expression by butyrate. Transgenic lines carrying
human ‘ y gene promoter truncations at position -141,
-201, -382, and -730 ‘y were treated with a amino butyric
acid (aABA), and effects on y globin expression were anaRNA level. No induction of y gene
l v e d at the m-nger
expression was observed in animals carrying promoters
truncated at positions -141, -201, or -382 ’y, suggesting
either that butyrate responseelements (BREI are not located
in the proximal y gene promoter or, if they were, they require
the cooperation of upstream sequences for y gene induction.
Two animals from one line carrying the -730 * y truncation
responded to aABA treatment with significant increases in
y gene expression, indicating that a BRE is located between
position -382 and -730 region of the ‘ y gene promoter.
Because the maximum induction by aABA is observed in
transgenic mica carrying a ‘ y gene promoter extending to
nucleotide -1350, it is likely that another butyrate responsive element is located between -730 and -1350 of the ‘y
gene promoter. These results indicate that the transgenic
mouse model can be used for identificationof DNA regions
that contain ciselementsinvolved in y globin gen6 inducibility.
0 7996 b y The American Society of Hemstology.
B
miniosmotic pump with a capacity of 200 pL. The small capacity
of the miniosmotic pump precluded the use of properly neutralized
sodium butyrate, at the doses needed for our study. In contrast,
aABA can be dissolved in normal saline, and the final volume of the
solution containing the desired dose can be readily accommodated in
the miniosmotic pump. aABA (Sigma Chemical CO,St Louis, MO),
250 mg/kg/d, was dissolved in normal saline and given subcutaneously over a 7-day period through a surgically implanted miniosmotic pump (Alza Corporation, Palo Alto, CA) according to the
manufacturer’s specifications.
Hematologic studies. Fifty microliters of blood was drawn from
the retroorbital space of the transgenic mice for the analyses described below. The reticulum in the red blood cells was precipitated
by incubating blood with 1% brilliant cresyl blue, and reticulocyte
counts were calculated by counting 1,OOO cells. Measurements of F
cells (hemoglobin F containing erythrocytes) were performed on
methanol-fixed blood smears labeled by an indirect immunofluorescent assay using a monoclonal anti-y-chain antibody and anti-mouse
F (ab‘)*-fluorescein isothiocyanate (Tag0 Diagnostic, Inc, Burlingame, CA).
RNA analysis. Total cytoplasmic RNA was prepared by the
method of Chomcynski and Sacchi.” RNA concentration and purity
was assessed with UV spectrophotometry.” Globin messenger RNA
(“A)
was analyzed by RNase protection with the following
probes: P T , ~(170).
~ linearized with BstEII, to give a 170-bp protected fragment, and pT,mousea (128) linearized with HindIII to
give a 128-bp protected fragment. RNA (200 ng) was hybridized
overnight at 45°C with lo6 cpm of each radiolabeled probe. After
digestion with RNase A, the protected fragments were separated on
a 6% polyacrylamide-8 m o m urea gel and autoradiographed without
an intensifying screen. Human Ay and murine a “A
was quanti-
UTYRATE AND its analogues have been shown to
induce embryonic globin gene expression in anemic
chicken,’ to inhibit the y to p switch in sheep,’ and to induce
fetal hemoglobin in baboon^,^" in transgenic mice carrying
y globin gene constructs: and in a proportion of patients
with sickle cell disease or homozygous
Butyrate also influences the expression of several other genes.”
It has been postulated that specific cis acting elements are
responsible for the induction of gene expression by butyrate,” and a search for such elements has been done in
various genes.”-’5Typically, the search for butyrate response
elements (BRE) is done by transfecting recombinant genes
into established cell lines and by measuring levels of gene
expression after treatment with butyrate.”-LSIn this study,
we search for BRE using an in vivo approach. Mice carrying
various y gene promoter truncations were treated with a
butyrate analogue, a amino butyric acid (aABA), which has
been previously shown to induce y globin gene expression
in human and baboon erythroid cells3as well as in transgenic
mice carrying a Ay gene promoter extending to position
-1350
If BRE were present along the y gene promoter, mice carrying truncated constructs lacking these elements were expected to fail to respond to aABA by increasing y gene expression. Mice carrying y gene promoters
containing a BRE were expected to respond to butyrate treatment by increasing y gene expression. Our results suggest
that this in vivo approach allows the identification of regions
of the y gene promoter in which BRE are located. The
approach may prove useful in the localization of cis sequences involved in induction of y globin genes by cytotoxic
drugs or other compounds inducing fetal hemoglobin.
MATERIALS AND METHODS
* y gene constructs. A diagram illustrating the truncations of the
Ay promoter used to make the recombinant DNA constructs is shown
in Fig 1. All y gene fragments are linked to a p locus control
region (pLCR) cassette containing core elements from DNAse I
hypersensitive sites of the PLCR. All Ay gene fragments have the
same 3’ end (the HindIII site) at position +1950. The 5’ ends are
at positions -141, -201,-382, and -730 relative to the * y gene
capsite. These constructs have been described previous1y.l’
Compounds. Induction of fetal hemoglobin in humans or baboons require the continuous intravenous administration of butyrate.
Continuous infusion of drugs to the mouse is achieved by using a
Blood, Vol88, No 3
(August I), 1996: pp 1079-1083
From the Department of Structural and Cellular Biology, University of South Alabama, Mobile; and the Department of Medicine,
University of Washington, Seattle.
Submitted June 14. 1995; accepted March 26, 19%.
Supported by National Institutes of Health Grant HL 20899 and
a Robert Woods Johnson grant to B.S.P.
Address reprint requests to Betty S. Pace, MO, Department of
Structural and Cellular Biology, MSB 2042, University of South
Alabama, Mobile, AL 36688.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advdsement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
OOO6-4971/96/8803~0Q36$3.00/0
1079
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ioao
-1350
I
PACE, LI, AND STAMATOYANNOPOULOS
-7?0
-3p2
-20 1
Fig 1. pLCR”y promoter truncations used for production of
transgenlc mice. Dlagramof the y globin promotershowing the position of transcriptionalmotifs and other regulatory elements. The 5’
end. of the “y promoter truncations ere indlcated with arrows and
a number showing the site of truncation. -141 is an NCOI site, -201
is an Apa I site, -382 is a shc I site, -730 is a Ssp I site, and -1350
is a H/ndlll site. Each of the five constructstested contain the 2.5 kb
pLCR ceuette and the human ”y globin gene (with a 3’ end at position +1950 ~Hhdlll&e)relative to the cap site). 8 corresponds to a
region containing e putetlve negativeelement identifiedwith studies
in transgenic mice.”
tated using a Molecular Dynamics PhosphorImager (Sunnyvale,
CA). Human y globin gene expression per copy of the transgene
and per copy of the endogenous murine a gene was calculated as
%ylMocu/copy.
RESULTS
Experimental approach. The truncations of the y globin
promoter used to make the five constructs are shown in Fig
1. Animals were treated with a continuous infusion of
aABA, 250 mgkg/d, for 7 days using a miniosmotic pump.
Peripheral blood samples were analyzed for y mRNA production, and F cells were measured using a fluorescent antiy chain antibody. y mRNA levels were expressed as a
percentage of murine a mRNA, after correction for the expression per copy of the transgene and per copy of the endogenous murine a gene. A consistent increase in y mRNA
production during several days of treatment was scored as
a positive response to butyrate treatment. Lack of an increase
in y mRNA production was not automatically scored as a
negative response because it could be due to a lack of biological effect of butyrate in the treated animals. As an independent measure of biological effect of butyrate in such animals,
we used the property of aABA to stimulate erythropoiesis
and induce reticulocytosis. Reticulocytes were counted for
several days after treatment, and a positive response to
aABA was scored when the level of peak reticulocytosis
was 50% higher than the baseline pretreatment value.
Mice carrying -141 *y constructs. Truncation of the Ay
promoter at position -141 conserves all of the essential
elements of the promoter (the duplicated CAAT box, TATA
box, etc) but it destroys the CACC site around nucleotide
-141. These mice express y globin in the fetal stage of
development but lack y expression in the adult stage, suggesting that a negative element, perhaps the site of binding
for y gene repressors, is located 3‘ to position -141 in the
Ay gene promoter.” cis elements in this region of the y
promoter previously shown to be involved in y gene developmental regulation include the distal CAAT box that is
mutated in the - 117AyGreek type of Hereditary Persistence
of Fetal Hemoglobinzo.zland a “stage selector element”
located in the -50 Ay region.” Because the Ay gene of the
-141 Ay construct is “on” in the transgenic fetuses, it was
reasonable to ask whether treatment of adult mice with butyrate will result in induction of y gene expression.
Six animals from three lines carrying this construct were
used. Treatment with aABA increased reticulocytes about
fourfold in animal 2 of line 134/1-5, 1.8-fold in animal 1 of
line 134/1-5, and 2.6-fold in animal 1 of line 134/5-6. In
these animals, pretreatment y mRNA levels were lower than
1% of murine a mRNA and essentially remained unchanged
(Table 1). These results suggest either that no cis elements
that can respond to in vivo administration of butyrate are
located 3‘ to nucleotide - 141 of the Ay promoter or that the
lack of response is due to the fact that the “ y gene of the
- 141 Ay mice is silenced in the adult.
Mice carrying -201 Ay or -382 Ay constructs. Addition
to the - 141 “ y construct of the sequence between -141 and
-201 of the Aypromoter results in the loss of developmental
regulation so that the level of y gene expression in adult
animals is equal to that of the fetuses. Therefore, transgenic
mice carrying a y gene promoter extending to position -201
Ayor -382 Ay display abundant y gene expression. Furthermore, in these animals, there is an excellent correlation between level of y gene expression and the number of the
integrated transgenes indicating that y gene expression is
Table 1. y Globin Gane Induction After Treatment With aABA
Transgenic M i Carrying the pLCR -141”y Construct
YlMOUl
Transgenic
Animal
Animal
13411-5
1
Copy
Day of
No.
Treatment
2
0
6
7
8
9
13411-5
2
2
0
6
7
a
13411-5
3
2
9
0
6
7
a
13413-2
13415-6
1
1
49
8
9
0
5
7
8
9
0
6
7
a
134/5-6
2
8
9
0
6
7
a
9
Reticulocytes
(%)
F Cells 1%)
2.5
3.5
4.2
4.5
4.2
1.8
4.8
7.5
5.7
6.1
2.3
1.5
3.9
1.5
5.6
2.6
2.0
4.0
4.4
4.2
2.2
3.7
4.4
5.3
5.8
3.0
5.1
3.0
5.4
5.6
0.6
1.7
1.5
1 .o
1 .o
0.3
0.2
0.8
0.5
0.2
0.3
0.6
0.2
0.4
0.8
Not done
1.7
0.6
0.7
0.8
0.9
0.1
0.2
0.1
0.1
0.1
COPY
(%)
0.08
0.07
0.08
0.14
0.12
0.20
0.20
0.20
0.17
0.24
0.10
0.14
0.18
0.12
0.10
0.49
0.50
0.53
0.43
0.19
0.31
-
0.20
0.20
0.43
0.52
0.56
0.52
0.50
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1081
IN VIVO SEARCH FOR BRE
copy-number dependent.17 These results have been interpreted to suggest that the -141 to -201 region of the Ay
promoter represents the site of interaction between the promoter and the LCR.I7If a cis element responsive to butyrate
is placed 3’ to -201 Ay, one would expect that y gene
expression will be induced when the -201 Ay or the -382
Ay transgenic mice are treated with a-ABA.
Two lines carrying the -201 Ay construct were used. The
baseline y mRNA level of line 136/3-4 was 25.5%of murine
a. Treatment with aABA increased reticulocytes by about
1 J-fold, but there was no further increase in y mRNA. The
mouse from line 136/4-2 had a baseline y mRNA level
17.4%of murine a;there was no y gene induction although
aABA stimulated production of reticulocytes (from a baseline value of 5% to a peak value of 8.5%) (Table 2).
The three lines carrying the -382 Ay promoter construct
(Table 3) responded to aAJ3A with consistent increases in
reticulocyte production (from 6.1% to 9.0%, line 135/1-2;
from 3.0%to 7.3% line 1394-2; from 4.0% to 6.0% line
135/4-8). Two lines (135/1-2 and 135/4-2) failed to change
y gene expression during treatment (Table 3). y mRNA
increased in line 135/4-8 from 18.9%to 29.2% on treatment
day 5, but it was 22.8% on day 6 and 23.1% on day 7,
suggesting that the higher y mRNA level of day 5 was due
to incorrect measurement.
Transgenic mice carrying the -730 A y construct. Extension of the Ay gene promoter to the -730 Ay site results in
two changes in Ay gene expression. First, Ay gene expression
becomes copy number independent, so that the excellent
correlation between the copy number of the transgene and
the level of Ay gene expression that is characteristic of the
-201 Ay and the -382 Ay transgenic mice disappear^.'^
This phenomenon indicates that the -382 to -730 sequence
renders the ’7 promoter sensitive to position effects. Second,
in several -730 Ay transgenic lines, there is developmental
regulation of y gene expression (ie, high Ay gene expression
in the fetus but very low expression in the adult).I7 These
observations were interpreted to indicate that the -382 to
-730 region contains a sequence that acts as a silencer,
hence the low y globin expression in the adult animals of
certain lines.
Eight transgenic animals from four lines were used (Table
Table 2. y Globin Gene Induction After Treatment With aABA
Transgenic Mice Carrying the pLCR -2OlAy Construct
F
COPY
Transgenic
Line
Animal
No.
pLCR-20lAy
13613-4
1
5
Day of
Treatment
Reticulocytes
(%)
0
6
7
2.6
1.6
3.4
4.0
4.4
5.0
4.2
8.5
4.4
5.9
8
13614-2
1
3
9
0
6
7
8
9
Cells
ylMout
Copy
(461
(%)
90
89.5
25.5
15.0
20.4
23.9
22.3
17.4
15.9
20.3
18.4
16.4
Table 3. y Globin Gene Induction Aftor Treatment With aABA
Transgenic Mice Carrying the pLCR - e y Construct
YmW
Transgenic
Line
pLCR-382Ay
13511-2
Animal
Copy
No.
Day of
Treatment
1
6
0
6
7
Reticulocytes F Calls
(%J
(%)
8
13514-2
1
5
13514-8
1
3
9
0
5
6
7
8
0
5
6
7
6.1
8.9
9.0
6.5
6.0
3.0
7.3
5.0
2.6
3.7
4.0
4.7
5.5
6.0
90.4
88.5
90
Copy
1%)
9.3
-
7
8.1
10.2
15.9
16.1
16.1
17.2
17.4
18.9
29.2
22.8
23.1
4). Line 137/1-2 has barely detectable y gene expression in
the adult; the two animals of this line failed to respond to
butyrate treatment by increasing y mRNA (although reticulocytosis occurred in one animal). y mRNA levels in line
137/2-4 range from 1.5%to 1.8% of murine a. Animal 1 of
this line responded to butyrate with brisk reticulocytosis but
increased y mRNA only slightly over baseline levels. aABA
did not induce significant reticulocytosis in animal 2. y
mRNA levels in line 137/5-2 is 6.4% of murine a;this line
failed to respond to butyrate treatment (Table 4). The three
animals from line 137/5-1 had y mRNA levels of 5.5%,
12.8%, and 16.7% of murine a.The study of animal 1 was
incomplete because “A
measurements were done only
on days 6 and 7; y mRNA did not increase above the baseline
level in these 2 days. Animal 2 increased y mRNA from a
baseline value of 12.8%to 22.4% by day 9 (Table 4 and Fig
2). Animal 3 increased y mFWA from baseline value of
16.7%to 51.1% by day 9 (Fig 2).
DISCUSSION
It has been previously shown that transgenic mice carrying
a y gene construct with Ay promoter extending to position
-1350 Ay increase y gene expression in response to treatment by 5 azacytidine, butyrate, erythropoietin, hydroxyurea, or Didox, an inhibitor of ribonucleotide red~ctase.’*~.*~
The molecular mechanisms whereby these compounds induce y globin expression remain unknown. 5-Azacytidine
presumably demethylates regulatory sequences, thus modulating the interaction between these sequences and DNA
binding proteins. A likely mechanism of action of the other
hemoglobin (Hb)F-inducing agents is through modulation
of proteins that interact with DNA regulatory sequences.
Nothing is known about the regulatory sequences of the y
globin gene through which these Hb F inducers presumably
exert their effects. In this report, we test whether regions
containing such sequences can be recognized with in vivo
studies in transgenic mice.
Truncations of the Ay gene promoter at position -141,
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PACE, LI, AND STAMATOYANNOPOULOS
1082
Table 4. y Globin Gene Induction After Treatment With aABA
Transgenic Mice Carrying the pLCR -73Py Construct
Transgenic
Line
13711-2
13711-2
13712-4
13712-4
13715-2
13715-1
13715-1
13715-1
Animal
Copy
No.
F
y/Moa/
Day of
Reticulocytes
Cells
Treatment
(%)
(%)
Copy
I%)
0
4
5
6
1
0
6
3.9
3.5
3.6
3.4
4.5
2.8
2.6
2.6
4.2
4.2
3.1
8.7
1.3
7.2
4.5
3.7
Rare
Rare
0.1
Rare
Rare
0.3
0.8
0.1
0.8
2.0
Rare
Rare
Rare
Rare
Rare
0.5
5.9
0.1
1
8
9
0
4
5
6
7
0
6
7
8
9
0
6
1
8
9
0
6
7
0
6
1
8
9
0
6
1
8
9
4.3
2.1
6.3
7.0
6.4
2.4
3.5
3.8
2.0
3.1
4.4
5.3
5.8
1.9
2.3
2.5
4.4
10.9
90
90
90
90
0.04
0.02
0.05
0.04
0.03
0.10
0.09
0.12
0.04
0.04
1.8
2.4
2.5
1.7
2.3
1.5
1.o
1.5
1.o
1.o
6.4
5.0
6.7
6.7
7.0
5.5
4.0
3.0
12.8
14.1
14.1
18.4
22.4
16.7
23.5
30.6
31.5
51.1
they require the cooperation of upstream elements to elicit
a response to butyrate.
Previous evidence suggests that the -382 to -730 region
of the Ay promoter contains a y gene silencer that is sensitive
to “position effects” (ie, its action depends on the position
of integration of the transgene). The mechanism of the position-dependence of this element remains speculative. Perhaps the -382 to -730 Ay silencer acts by influencing the
conformation of the chromatin of the upstream y promoter
in a manner inhibiting the interaction between y promoter
and the LCR. Under this hypothesis, “position effects” arise
because the position of integration of the transgene affects
the conformation of this region and secondarily influences
the interaction between the silencer and the LCR. Of the
four -730 Ay transgenic lines treated with aABA, only two
animals of one line responded by increasing y gene expression by twofold to threefold. The fact that even two animals
responded to butyrate with significant increases in y mRNA
levels suggests that a BRE is located in the -382 to -730
Ay region. The lack of response in the other lines can be
attributed to position effects. Butyrate may activate y globin
expression by modifying DNA binding proteins and the interaction of such proteins with the y silencer may be modu-
A
30
B 601
-201 or -382 abolish the ability of butyrate to induce y
gene expression. These results make it unlikely that a butyrate responsive element is located 3’ to nucleotide -382 of
the Ay gene promoter. It could be argued, however, that the
lack of y gene induction in these mice is not due to absence
of a BRE in the proximal promoter but to the fact that the
Ay genes of the -201 and -382 animals have reached a
maximal level of expression and aABA could not induce y
gene expression further. This is not a strong argument, however, because levels of Ay gene expression higher than those
of -201 Ay, and -382 * y mice have been reached when
animals carrying a promoter extending to -730 Ay (animal
137/51-3 of Table 4) or to -1350 Ay’6 were treated with
butyrate. The lack of induction of the y gene of the -201
Ay and -382
transgenic mice suggests either that the y
globin BRE are located upstream of position -382 of the
Ay promoter or that if BRE are located 3‘ to position -382,
1371 5-11 2
1 13715-113
Days of Treatment
Fig 2. Induction of ‘ y mRNA production in two pLCR -730 ‘y
mice treated with oABA.
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IN VIVO SEARCH FOR BRE
lated depending on the position of integration of the
transgene.
The highest degree of induction of
gene expression by
butyrate has so far been observed in transgenic mice that
carry a Ay gene construct extending to position -1350 in
the
promoter?*16Although only one such pLCR
line
with promoter extending to - 1350 is available, multiple
generations of this line have been treated with butyrate and
induction of y globin expression has been consistently ob~ e r v e d . ~These
. ' ~ results suggest that another BRE is located
between positions -730 and - 1350of the * y gene promoter.
Our results demonstrate that in vivo studies in transgenic
mice allow identification of promoter regions that are involved in butyrate inducibility. This approach can be used
in the delineation of the cis elements involved in the induction of hemoglobin by other agents, such as hydroxyurea,
erythropoietin, or 5-azacytidine. Although the in vivo approach we describe here allows the localization of regions
containing BRE, further use of transgenic mice for pinpointing the structure of the localized BRE is prohibited by
the expense of producing and maintaining transgenic mice.
Studies using stable or transient expression systems are more
appropriate for that purpose. Once a BRE sequence has been
identified with in vitro assays, studies in transgenic mice can
be used to verify the in vivo relevance of the in vitro findings.
ACKNOWLEDGMENT
We thank Bonnie Lenk and Shem Brenner for their help in the
preparation of this manuscript.
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1996 88: 1079-1083
In vivo search for butyrate responsive sequences using transgenic
mice carrying A gamma gene promoter mutants
BS Pace, Q Li and G Stamatoyannopoulos
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