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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4383-4393
Coinduction of Embryonic and Adult-Type Globin mRNAs by Sodium
Butyrate and Trichostatin A in Two Murine Interleukin-3-Dependent Bone
Marrow-Derived Cell Lines
By
Kimiko Ishiguro and
Alan C. Sartorelli
From the Department of Pharmacology and Developmental Therapeutics
Program, Cancer Center, Yale University School of Medicine, New Haven,
CT.
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ABSTRACT |
Using an RNase protection assay, globin mRNA species expressed in
clones derived from Ba/F3 and B6SUtA cells transfected with the
erythropoietin receptor (EpoR) and selected with erythropoietin (Epo)
were compared with globin mRNA species induced in corresponding parental cells by sodium butyrate (SB) and trichostatin A (TSA).  Major/minor- and  -1/-2-globin
mRNAs were the major species, with trace amounts of  -globin mRNA,
formed in Epo-stimulated EpoR+ Ba/F3 clones, whereas SB
and TSA allowed expression of all species of globin mRNAs, ie, ,
h1, major/minor,  , and -1/-2,
in parental Ba/F3 cells. In contrast, - and -1/-2-globin
mRNAs were the major species present in Epo-stimulated EpoR+ B6SUtA clones, whereas SB and TSA activated -,
h1-, S/T-, and -1/-2-globin
genes in parental B6SUtA cells; -globin mRNA was not detected in SB-
and TSA-treated B6SUtA cells. Because TSA is a specific inhibitor of
histone deacetylase, the mimicry of action exhibited by SB and TSA
suggests that the effects of SB are mediated through its ability to
inhibit histone deacetylase and that histone deacetylase is an integral
part of the repression of globin genes in these
interleukin-3-dependent cells. Efficient coinduction of embryonic and
adult types of globin mRNA in bone marrow cell lines derived from adult
mice indicates that adult hematopoietic precursors possess an embryonic
nature. These cell lines are useful models to study the mechanism(s) of
developmental globin gene switching.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
MURINE INTERLEUKIN-3 (IL-3)-dependent
bone marrow-derived cell lines are considered to be immature because
of their dependency on IL-3 and to be "untransformed" because of
their dependency on a cytokine for survival and growth.1
These cells differ from cell lines established from hematopoietic
malignancies, which proliferate in the absence of cytokines and whose
movement along the pathway to maturity is impaired by alterations in
regulatory mechanisms. Two murine IL-3-dependent cell lines,
Ba/F32,3 and B6SUtA,4 acquire responsiveness to
the mitogenic and differentiation-inducing properties of erythropoietin
(Epo) on introduction of the Epo receptor (EpoR) by transfection.
Epo-induced differentiation is shown by accumulation of
major-globin mRNA in EpoR+ Ba/F3
cells2,3 and by accumulation of hemoglobin in B6SUtA cells.4 Whereas some leukemia cell lines have the ability
to terminally differentiate in vitro in response to a variety of nonphysiological chemical agents,5,6 the capacity of
factor-dependent cells to respond to chemical inducers of
differentiation has not been well studied. We have been interested in
establishing model systems in which signal molecules involved in the
differentiation pathways used by both Epo and chemical inducers can be
dissected. To this end, we have evaluated a wide range of potential
chemical inducers in B6SUtA cells using benzidine positivity to measure hemoglobin accumulation and have identified sodium butyrate, diazepam, and hemin as positive initiators of maturation.4 In this
report, we have analyzed in depth the globin mRNA species expressed in Epo-stimulated EpoR+ clones of Ba/F3 and B6SUtA cells and
have compared these with the globin mRNA species induced in parental
cells by chemical inducers. Furthermore, to determine whether sodium
butyrate (SB) exerts its action through an ability to inhibit histone
deacetylase, comparative studies were conducted with trichostatin A
(TSA), a specific inhibitor of histone deacetylase.7 These
investigations collectively revealed unique features characteristic of
these IL-3-dependent cell lines.
The murine diffuse haplotype -globin locus (in strains such as
BALB/c and DBA/2) contains five genes, including  y, h0, h1,
major, and minor, whereas the murine
single haplotype -globin locus (in strains such as C57BL/6) contains
y, h1, S, and T.8 The
majority of the h0 gene is deleted in the single haplotype -globin cluster.9 The h0 gene in the diffuse
haplotype has been proposed to be a pseudogene.9 The murine
-globin locus contains three functional genes (, -1, and
-2).10,11 Whereas the human exhibits embryonal to fetal
and fetal to adult hemoglobin switches, the mouse shows only one
switch, from embryonic to adult hemoglobin.12 In the mouse,
the embryonic to adult switch takes place at 10.5 days of gestation.
The primitive nucleated erythroid cells in the yolk sac contain two
embryonic -globins, h1 and y, and -globins, and
-1/-2. The definitive erythrocytes from fetal liver, spleen, and
bone marrow contain adult-type
-globins-major/minor
(S/T), although there is a report which
shows that y-globin mRNA is produced not only in the yolk sac but
also in the fetal liver during midgestation, suggesting that y gene
expression is analogous to that of the fetal globin genes in the
human.13 The -1/-2 genes are expressed throughout
development and are the only genes expressed during adult
life.12
The mechanism by which the developmental switching of globin genes is
executed and the mechanism by which the induction of globin genes by
the physiological cytokine Epo, as well as by chemical inducers, occurs
is far from complete. One of the obstacles to the understanding of
these phenomena is the limitations of in vitro model systems for
molecular analysis. Thus, the availability of tissue culture cell lines
in which expression of both embryonic and adult-type globins becomes
permissive on exposure to inhibitors of histone deacetylase would
provide a first step toward the identification of factors involved in
repression and derepression of globin genes. The results of our
previous2-4 and current studies point to the utility of
IL-3-dependent bone marrow cell lines as model systems for (1) the
dissection of growth versus differentiation signals transduced by Epo,
and (2) the dissection of the differentiation pathways used by the
physiological cytokine Epo and inhibitors of histone deacetylase.
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MATERIALS AND METHODS |
Cells.
Murine IL-3-dependent Ba/F3 cells were obtained from Dr H. F. Lodish
of the Whitehead Institute for Biomedical Research (Cambridge, MA).
They were established from the bone marrow of an adult BALB/c mouse
(-globin locus diffuse haplotype),9 and were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and
10% conditioned medium from WEHI-3B murine myelomonocytic cells
(WEHI-CM) as a source of IL-3.14 Clones of Ba/F3 cells expressing the EpoR were obtained by transfection of the eukaryotic expression plasmid for the EpoR, p75/15-EpoR,4 which
contained the EpoR cDNA under the control of the human metallothionein
IIA promoter (clones Ba-ER-1 to -4). These clones were developed in 0.8% methylcellulose medium containing 15% FBS and 1 U/mL of Epo (human recombinant Epo generously donated by the R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ). The E-7
clone was obtained from Ba/F3 cells by transfection of p75/15-EpoR, followed by selection with 0.8 mg/mL of G418 in medium containing 0.8%
methylcellulose, 15% FBS, and 10% WEHI-CM. The E-7 clone shows
Epo-dependent accumulation of maj/min globin mRNA on
exposure to the cytokine.2
Murine IL-3-dependent B6SUtA cells,15 kindly provided by
Dr J. S. Greenberger of the University of Massachusetts Medical Center (Worcester, MA), were adapted to replicate in RPMI
1640 medium containing 10% FBS and 10% WEHI-CM in this
laboratory.4 B6SUtA cells were established from the bone
marrow of an adult B6.S mouse15 (B6.S has the genetic
background of C57BL/6 mice which belong to the -locus single
haplotype).8,9 Clones of B6SUtA cells expressing the EpoR
(B6-ER-1 to -4) were obtained by transfection of p75/15-EpoR, followed
by selection with 1 U/mL of Epo and 0.7 mg/mL of G418 in medium
containing 0.8% methylcellulose and 15% FBS. Because of the
overgrowth of nontransfected and/or transiently transfected
cells, which interfered with the formation of discrete colonies of
stable EpoR transfected cells when clones were selected with Epo as the
sole selecting agent, it was necessary to select EpoR+
B6SUtA clones in the presence of both Epo and G418.
Friend murine erythroleukemia (MEL) 745-PC4 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 15% FBS.
MEL745-PC4 was derived from a DBA/2 mouse which belongs to the
-globin locus diffuse haplotype.16 Eleven and
one-half-day-old DBA/2 mouse embryonal blood was prepared as described
by Kovach et al.17
MEL-PC4 and Ba/F3 cells at an initial cell density of 4 × 104 cells/mL, and B6SUtA cells at an initial density of 6 × 104 cells/mL were treated with various chemical
agents for 3 days before RNA extraction. The percent of inhibition of
cell growth was calculated according to the following equation:
[log(final cell density of the control culture)  log(final cell
density of the treated culture)]/[log(final cell density of the
control culture) log(initial cell density of the control
culture)] × 100.
RNA extraction.
Total cellular RNA was isolated by a guanidinium/CsCl gradient
sedimentation procedure,18 or extracted using TRIZOL
reagent (GIBCO-BRL, Rockville, MD) from 1 to 4 × 107 cells according to the instructions of the
manufacturer.
Northern hybridization.
Total cellular RNA (10 µg) was separated by electrophoresis,
transferred to a nitrocellulose membrane, and hybridized with cDNA
probes as described previously.2 To probe for different types of globin mRNA, duplicate blots were made rather than stripping and reprobing the same blot to avoid incomplete stripping.
The nitrocellulose filters were washed at 55°C in 0.2 × SSC
(0.15 mol/L sodium chloride/0.015 mol/L sodium citrate, pH
7.0). To obtain a murine major-globin cDNA
probe, a 457-bp fragment containing the entire murine major-globin cDNA19 was amplified by reverse
transcription polymerase chain reaction (RT-PCR) from dimethyl
sulfoxide (DMSO)-treated MEL 745-PC4 cell RNA and cloned into pRc/CMV
(Invitrogen, San Diego, CA). pBluescript KS containing a
part of embryonic y2 cDNA20 was supplied by
Dr Ajay Bhargava of Yale University (New Haven, CT) and a 0.25-kb cDNA insert was excised by SpeI/EcoRV digestion. The
actin probe was a 2-kb PstI fragment of chicken -actin from
pA1.21
RNase protection assays.
In vitro transcription kits (MAXIscript) and ribonuclease protection
assay kits (RPA II) were purchased from Ambion, Inc (Austin, TX).
Detailed procedures provided by the manufacturer were followed. Antisense probes were made by labeling 1 µg of linearized plasmid DNA
with 50 µCi (3.125 µmol/L) of [-32P]UTP (800 Ci/mmol) in a volume of 20 µL. The transcription mixture was
separated on a 5% polyacrylamide/8 mol/L urea gel, and the antisense
probe corresponding to a full-length transcript was eluted from the
gel. Three kinds of antisense probes (, h1, and
maj/min or , -1/-2, and actin), each of which
contained 0.9 to 1.8 × 105 cpm, were combined and
hybridized with 10 µg of total cellular RNA according to the
streamlined procedure provided by the manufacturer. The RNA-RNA hybrids
were treated with a mixture of RNase A and RNase T1, and
the digested fragments were analyzed on a 5% polyacrylamide/8 mol/L
urea gel.
Murine globin probes for RNase protection assays.
A 17-base difference exists between major-
and minor-globin coding regions.22 The
antisense maj/min-globin probe was constructed by
amplifying a 113-bp fragment encompassing parts of exon l and exon 2 by
PCR using pRc/CMV-major-globin containing the entire
major-globin cDNA described above as a template and
subcloning the fragment into pCR2.1 by TA cloning (Invitrogen). This
portion was selected because of identity in the nucleotide sequence
between these two homologous globin genes.22
pBluescript-y2 was described above. PBSmT96
containing an embryonic/adult-type -globin DNA fragment, and
pSP64M10,23 were gifts of Drs Murat Arcasoy and Bernard
Forget of Yale University. pSP65h124,25 was provided by
Dr Thalia Papayannopoulou (University of Washington, Seattle, WA). All globin probes were subjected to DNA sequencing.
Three bases were different in the 96-base protected region between the -1-26 and -2-globin genes (the murine -2-globin
DNA sequence was kindly provided by Dr Aya Leder of Harvard Medical
School, Boston, MA). The probe in PBSmT96 contained
1 base corresponding to the -1 sequence, 1 base corresponding to the
-2 sequence, and 1 base unrelated to either sequence.
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RESULTS |
Murine IL-3-dependent Ba/F3 and B6SUtA cells produced subsets of
clones with different phenotypes on transfection of an EpoR expression
plasmid followed by selection with a drug resistance marker, one
subtype being sensitive only to the mitogenic activity of Epo, and the
other sensitive to both the mitogenic and differentiation inducing
properties of the cytokine.2,4 In contrast, Ba/F3 clones
transfected with the EpoR expression plasmid and selected with Epo were
uniformly positive for differentiation, expressing major-globin mRNA; furthermore, an inverse relationship
existed between the content of -globin mRNA and the level of EpoR
mRNA.3 RNA prepared from EpoR+ Ba/F3 clones
(Ba-ER-1 to -4) and EpoR+ B6SUtA clones (B6-ER-1 to -4)
that were selected by Epo-dependent growth was hybridized with
major- and -globin cDNA probes
(Fig 1). Although the Ba/F314
and B6SUtA15 cell lines were established from adult BALB/c
and B6.S mouse bone marrow, respectively, with IL-3 as a stimulant for cell growth, developmentally different types of globin mRNA were expressed in their respective EpoR+ clones. The levels of
major-globin mRNA were markedly increased in
EpoR+ Ba/F3 clones compared with parental Ba/F3 cells, with
the content of major-globin in EpoR+ Ba/F3
clones being comparable with the levels observed in DMSO-treated Friend
MEL cells. In contrast, significant amounts of embryonic type
-globin mRNA were present in parental B6SUtA cells, and -globin
gene expression seemed to be enhanced in EpoR+ B6SUtA
clones. Despite a 74% nucleotide sequence similarity between the
coding regions of major- and -globin
genes,20 cross-hybridization between the heterologous globins appeared to be minimal under the conditions used for the northern hybridizations.
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| Fig 1.
Expression of developmentally different -globin mRNA
species in Epo-stimulated EpoR+ clones derived from
B6SUtA and Ba/F3 cells analyzed by northern hybridization. B6-ER-1 to
-4 and Ba-ER-1 to -4 were EpoR+ clones derived from
B6SUtA and Ba/F3 cells, respectively. Friend MEL cells exposed to 1.5%
DMSO for 0 to 4 days served as a control. RNA was probed with
major-globin, -globin, and -actin cDNAs.
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To clarify further the type of globin mRNA expressed in
EpoR+ clones of Ba/F3 and B6SUtA cells, RNase protection
assays capable of distinguishing between different mRNA species were
used. The lengths of the probe and protected fragment for each globin
mRNA are summarized in Table 1. Total
cellular RNA was hybridized separately with a mixture of , h1,
and maj/min antisense probes or with a mixture of ,
-1/-2, and actin antisense probes. The globin mRNA species
expressed in Epo-stimulated EpoR+ Ba/F3 and B6SUtA clones,
corresponding parental cells, and day 11.5 DBA/2 mouse embryonal blood
are shown in Fig 2. In accord with the
results of northern hybridizations, the levels of
maj/min-globin mRNA in EpoR+
Ba/F3 clones (Ba-ER-1 and -2) were elevated relative to that of
parental cells. The levels of -1/-2-globin mRNAs were also increased in these clones. Trace amounts of -globin mRNA were also
detected. However, h1- and -globin mRNAs were not detectable. Also consistent with the results of northern hybridizations, parental B6SUtA cells contained significant amounts of -globin mRNA. In addition, -1/-2-globin mRNAs were also present in parental
B6SUtA cells. The levels of both - and -1/-2-globin mRNAs
were elevated in EpoR+ B6SUtA clones (B6-ER-1 and -2)
compared with that of parental cells. However, neither parental B6SUtA
cells nor EpoR+ B6SUtA clones contained mRNAs for
adult-type S/T-globins and embryonic h1- and
-globins. It should be noted that the size of the protected fragment
of h1-globin mRNA in B6SUtA cells was 180 bases instead of 245 bases
in day 11.5 DBA/2 embryonal blood as discussed below. Although Epo has
been reported to induce both embryonic and adult-type -globin mRNAs
in parental B6SUtA cells in the absence of cell growth, with a
preferential augmentation of adult -globin mRNA as a function of
time,24 adult-type -globin mRNAs were not detected in
our clones of B6SUtA cells transfected with the EpoR and selected with
Epo.
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| Fig 2.
Types of globin mRNA expressed in Epo-stimulated
EpoR+ clones of Ba/F3 and B6SUtA cells analyzed by RNase
protection assays. Total cellular RNA (10 µg) was hybridized
separately with a mixture of , h1, and maj/min
probes and with a mixture of , -1/-2, and actin probes.
Ba-ER-1 to -2 and B6-ER-1 to -2 are described in Fig 1. RNA
from DBA/2 mouse day 11.5 embryonal blood (d11.5 emb, lane 1) served as
controls for embryonal globin mRNAs. Molecular weight markers (MWM,
lane 8) are Sau3A-digested 32P-labeled pUC19 DNA.
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Given the specificity of globin mRNAs stimulated by Epo in
EpoR+ Ba/F3 and B6SUtA clones, it was of interest to
determine the types of globin mRNA induced by the chemical initiators
of maturation. Although Ba/F3 cells have been reported to express
erythroid-specific transcription factors such as GATA-1, NF-E2, and
EKLF,27,28 and Epo-stimulated EpoR+ clones
express both - and -globin mRNAs, these cells, unlike B6SUtA
cells, do not readily become benzidine positive,2,3 suggesting a defect(s) in the hemoglobin synthesizing machinery. Thus,
it was necessary to monitor the sensitivity of Ba/F3 cells to chemical
inducers at the globin mRNA level. To determine whether SB exerts its
activity through a solvent effect, inhibition of histone deacetylase,
or a combination of these actions, both DMSO and the specific inhibitor
of histone deacetylase, TSA,7 were evaluated for their
capacity to induce globin mRNA. Thus, the chemical inducers used
included DMSO, SB, TSA, diazepam (D2), and hemin. Although
Friend MEL 745 cells are known to respond to all of these agents by
attaining benzidine positivity, information on the type of globins
induced at the mRNA level by different initiators of differentiation is
limited. Therefore, MEL 745-PC4 cells were used to compare the profiles
of globin mRNAs produced by these cells with those by IL-3-dependent
cell lines. Total cellular RNA was prepared from the three cell lines
after exposure to two concentrations (relatively low and high) of
chemical inducers which gave comparable degrees of cell growth
inhibition within the same cell type. A summary of the degree of cell
growth inhibition calculated from the initial and final (day 3) log
cell densities and the percent benzidine positive cells is shown in
Table 2. The globin mRNA species induced in
MEL-PC4, Ba/F3, and B6SUtA cells by these inducers are shown in
Figs 3, 4, and
5, respectively. In MEL cells, DMSO and
D2 were stronger inducers than SB and TSA, as measured by
both benzidine assays (Table 2) and RNase protection assays (Fig 3).
The globin mRNAs found in MEL cells exposed to DMSO and D2
were only of the adult type (ie, maj/min and
-1/-2). In contrast, benzidine-positive cells were produced in
Ba/F3 cells by SB and TSA, but not by DMSO and D2 (Table
2). Remarkably, treatment of Ba/F3 cells with SB and TSA resulted in
the expression of all species of globin mRNAs (embryonic types: ,
h1, and ; and adult types: maj/min and
-1/-2), although the relative concentrations of the mRNAs induced
by the two agents differed slightly (Fig 4). Thus, SB caused more
prominent induction of and -1/-2 than TSA, whereas TSA caused
preferential induction of , h1, and maj/min. In a
manner analogous to Ba/F3 cells, exposure to SB and TSA caused a
significant increase in the percent of benzidine-positive B6SUtA cells
(Table 2). Furthermore, these agents caused accumulation of significant
amounts of S/T- and h1-globin mRNAs in B6SUtA cells
(Fig 5). However, -globin mRNA, which was noticeably present in
SB-treated Ba/F3 cells, was not induced in B6SUtA cells exposed to
either SB or TSA.
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Table 2.
Inhibition of Cellular Growth and Induction of
Benzidine-Positive Cells by Chemical Inducers in the Cell Lines
Used as Sources of RNA for RNase Protection Assays
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| Fig 3.
Globin mRNA species induced in Friend MEL745-PC4 cells by
various chemical inducers analyzed by RNase protection assays.
Treatment of cells with chemical inducers, RNA preparation, and RNase
protection assays are described in Materials and Methods. The
concentrations of agents used (L for low and H for high) are as listed
in Table 2. The abbreviations of the inducers used are given in the
legend of Table 2. Each RNA sample (10 µg) was hybridized separately
with a mixture of , h1, and maj/min probes and
with a mixture of , -1/-2, and actin probes as in Fig 2.
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| Fig 4.
Globin mRNA species induced in Ba/F3 cells by various
chemical inducers analyzed by RNase protection assays. RNA samples
prepared from nontreated and chemically induced Ba/F3 cells were
analyzed as described in Fig 3. Note that Ba/F3 cells belonging to the
diffuse haplotype produced the 245-base-protected fragment of h1
mRNA, which is longer than the protected fragment derived from B6SUtA
cells, as discussed in detail in the text.
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| Fig 5.
Globin mRNA species induced in B6SUtA cells by various
chemical inducers analyzed by RNase protection assays. RNA samples
prepared from nontreated and chemically induced B6SUtA cells were
analyzed as described in Fig 3. Note that B6SUtA cells belonging to the
single haplotype produced a 180-base-protected fragment of h1 mRNA,
shorter than that produced by Ba/F3 cells and 11.5-day-old DBA/2 mouse
embryo blood as discussed in the text.
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Because the concentrations of SB and TSA were chosen for their
equivalence to DMSO in the extent of cell growth inhibition, and the
induced levels of maj/min-globin mRNA in MEL PC4 cells
by SB and TSA at these concentrations were low (Fig 3), a question
remained as to whether these agents at higher concentrations had the
ability to induce higher amounts of maj/min-globin mRNA,
as well as to induce the embryonic species of globin mRNAs. SB at
concentrations of 2.5 and 3.0 mmol/L caused 51% and 69% of cell
growth inhibition, respectively, and induced
maj/min-globin mRNA at a level comparable with that
produced by 2.0% DMSO. Under these conditions, SB induced trace
amounts of embryonic species of globin mRNAs in MEL 745-PC4 cells.
Quantification of the autoradiogram by densitometry, with conversion
based on the specific activity of the probes and the length of the
protected fragments, showed that embryonic species of globin mRNAs were less than 1% of the maj/min-globin mRNA on a molar
basis (data not shown). None of the globin mRNA species were detected
in murine WEHI-3B myelomonocytic leukemia cells exposed to SB or hemin
(data not shown). All of the cell lines, including WEHI-3B cells,
treated with hemin for more than 3 days became benzidine positive. The
absence of globin mRNAs in hemin-treated Ba/F3 and WEHI-3B cells
suggests that the positive reaction with benzidine is caused by the
intracellular accumulation of hemin.
The h1-globin antisense probe included parts of exon III and
3 UTR constructed from BALB/c (diffuse haplotype) mouse DNA. To
show that the length of the protected fragment (180 bases) produced by
cells that belong to the single haplotype is shorter than that (245 bases) produced by cells that belong to the diffuse haplotype, RNA from
TSA-treated Ba/F3 cells (diffuse haplotype), SB-treated B6SUtA cells
(single haplotype), and day 11.5-old DBA/2 embryonal blood (diffuse
haplotype) were hybridized with a h1 probe alone
(Fig 6). The same set of RNA samples were
also hybridized with an probe alone, to show that the protected
fragment of -globin mRNA (253 bases) is equal in size among
different haplotypes and is distinguishable from the longer h1
protected fragment (245 bases). The difference in the protected
fragment of h1-globin mRNA in the two haplotypes is presumably
because of the reported diversion in the nucleotide sequence in the
3UTR region of the different mouse strains.25
Although Figs 2 through 5 were derived from RNase protection assays
using multiple antisense probes, RNA samples from different cell types
were analyzed by hybridization with a single antisense probe to ensure
that the protected fragments of all globin mRNAs, with the exception of
h1-globin mRNA, were equal in size regardless of the cell type, as
well as to rule out the possibility that the absence of detection of a
particular mRNA was due to overlapped fragments in the hybridization
with multiple probes.
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| Fig 6.
Different sizes of the protected fragment of h1-globin
mRNA produced by cells with different haplotype origins. RNA samples
from Ba/F3 cells treated with 15 nmol/L TSA (Ba/F3-TSA, L), B6SUtA
cells treated with 1.0 mmol/L SB (B6SUtA-SB, H), and 11.5-day-old DBA/2
mouse embryonal blood (d11.5 emb) were hybridized with the h1-globin
probe alone (lanes 4, 5, and 6). The same set of RNA samples was
hybridized with the -globin probe alone (lanes 1, 2, and 3).
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Ba/F3 cells and EpoR+ Ba/F3 clones grew in IL-3-containing
medium with a doubling time of 8.9 hours. Treatment of BaF3 cells with
SB, or switching of the growth factor from IL-3 to Epo in EpoR+ Ba/F3 clones, caused retardation of cell
growth.3 The time course of induction of
maj/min-globin mRNA in Ba/F3 cells by SB was compared
with that by Epo in the EpoR+ Ba/F3 clone E-7,2
in which the induction of maj/min-globin mRNA was
dependent on Epo. A significant increase in -globin mRNA content was
detected in Ba/F3 cells treated with SB and in clone E-7 treated with
Epo at 24 and 12 hours, respectively (Fig 7). The time course of induction of -globin mRNA in Ba/F3 cells by
SB was similar to that of maj/min-globin mRNA. One of
the possible mechanisms responsible for the 12-hour delay in the
induction of the globin gene by SB relative to that by Epo was that SB
induction required newly synthesized protein(s). To evaluate this
possibility, cycloheximide was added to the medium for the first or
second 5 hours of the incubation period together with SB according to
the schedule shown in Fig 8. Quantification
of the autoradiogram by densitometry, with a correction based on the
actin control, showed that the copresence of cycloheximide for the
first (lane 4) and second (lane 7) 5 hours inhibited the accumulation
of maj/min-globin mRNA by 27% and 39%, respectively,
compared with respective controls (lanes 5 and 8). The moderate degrees
of inhibition manifested by cycloheximide suggest that protein
synthesis in the early part of the incubation period did not have a
major role in the induction of maj/min-globin mRNA by
SB. With respect to the requirement for protein synthesis in the
induction of gene expression by SB, opposite results have been
reported.29,30 In the J2E erythroid cell line, which is
also known to be responsive to both Epo and SB, the time course of the
appearance of major-globin mRNA has been shown to be
faster in SB-treated cells than in Epo-treated cells.31
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| Fig 7.
Time course of the induction of -globin mRNAs by SB
and Epo. Parental Ba/F3 cells were treated with 1 mmol/L SB. E-7, a
clone of Ba/F3 cells transfected with the EpoR and selected with G418,
responds to both the mitogenic and differentiation-inducing effects of
Epo.2 E-7 cells in IL-3-containing medium were washed
three times with RPMI 1640 medium containing 10% FBS and exposed to
0.5 U/mL of Epo. RNA was subjected to northern hybridization with
major-globin, -globin, and actin probes.
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| Fig 8.
The effects of cycloheximide (CH) on the induction of
major-globin mRNA by SB. Ba/F3 cells were exposed to SB
and/or CH for the first or second 5-hour period according to
the treatment schedules shown in the top of the figure. In panels 4 and
5, and in panels 7 and 8, SB was added back to the medium after cells
were washed at 5 and 10 hours of the incubation period, respectively.
RNA was extracted at 48 hours and subjected to northern hybridization
(bottom of the figure). The cell densities at 48 hours are shown. The
concentrations of SB and CH used were 1 mmol and 10 µmol/L,
respectively. CH at this concentration inhibited protein synthesis,
measured by incorporation of 35S-methionine into the
acid-insoluble fraction, by more than 95% (data not shown).
|
|
| |
DISCUSSION |
The IL-3-dependent Ba/F3 and B6SUtA cells used in this study were
derived from the adult bone marrow of BALB/c14 and
B6.S15 mice, respectively. Whereas basal levels of globin
mRNA of all types were negligible in Ba/F3 cells, the constitutive
expression of - and -1/-2-globin mRNAs was observed in B6SUtA
cells. The expression of embryonic/fetal type globins in B6SUtA cells
suggests that the origin of this cell line is from a primitive lineage of erythroid precursors. It is also possible that during the
establishment or propagation of this cell line they may have been
exposed to an inductive environment, ie, exposure to factors such as
Epo and/or stem cell factor (SCF). Once the globin gene is
activated, expression of the message persists after withdrawal of the
stimulus. This phenomenon is exemplified by Ba/F3 cells, where the
levels of major-globin mRNA persist in the absence of
Epo in EpoR+ Ba/F3 cells once the -globin gene is
activated by the cytokine.2,27
Epo predominantly induced adult types (maj/min and
-1/-2) of globin mRNAs in EpoR+ Ba/F3 clones, whereas
more striking effects on globin gene expression were obtained in
parental Ba/F3 cells exposed to SB and TSA. Thus, SB and TSA allowed
the expression of all species of globin mRNAs. Furthermore, as opposed
to the augmentation of the preexisting program (ie, expression of -
and -1/-2-globin genes) by Epo in EpoR+ B6SUtA
clones, SB and TSA activated a latent phenotype (ie, expression of
S/T- and h1-globin genes) in parental B6SUtA cells.
The difference in the profile of globin mRNAs displayed in
Epo-stimulated EpoR+ cells and in SB- and TSA-treated
parental cells suggests that the mechanism by which Epo stimulates
expression of globin genes is different from that used by SB and TSA.
The types and amounts of globin mRNAs induced in B6SUtA cells by SB and
TSA were comparable (Fig 5), whereas the spectrum of globin mRNAs in
Ba/F3 cells induced by SB and TSA were slightly different (Fig 4).
Thus, SB was more prominent in inducing globin genes in the -cluster
(-1/-2 and ) than TSA, whereas TSA induced -globin mRNAs
(, h1, and maj/min) more efficiently than SB.
Because the RNA samples were prepared from cells exposed to TSA and SB
at the concentrations that led to comparable degrees of growth
inhibition and benzidine positivity (Table 2), the difference in the
spectrum of globin mRNAs observed in Ba/F3 cells exposed to SB and TSA
may be caused by competition between different globin loci for a
factor(s) necessary for gene activation. Overall, the effects of SB and
TSA were strikingly similar in these two types of IL-3-dependent cell
lines, in that both agents allowed simultaneous activation of embryonic
and adult globin genes, despite a difference in their effective
concentrations in vitro by approximately five orders of magnitude (1 mmol/L SB v 10 nmol/L TSA). The mimicry of action shared by SB
and TSA implies that (1) SB activates globin genes through an
inhibition of histone deacetylase, and that (2) histone deacetylase is
involved in the repression of both adult and embryonic globin genes in
these IL-3-dependent cells.
The significance of the acetylation of core histones on the
transcriptional activity of chromatin has begun to be recognized recently, with components of the transcriptional complex, which control
the activation and repression of genes, being identified as histone
acetylase and histone deacetylase, respectively.32-34 Because a global increase in acetylation of core histones produced by
inhibitors of histone deacetylase does not result in the widespread activation of genes,35 and information regarding which
specific genes are under the control of histone deacetylase is limited, the present study, which identifies embryonic and adult globin genes in
IL-3-dependent cells as genes under the control of histone deacetylase, is significant. Identification of additional components that tether histone deacetylase to the promoter region of globin genes
is required. In this context, it is noteworthy that the retinoblastoma
protein has been shown to recruit histone deacetylase to repress
transcription of a subset of genes which are involved in progression
from G1 to the S phase of the cell cycle and are controlled by the E2F
family of transcription factors.36,37 Examination of
whether the retinoblastoma protein is also involved in repression or
derepression of globin genes and identification of an
erythroid-specific transcription factor(s) which participates in these
processes form the next focus of investigations. In connection with
acetylation of chromatin and gene activation, it is interesting to note
that signal transducers and activators of transcription, which bring
about altered gene expression in response to cytokines, have been
proposed to cooperate with p300/CBP,38,39 which possesses intrinsic histone acetylase activity.34
Heavily based on the types of hemoglobins produced in these cells by
DMSO, it is generally accepted that Friend MEL cells possess an
adult-type environment for globin gene expression, with this
generalization forming the basis of many cell fusion experiments.
Consistent with this notion, globin mRNAs in MEL 745-PC4 cells induced
by DMSO were confined to adult types. In contrast to induction of adult
types of globin mRNAs by DMSO, other groups have shown the induction by
SB of small amounts of -globin in these cells.40-42 In
our hands, embryonic species of globin mRNAs were detectable in
SB-treated MEL 745-PC cells; however, the fraction of -globin was
extremely low, estimated to be less than 1% of the
major/minor-globins. In contrast to Friend MEL 745 cell
lines, the Friend MEL GM979 cell line, renamed after the T-3-Cl-2 clone
originally isolated by Ikawa et al,43 contained -,
h1-, and major/minor-globins in the uninduced
state.16,25,41 Treatment of these cells with SB resulted in
induction of large amounts of -globins.42,44 Thus,
Friend MEL cell lines have varying environments for globin gene
expression depending on the isolate.
Permissiveness for activation of embryonic as well as of adult globin
genes on treatment with SB in the two IL-3-dependent cell lines used
in this study has important implications in the assignment of the
origin of these cells in the developmental stage. Coexpression of
hemoglobins of different developmental stages has been shown in
numerous systems. Thus, (1) small amounts of fetal hemoglobin are
consistently present in the blood of normal human adults12;
(2) burst-forming unit-erythroid colonies developed from
the human yolk sac, as well as from fetal liver erythroid precursors,
coexpress embryonic, fetal, and adult globins45; (3)
embryonic and adult hemoglobins are produced in erythroid colonies from
mouse embryos at an early gestational stage46; and (4)
blast colonies of embryonic stem cells formed in response to vascular
endothelial growth factor and SCF express h1- and major-globin mRNAs.47 These findings are
interpreted as indications that (1) globin expression in erythroid
cells is not strictly stage specific, (2) erythroid cells in a
transitional state at the time of the hemoglobin switch have the
potential to coexpress hemoglobins of different developmental origins,
and (3) primitive and definitive erythropoiesis are derived from a
common precursor. Human erythroid cell lines, such as K562 and HEL,
established from adult leukemias with an erythroleukemic phenotype,
characteristically express fetal and some embryonic globin
chains.12 Deduced from the program of globin gene
expression and the methylation pattern of globin genes in these cell
lines, Enver et al48 have speculated that expression of
fetal and/or embryonic globins in leukemia cell lines is not an
aberration of neoplastic transformation, but is indicative of fetal or
embryonic potential in normal adult hematopoietic progenitors. The fact
that both Ba/F3 and B6SUtA cells are derived from adult mouse bone
marrow and yet are permissive for the expression of both adult and
embryonic globin genes adds direct evidence in support of this view,
and suggests that (1) multipotent stem cells in adult bone marrow
undergo a program of hemoglobin switching during erythroid
differentiation analogous to that of developmental switching during
ontogeny, and (2) these IL-3-dependent cells possess the
characteristics of erythroid precursors at the transitional
developmental stage. In contrast to human erythroid leukemia cell
lines, which invariably express fetal/embryonic globin mRNAs to various
extents in the uninduced state,12,48 the Ba/F3 mouse bone
marrow cells described in this report are devoid of background globin
expression, and fetal/adult globin mRNAs are induced only after
exposure to inhibitors of histone deacetylase. Thus, the Ba/F3 cell
line is a relatively clean model system to study the mechanism of
activation of fetal/adult globin genes. Previous investigations have
shown that methylation of genes is an important determinant of the
control of activity of globin genes.48,49 Methylation of
genes and hypoacetylation of chromatin may be coupled to contribute to
the mechanism of repression of globin genes, because a protein that
selectively binds to methylated DNA sequences has recently been shown
to exist as a complex with histone deacetylase.50,51
In previously known cell systems where developmentally incorrect forms
of globin(s) are expressed, the ratio of the incorrect form of globin
relative to total globin is generally low.12,45,46 In
contrast, SB and TSA simultaneously and efficaciously activated embryonic as well as adult-type -globin genes in IL-3-dependent cells. Extensive investigations on tissue specific and developmental stage-specific regulation of globin genes have shown that sequences local to the individual globin genes are sufficient to direct developmental stage-specific and tissue-specific expression, whereas high-level expression of the globin genes depends on the distal regulatory elements LCR.52,53 Studies have also indicated
that the LCR can only activate one gene at a time and that the LCR interacts directly with the gene it is enhancing through a looping mechanism. Thus, it is believed that the developmental switching of
globin gene expression is regulated, at least in part, through a
competition between the genes for interaction with the
LCR.52 The occurrence of the phenomenon observed in this
study, ie, efficacious activation of all of the genes (, h1,
major/minor) in the -cluster in
SB-treated B6SUtA cells, thus seems contradictory with this model.
However, using erythroid cells of human embryonic liver origin, which
coexpress  - and -globins, Wijgerde et al54 have shown
that the LCR can interact with these two genes by a flip-flop
mechanism. Based on in vivo dimethyl sulfate foot printing, Reddy and Shen55 have constructed three possible models for DMSO-induced globin gene expression, with or without the involvement of
the LCR. Overall, the precise role of the LCR in the activation of
globin genes by DMSO and SB is unclear.
In summary, our previous2-4 and current studies have shown
the versatility of murine IL-3-dependent bone marrow-derived Ba/F3 and B6SUtA cells as models for comparative studies of growth and differentiation controlled by the physiological cytokine Epo and by
chemical inducers in several ways. First, both cell lines yield two
subsets of clones on transfection of the EpoR, one with sensitivity to
the mitogenic action of Epo and the other with sensitivity to both the
mitogenic and differentiation-inducing effects of Epo.2,4
Therefore, these clones are capable of serving as model systems for the
dissection of Epo-dependent growth and differentiation signals.
Secondly, because these cell lines respond to both Epo and inhibitors
of histone deacetylase by undergoing differentiation, they are
excellent models to use to delineate identities and differences in
differentiation signals utilized by these agents. Most importantly, the
identification of histone deacetylase as an integral part of repression
and derepression of globin genes in IL-3-dependent bone
marrow-derived cells provides the initial step toward identification of factors involved in these processes which could serve as targets of
therapeutics to correct hemoglobin disorders.
| |
ACKNOWLEDGMENT |
We are grateful to Drs Harvey Lodish and Joel Greenberger for gifts of
the Ba/F3 and B6SUtA cell lines, respectively. We also thank Drs Murat
Arcasoy and Bernard Forget for gifts of PBSMT96 and pSP64M and
for helpful discussions. We are indebted to Drs Ajay Bhargava and
Thalia Papayannopoulou for provision of pBluescript-y2 and pSP64-, respectively. We are also grateful to Dr Aya
Leder for providing the unpublished DNA sequence of the murine
-2-globin gene. We wish to thank the R. W. Johnson Pharmaceutical
Research Institute for supplying human recombinant Epo.
| |
FOOTNOTES |
Submitted July 17, 1997;
accepted July 20, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Alan C. Sartorelli, PhD,
Department of Pharmacology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520.
| |
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Abstract
Introduction