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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3459-3466
Erythroid Maturation and Globin Gene Expression in Mice With Combined
Deficiency of NF-E2 and Nrf-2
By
Florence Martin,
Jan M. van Deursen,
Ramesh A. Shivdasani,
Carl W. Jackson,
Amber G. Troutman, and
Paul A. Ney
From the Departments of Biochemistry, Genetics, and Experimental
Hematology, Saint Jude Children's Research Hospital, Memphis, TN; and
Dana-Farber Cancer Institute, Children's Hospital, Harvard Medical
School, Boston, MA.
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ABSTRACT |
NF-E2 binding sites, located in distant regulatory sequences, may be
important for high level  - and  -globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either
little or no effect on erythroid maturation in mice. For p18 NF-E2,
this lack of effect is due, at least in part, to the presence of
redundant proteins. For p45 NF-E2, one possibility is that
NF-E2-related factors, Nrf-1 or Nrf-2, activate globin gene expression
in the absence of NF-E2. To test this hypothesis for Nrf-2, we
disrupted the Nrf-2 gene by homologous recombination. Nrf-2-deficient
mice had no detectable hematopoietic defect. In addition, no evidence
was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in
fetal liver cells deficient for either factor. Fetal liver cells
deficient for both NF-E2 and Nrf-2 expressed normal levels of - and
-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did
not exhibit a defect in erythroid maturation beyond that seen with loss
of NF-E2 alone. Thus, the presence of a mild erythroid defect in
NF-E2-deficient mice is not the result of compensation by Nrf-2.
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INTRODUCTION |
HIGH-LEVEL EXPRESSION from the -like
globin genes depends on far upstream regulatory sequences known as the
locus control region (LCR).1-3 The -globin LCR acts, in
part, by establishing an open chromatin domain over the entire
-globin cluster.3,4 However, the complete mechanism of
action of the LCR remains elusive. The -globin LCR contains four
core elements, which are marked by erythroid specific DNase I
hypersensitive sites (HS1 to HS4). Individual core elements do not
retain the activity of the full LCR, but are sufficient to confer high
level, tissue-specific expression on a linked -globin gene in
transgenic mice.5-7 These core elements contain numerous
binding sites for erythroid-specific and ubiquitous transcription
factors, including NF-E2, GATA, and GGTGG/CACC motifs. In contrast to
other binding motifs, the distribution of NF-E2 binding sites in globin
regulatory sequences is limited to core-elements in the -globin LCR
and the -globin positive regulatory element (PRE).8,9
This distribution, along with the evolutionary conservation of these
sites,10-14 suggests an integral role for NF-E2 sites in
LCR function.
NF-E2 is a heterodimeric member of the basic-leucine zipper (BZIP)
transcription factor family, composed of 45- and 18-kD subunits (p45 and p18 NF-E2).15-18 Targeted disruption of
p45 NF-E2 causes a maturation defect in megakaryocytes, severe
thrombocytopenia, and perinatal mortality.19 NF-E2 per se
is not required for terminal erythroid differentiation; targeted
disruption of each subunit has either little (p45), or no (p18) effect
on erythroid maturation.19-21 For p18 NF-E2, this lack of
effect can be attributed to the presence of redundant proteins. For p45
NF-E2, the role of redundant factors is less clear.
p45 NF-E2 is a member of the cap'n collar (CNC) subfamily of BZIP
transcription factors, named for the prototype protein CNC in
Drosophila.22 These proteins share a conserved
region of 43 amino acids, which lies immediately N-terminal of the BZIP domain. This region is required for activation of globin gene expression by NF-E2.23 Besides NF-E2, CNC family members in man and mouse include NF-E2 related factors-1 and -2 (Nrf-1/LCR-F1 and
Nrf-2).24-27 Both Nrf-1/LCR-F1 and Nrf-2 have been shown to activate reporter genes in transient assays.25,26 Nrf-2 is the homolog of a chicken protein, erythroid protein with CNC homology (ECH).28 Interestingly, ECH, rather than a homolog of
NF-E2, was the predominant isolate from an anemic chicken erythrocyte library screened with the BZIP domain of murine NF-E2. We hypothesized that the mild erythroid defect in NF-E2-deficient mice might reflect the ability of Nrf-2 to compensate for the loss of NF-E2. To test this
hypothesis, we examined the effect of combined NF-E2 and Nrf-2
deficiency on globin gene expression in mice.
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MATERIALS AND METHODS |
Cloning of Nrf-2.
A partial cDNA clone of human Nrf-2 was provided by S. Ruben (Human
Genome Systems, Rockville, MD). This was used to screen a
murine pre-B-cell library and obtain a full-length cDNA
clone. The murine Nrf-2 cDNA was used to screen a 129 strain
(CCE) embryonic stem cell (ES) library in EMBL3. Three
genomic clones were obtained, of which two showed evidence of
rearrangement. The third clone was 17.5 kb and contained exons 2-5 of
the Nrf-2 gene.
Targeting of the Nrf-2 gene.
A 1.9-kb Cla I-Hind III fragment from the second intron to the fifth
exon and a 4.5-kb Hind III-Eco RV fragment from the fifth exon into
3 flanking sequences were subcloned into targeting vector PPNT
(gift from S. Orkin, Children's Hospital, Boston, MA).29 The targeting vector was linearized at
a unique Not I site and transfected into RW4 ES cells (Genome Systems
Inc, St Louis, MO). ES cells were cultured on SNLH9 feeder
cells,30 and the media was supplemented with 1,000 U/mL of
leukemia inhibitory factor (LIF) (ESGRO; Life Technologies,
Gaithersburg, MD). Clones were selected in G418 (Geneticin, 350 µg/mL
active, Life Technologies, and
1(1-2-deoxy-2-fluoro--Darabinofuransyl)-5-iodouracil (FIAU, 0.2 µmol/L), and screened for targeted disruption of Nrf-2 by Southern
blot analysis. The probe used in the Southern analysis was a 0.7-kb Cla
I-Bgl II fragment of exon 2 and intron 2, which was 5 of the
homologous sequences used in the targeting construct. Six clones with
homologous recombination of the Nrf-2 gene were karyotyped. Four clones
with a normal karyotype were injected into C57BL/6J blastocysts and
implanted into pseudopregnant foster mothers, as previously
described.31 Male chimeras were mated with C57BL/6J females
and germline transmission confirmed by agouti color in the F1 animals.
Agouti offspring were tested for mutation of Nrf-2 by Southern blot
analysis of tail DNA.
Breeding of mice with combined deficiency of NF-E2 and Nrf-2.
Nrf-2 (-/-) mice on a
mixed 129/SvJ-C57BL/6J background were mated to inbred 129/Sv NF-E2
(+/-) mice (gift from S. Orkin).19 Compound heterozygotes
from this mating were bred to obtain double homozygous mutant embryos,
for analysis, at a frequency of 1/16. Nrf-2 (-/-)/NF-E2 (+/-) mice,
generated from the mating of compound heterozygotes, were bred to
obtain double homozygous progeny at a frequency of 1/4.
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| Fig 1.
Nrf-2/p18 heterodimers have the same DNA binding
specificity as NF-E2. A gel mobility shift assay was performed on in
vitro-translated p45/p18 NF-E2 (lanes 1 and 2) and Nrf-2/p18
heterodimers (lanes 3 and 4) and on nuclear extract from untransfected
COS cells, as a source of AP-1 binding activity (lanes 5 and 6). The
discriminatory mutant probe (M) differs from wild-type (WT) at one
nucleotide.39
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| Fig 2.
Gene targeting strategy. (Top) Genomic clone containing
exons 2-5 of Nrf-2 in EMBL3. Restriction sites indicated are Hind III
(H) and Cla I (C). The exon 2 probe used to screen Southern blots is
shown by a black box. The junction (J) with vector sequences (wavy
lines) is shown, as is the germline Hind III fragment detected with the
exon 2 probe. (Middle) Targeting vector showing insertion of the
Neomycin resistance gene (Neo) into the fifth exon, in the reverse
orientation relative to Nrf-2. An inactivated Hind III site is
indicated by parentheses. The thymidine kinase gene (tk) is also in
reverse orientation and lies outside the region of homology. (Bottom)
Structure of the targeted allele after homologous recombination. The
thymidine kinase gene has been lost as a result of homologous
recombination. A rearranged 5.8-kb Hind III fragment is shown.
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| Fig 3.
Targeting of the Nrf-2 gene by homologous recombination.
(A) Targeted disruption of one Nrf-2 allele. A representative Southern blot is shown hybridized with the Nrf-2 exon 2 probe (top) or a
Neomycin resistance cDNA probe (bottom). The germline Hind III band of
4.0 kb is present in all clones. The 5.8-rearranged band, specific for
homologous recombination, was present in about 60% of the clones. (B)
Southern blot of tail DNA from the first F2 litter with controls. (C)
Targeted disruption of both Nrf-2 alleles. A Southern blot is shown
hybridized with the Nrf-2 exon 2 probe (top) or a hygromycin resistance
cDNA probe (bottom). The second allele, disrupted by the hygromycin
resistance gene, gives a 6.0-kb rearranged Hind III band and the
appearance of a doublet on shorter exposures.
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In vitro differentiation of Nrf-2 (-/-) embryoid bodies.
The targeting construct was modified by substituting a hygromycin
resistance gene for neomycin resistance. This construct was transfected
into ES clone 5.18 with one targeted Nrf-2 allele. ES clones were
selected in hygromycin (350 µg/mL; Boehringer Mannheim, Indianapolis,
IN), G418 (350 µg/mL), and gancyclovir (2 µmol/L), and
resistant clones screened for targeted disruption of both Nrf-2 alleles
by Southern blot analysis. For the in vitro differentiation assay, ES
clones were adapted to growth without feeder cells on gelatin-coated
plates in the presence of LIF.32 Gelatin-adapted ES cells
were plated at various concentrations in 1.1% methylcellulose (Stem
Cell Technologies Inc, Vancouver, Canada), Iscove's
modified Dulbecco's medium (IMDM), 15% fetal bovine
serum, iron-saturated transferrin (300 µg/mL; Boehringer Mannheim),
insulin (10 µg/mL; Sigma, St Louis, MO), hemin (10 µg/mL; Sigma), monothioglycerol (450 µmol/L), erythropoietin (EPO,
1 U/mL; Amgen, Thousand Oaks, CA), murine interleukin-3
(IL-3, 25 U/mL; Collaborative Biomedical Products,
Bedford, MA), and murine stem cell factor (SCF, 25 ng/mL; R & D
Systems, Minneapolis, MN). Embryoid bodies were assessed by light microscopy for evidence of hemoglobinization and by benzidine staining as previously described.33
Analysis of mice.
Peripheral blood (150 µL) was obtained from anesthetized mice by
retro-orbital puncture using heparinized microhematocrit tubes. One
tube was used to determine the hematocrit. Red and white blood cell
counts were determined on 20 µL of blood using an automated cell
counter (Multisizer II; Coulter, Miami, FL). Hemoglobin
concentration was determined on 10 µL of blood by Drabkin's method.34 Platelet counts were determined on 20 µL of
blood, by phase-contrast microscopy. Peripheral blood smears were
treated with Wright-Giemsa stain and erythrocyte morphology examined by light microscopy. Bone marrow hematopoietic progenitors were cultured in methylcellulose with IL-3, IL-6, SCF, and (+/-) EPO (MethoCult M3434, and M3534; Stem Cell Technologies Inc). Colony-forming unit-erythroid (CFU-E) were quantitated 3 days after
plating and colony-forming cells (CFC) (colony-forming
unit-granulocyte [CFU-G], colony-forming unit-granulocyte-macrophage
[CFU-GM], and colony-forming unit-macrophage
[CFU-M]) and burst-forming unit-erythroid
(BFU-E) were quantitated 8 days after plating the cells.
RNA analysis.
Total RNA was extracted from the bone marrow of Nrf-2 (+/+) and Nrf-2
(-/-) mice by the method of Chomczynski and Sacchi35 (RNazol B, Tel-Test). Northern blot analysis was performed on bone
marrow RNA by the method of Sambrook et al.36 The blot was
probed first with full-length Nrf-2 cDNA, stripped, then reprobed with
a human beta-actin cDNA probe (Clontech, Palo Alto, CA). Total RNA was extracted from fetal livers of day 13.5 to 15.5 embryos
and analyzed by ribonuclease protection assay, as previously described.23 The -globin, -globin, and -actin
riboprobes protected 245, 128, and 150 bp fragments,
respectively.23,37 Test samples fell within standard curves
for all three probes.
Protein assays.
In vitro transcription and translation of p18 and p45 NF-E2 and Nrf-2
was performed with the rabbit reticulocyte lysate (Promega, Madison,
WI) and [35S]methionine in accordance with
the manufacturer's instructions. NF-E2 and Nrf-2 were expressed in the
in vitro transcription/translation vector pCITE4a (Novagen, Madison,
WI), as previously described.23 COS cell
nuclear extracts were made by the method of Dignam.38 Gel
mobility shift assays were performed as previously
described.23 DNA sequence of the wild-type NF-E2 probe was
GGAACCTGTGCTGAGTCACTGGAGG. Sequence of the mutant
NF-E2 probe was
GGAACCTGTTCTGAGTCACTGGAGG.39 Gel shift
probes were labeled with deoxycytidine triphosphate (dCTP) and Klenow enzyme (Boehringer Mannheim).
Immunoprecipitations were performed in radioimmune precipitation assay
(RIPA) lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris [pH 8.0], 1%
NP-40, 0.1% sodium dodecyl sulfate [SDS], and 0.5% deoxycholate) by
the method of Harlow and Lane.40 Fetal livers were
harvested from day 13.5 to 15.5 embryos and processed separately. A
single cell suspension of fetal liver cells (FLCs) was made by repeated
passage of livers through a 21-gauge needle. FLCs were washed and
resuspended in methionine-free Dulbecco's modified Eagle medium
(DMEM). FLCs were starved for 1 hour (37°C, 5% CO2), followed by addition of [35S] methionine to a final
concentration of 100 µCi/mL, and 4 more hours of culture. FLCs were
washed once in phosphate-buffered saline (PBS) and resuspended in 0.5 mL of RIPA buffer with protease inhibitors. The lysate was incubated,
while rocking, at 4°C for 30 minutes and centrifuged for 15 minutes
at RCFmax=156,800 in a TLA-100.2 rotor (Beckman). The
supernatant was precleared and incubated for 1 hour at 4°C with
specific antibodies, followed by Protein A-Sepharose (Pharmacia,
Uppsala, Sweden). Anti-p45 NF-E2 antibody was raised to a synthetic
peptide (NVPSETSFEPQAPTPY) as previously described.23
Anti-Nrf2 antibody was raised to a synthetic peptide near the
N-terminus (LQKEQEKAFFAQFQLDE) by the same method (Rockland Inc,
Boyertown, PA). Anti-Sp1 antibody was raised to a synthetic peptide
corresponding to amino acid residues 436-454 of human Sp1 (Santa Cruz
Biotechnology, Santa Cruz, CA).
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RESULTS |
Gene targeting of Nrf-2.
Like p45 NF-E2, Nrf-2 is able to form a heterodimer with p18 NF-E2 and
bind NF-E2 sites (Fig 1). NF-E2 binding sites consist of
an extended AP-1 site, such that a mutation 5 of the AP-1 core
specifically interferes with NF-E2 binding.39 This
specificity is conferred by the small subunit of NF-E2, p18
NF-E2.18 Nrf-2/p18 heterodimers exhibit this same binding
specificity suggesting that, in cells expressing both proteins, NF-E2
and Nrf-2 could have overlapping effects on gene expression. The slower
mobility of Nrf-2/p18 relative to NF-E2 raises the possibility that
Nrf-2/p18 heterodimers could be present in a slowly migrating complex
in extracts from fetal liver cells.20 This complex, which
includes proteins from the AP-1 family, is unaffected by the loss of
NF-E2.
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| Fig 4.
Effect of gene targeting on Nrf-2 transcript size.
Northern blot analysis of total RNA from bone marrow (Nrf-2 (+/+),
13.0 µg RNA; Nrf-2 (-/-), 9.6 µg RNA). The blot was probed with
full-length Nrf-2 cDNA (top) or -actin cDNA as a control (bottom).
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To determine the significance of Nrf-2 for globin gene expression, we
inactivated the Nrf-2 gene by homologous recombination. The murine
Nrf-2 gene contains five exons (Fig 2). The large
terminal exon encodes two-thirds of the protein, including the BZIP
domain. To inactivate the Nrf-2 gene, a neomycin resistance cassette
was inserted in reverse orientation into exon 5. This mutation
introduced stop codons in all three reading frames and prevented
translation of the essential dimerization and DNA binding domains. The
frequency of homologous recombination in embryonic stem (ES) cells was
approximately 60% (Fig 3A). Single knock-out ES cells
were injected into C57BL/6J strain blastocysts and implanted into
pseudopregnant females.31 After passage of the mutation in
the germline, heterozygous mice were mated, producing viable homozygous
progeny (Fig 3B). In a sample of 164 progeny, Nrf-2 (-/-) mice were
present in the predicted Mendelian proportion, indicating that Nrf-2
deficiency is not associated with embryonic or neonatal lethality.
Nrf-2-deficient mice had no apparent developmental defects.
NF-E2-deficient mice are severely thrombocytopenic due to a defect in
megakaryocyte maturation.19 In contrast, the hematologic
parameters of Nrf-2-deficient mice were normal, including the platelet
count (Nrf-2 (+/+) mice, 1.24 ± 0.32 × 106/µL
[n = 12]; Nrf-2 (-/-) mice, 1.33 ± 0.34 × 106/µL [n = 12]). Bone marrow hematopoietic progenitors
(CFU-E, BFU-E, and CFC) were also present in normal numbers in adult
Nrf-2 (-/-) mice (data not shown).
To verify that the Nrf-2 gene had been disrupted, we performed Northern
analysis of bone marrow RNA from wild-type and Nrf-2 (-/-) mice. Nrf-2
transcripts from Nrf-2 (-/-) cells migrated slower than those from
wild-type cells, consistent with insertion of the neomycin resistance
gene (Fig 4). RNA polymerase chain reaction (PCR) and
sequencing confirmed that this insertion resulted in multiple in-frame
stop codons.
Next, we considered the possibility that expression of NF-E2 and Nrf-2
might be cross-regulated, as previously shown for GATA-1 and
GATA-2.41 To examine this, we immunoprecipitated p45 NF-E2 from the fetal liver cells of Nrf-2 heterozygous and homozygous knock-out embryos. No evidence was found for a change in p45 NF-E2 protein levels in Nrf-2-deficient erythroid cells
(Fig 5). Conversely, Nrf-2 protein was
undetectable in both wild-type and NF-E2-deficient fetal liver cells
(data not shown). Thus, if NF-E2 and Nrf-2 complement one another, this
occurs without a demonstrable change in expression of either protein.
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| Fig 5.
NF-E2 expression in Nrf-2 (+/ ) and Nrf-2 (-/-) fetal
liver cells. NF-E2 was immunoprecipitated from fetal liver cells of day
13.5 to 15.5 embryos as described in Materials and Methods. Four fetal
livers are shown from the embryos of one litter. Fourteen fetal livers
from two litters were studied, and these results are representative.
Nrf-2 genotype is indicated at the top with preimmune (PI) and immune
(I) antisera. The extracts were serially immunoprecipitated with
anti-Sp1 antibody (Santa Cruz) as a control. Sp1 peptide was added to
one reaction as a negative control.
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In vitro differentiation of Nrf-2 (-/-) ES cells.
Because compensatory mechanisms may exist in vivo that do not exist in
vitro, we also studied the effect of Nrf-2 deficiency on erythroid
differentiation in vitro. Through the use of a second gene targeting
vector, which contained a hygromycin resistance gene instead of a
neomycin resistance gene, we disrupted the second Nrf-2 allele at
approximately a 60% frequency (Fig 3C). ES cells were adapted to
growth without feeders and allowed to differentiate in vitro, as
previously described. 32 Embryoid bodies derived from
wild-type or Nrf-2 (-/-) ES cells showed no impairment in their ability
to undergo terminal erythroid differentiation
(Fig 6).
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| Fig 6.
In vitro differentiation of Nrf-2 (-/-) embryoid bodies.
(A) Light microscopy of two Nrf-2 (-/-) embryoid bodies, one showing terminal erythroid differentiation. (B) Light microscopy of Nrf-2 (-/-)
embryoid bodies after staining with benzidine.33
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Immunoprecipitation experiments with Nrf-2 (-/-) ES cells confirmed
that insertion of neomycin and hygromycin resistance genes functionally
inactivated the Nrf-2 gene. Antibody raised to an N-terminal epitope of
Nrf-2 (amino acids 46-62) efficiently precipitated in vitro translated
Nrf-2, as well as Nrf-2 protein expressed in COS cells
(Fig 7A and B). Endogenous Nrf-2 protein,
which has not been previously shown, was present in low amount in
wild-type ES cells (Fig 7C). Nrf-2 (-/-) ES cells did not express
detectable full-length Nrf-2. Furthermore, there was no evidence of a
premature termination product, suggesting that a truncated protein, if
expressed, was not stable.
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| Fig 7.
Absence of Nrf-2 protein in Nrf-2 (-/-) embryonic stem
(ES) cells. (A) Immunoprecipitation of [35S]-labeled
Nrf-2 from in vitro transcription/translation reaction showing the
starting material, immunoprecipitation with preimmune serum, and
immunoprecipitation with anti-Nrf2 antibody. The 98-kD marker is
indicated. Nrf-2 migrates at 96 kD.26 (B)
Immunoprecipitation of [35S]-labeled Nrf-2 from whole
cell extracts of 1 × 107 COS cells transfected with
vector alone, or with Nrf-2 cDNA in COS expression vector pEUK-1
(Clontech). (C) Immunoprecipitation of [35S]-labeled
Nrf-2 from whole cell extracts of 4 × 107 Nrf-2 (+/+)
ES cells and Nrf-2 (-/-) ES cells.
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Combined deficiency of NF-E2 and Nrf-2.
To determine the effect of combined deficiency of Nrf-2 and NF-E2 on
globin gene expression, mice deficient in the two factors were
interbred. Compound heterozygotes from the first round of breeding were
mated to obtain mice deficient in both factors. Fetal liver cells from
embryos of this mating were examined for globin gene expression by
ribonuclease protection assay. Day 13.5 to 15.5 embryos deficient for
both NF-E2 and Nrf-2 expressed normal levels of -and -globin
(Fig 8). Mature mice deficient for both factors showed the same hematologic profile as mice deficient for NF-E2
alone (Table 1). NF-E2-deficient mice are
mildly anemic, possibly due to hemorrhage or defective erythroid
maturation.20 However, combined deficiency of NF-E2 and
Nrf-2 was not associated with a more severe erythroid defect.
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| Fig 8.
Globin gene expression in embryos with combined
deficiency of NF-E2 and Nrf-2. Compound heterozygous mice were mated
and total RNA was extracted from the fetal livers of day 13.5 to 15.5 embryos. Genomic DNA was extracted from the heads of the embryos and
analyzed for NF-E2 and Nrf-2 genotype. - and -globin and
-actin expression was analyzed by ribonuclease protection assay (1 µg RNA). The size of the protected transcripts was 245, 128, and 150 bp, respectively. As indicated at the top, comparisons were made
between samples from the same litter. A standard curve was performed
with RNA from murine erythroleukemia cells for each of the probes.
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DISCUSSION |
NF-E2 binding sites contribute to the activity of erythroid-specific
elements in the -globin LCR and the -globin PRE. Evidence exists
that NF-E2 itself is a regulator of globin gene expression through
these sites. Murine erythroleukemia cells deficient in p45 NF-E2 have a
defect in globin gene expression, which can be rescued by introduction
of p45 NF-E2.42,43 However, the lack of a significant
defect in vivo raises the possibility that there are other activators
of globin gene expression that function through these sites. We tested
the hypothesis that Nrf-2 compensates for the loss of NF-E2 by
generating mice with combined deficiency of NF-E2 and Nrf-2.
Mice deficient in Nrf-2 had no apparent abnormalities. This result,
which confirms the findings of Chan et al,44 indicates that
Nrf-2 is not required for terminal erythroid differentiation. This
result was expected, as Nrf-2-deficient mice still express normal
levels of p45 NF-E2. No evidence was found for reciprocal upregulation
of expression of NF-E2 or Nrf-2 in fetal liver cells deficient for
either protein. Moreover, mice deficient in both NF-E2 and Nrf-2 failed
to show an erythroid defect beyond that seen with NF-E2 deficiency
alone. These results indicate that the mild erythroid phenotype seen in
NF-E2-deficient mice is not the result of compensation by Nrf-2.
Recent experiments indicate that the function of Nrf-2 may not be in
hematopoiesis, but in the induction of antioxidant enzymes through
NF-E2-like binding sites.45
Because Nrf-2 does not compensate for deficiency of NF-E2 in mice, what
accounts for the mild erythroid defect? One possibility is that other
members of the CNC family can substitute for NF-E2. Targeted disruption
of Nrf-1/LCR-F1 is not associated with a defect in erythroid maturation
in chimeric mice.33 The effect of combined NF-E2 and
Nrf-1/LCR-F1 deficiency on erythropoiesis will be difficult to assess
due to the early embryonic death of Nrf-1/LCR-F1 knock-out mice. The
description of two new CNC-related proteins, Bach-1 and Bach-2, further
complicates resolution of this issue.46
A second possibility is that redundancy exists at the level of
cis-acting sequences, bypassing the need for NF-E2 sites or proteins
that bind to these sites. Ablation of individual core elements HS2 or
HS3 is associated with a modest 30% decrease in -globin
expression.47,48 However, because NF-E2 sites are associated with each of the three most active core elements (HS2-4), these experiments do not address the requirement for NF-E2 sites in the
LCR. In contrast to the -globin LCR, there appears to be a single
core element upstream of the -globin cluster (HS-40 in man, HS-26 in
mouse). 9,13,49,50 Although this element is not sufficient
for normal regulation of -globin expression in transgenic mice, it
does seem to be required for expression of the endogenous -globin
gene.51 It will be interesting to determine the role of
tandem NF-E2 sites present in this element on -globin expression.
Finally, studies in thalassemic patients suggest that a mechanism may
exist to compensate for a balanced defect in globin gene expression.
Whereas patients with -thalassemia trait and a normal complement of
-globin genes have microcytic anemia, patients who coinherit
-thalassemia trait and deletion of two -globin genes (out of four
-globin genes) are not anemic and have near normal red
blood cell indices.52-54 Similarly, a
balanced defect in globin gene expression may exist in NF-E2-deficient mice. NF-E2 deficiency in cell lines is associated with a defect in
both -and -globin expression.42,43 Moreover, studies
in NF-E2-deficient mice suggest the presence of an ongoing
compensatory process. Erythrocytes in NF-E2-deficient mice are
hypochromic and microcytic, despite the presence of adequate iron
stores.20 Spleen weight is increased fivefold to sixfold as
a consequence of expanded, and possibly ineffective, erythropoiesis. In
contrast to GATA-1 deficiency, which causes a maturation arrest at the proerythroblast stage,55 or erythroid Krüppel-like
factor (EKLF) deficiency, which causes a marked imbalance in the ratio
of - to -globin expression,56-57 this phenotype is
consistent with a specific and balanced defect in - and -globin
expression. Further studies are needed to test this hypothesis.
The contribution of NF-E2, or other proteins that bind to NF-E2 sites,
to high level globin gene expression remains an important question in
tissue-specific gene regulation. Identification of the proteins that
activate globin gene expression through these sites will be a necessary
step towards understanding the mechanism of action of the -globin
LCR. Our results indicate that Nrf-2 does not make an important
contribution to globin gene expression in NF-E2-deficient mice.
Several alternative hypotheses are under consideration.
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FOOTNOTES |
Submitted September 16, 1997;
accepted December 10, 1997.
Supported by National Institutes of Health (NIH) Cancer Center
Support CORE Grant No. P30 CA21765 (Bethesda, MD) and the American Lebanese Syrian Associated Charities (ALSAC) (Memphis,
TN).
Address reprint requests to Paul A. Ney, MD, Department of
Biochemistry, Room 4064, Thomas Tower, 332 N Lauderdale, Memphis, TN
38101.
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.
| |
ACKNOWLEDGMENT |
The authors thank Melanie Loyd, Teresa Bean, and Shirley Steward for
expert technical assistance. The authors also thank Steve Ruben (Human
Genome Systems) for the human Nrf-2 cDNA clone and Stuart Orkin for
sending NF-E2-deficient mice in advance of publication.
| |
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Abstract
Introduction
Abstract