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PLENARY PAPER
From the Department of Biochemistry and Molecular
Genetics, University of Alabama at Birmingham.
Activating transcription factor (ATF) 4 is a ubiquitous basic
leucine-zipper transcription factor that is a member of the ATF/cyclic
adenosine monophosphate responsive element-binding (CREB) protein
family. To determine the in vivo function of ATF4, the ATF4 gene in
murine embryonic stem cells was deleted and homozygous mutant mice were
generated. ATF4 null fetuses were severely anemic because of an
impairment in fetal-liver definitive hematopoiesis; the hematocrit in
15.5-day mutant fetuses was 0.15, whereas that in controls was
0.35. The fetal livers in homozygous ATF4 mutants were pale and
hypoplastic. In vitro culture of fetal-liver cells showed fewer
hematopoietic progenitors per embryo and a dramatic decrease in the
size of progenitor colonies. Culture of primary murine embryonic
fibroblasts showed a proliferative defect. These results suggest that
ATF4 is critical, in a cell-autonomous manner, for normal cellular
proliferation, especially for the high-level proliferation required
during fetal-liver hematopoiesis.
(Blood. 2002;99:736-745) The activating transcription factor
(ATF)/cyclic adenosine monophosphate responsive element-binding (CREB)
family consists of transcription factors that bind the cyclic adenosine
monophosphate (cAMP) response element (CRE) in vitro through
highly related, carboxy-terminal basic leucine-zipper (bZip)
dimerization domains. ATF4 was initially isolated in a human Murine ATF4 is a 381-amino acid protein containing 3 acidic regions
similar to acidic transcriptional activation domains in other
transcription factors and a carboxy-terminal bZip motif involved in DNA
binding and dimerization. The murine and human complementary DNAs
(cDNAs) are 85% homologous in nucleotide sequence in the region
encompassing the open reading frame, and they encode proteins that are
80% similar overall, with a 98.6% amino acid identity in the bZip
region.4 The ATF4 gene is present as a single copy on
mouse chromosome 15.5 Northern blotting with ATF4 cDNA as
a probe detected a single 1.7-kilobase (kb) band in mouse liver,
spleen, kidney, heart, lung, muscle, thymus, testis, and
brain.2,6 ATF binding sites are present in the promoters of a wide variety of genes. Most are thought to be involved in responses to environmental stimuli (eg, mitogens, phorbol esters, viral
infection, and peptide hormones that elevate cAMP levels) and
include transforming growth factor ATF4 has been shown to form a homodimer in vitro and to heterodimerize
with Jun, Fos, and Fra-1,12,13 HTLV-1 Tax,14
GPE1-binding protein,15 IGEBP1,16 nuclear
factor-interleukin 6 (NF-IL-6) (CCAAT/enhancer-binding protein
[C/EBP] Tanaka et al19 and Hettmann et al27
independently described the effect of ATF4 inactivation on the
developing eye in mice. They found that a deficiency in ATF4 results in
a defect in embryonic lens formation leading to severe microphthalmia
in adults. The effect of inactivation of ATF4 in murine tissues other
than the eye has not been determined.
We isolated ATF4 in a yeast 2-hybrid screen with locus control region
factor 1 (LCR-F1). LCR-F1 is a bZip transcription factor identified in
2 independent screens for erythroid proteins capable of binding the
tandem activator protein 1-like sites (also designated NF-E2 sites) in
deoxyribonuclease I hypersensitive site 2 of the human In the current study, we examined the function of ATF4 by deleting the
ATF4 coding region by means of homologous recombination. We found that,
like LCR-F1 mutant mice, ATF4-deficient mice are severely anemic during
fetal development, apparently because of an impairment in definitive hematopoiesis.
Construction of the ATF4 targeting vector
Transfection of embryonic stem cells and generation of
ATF4-deficient mice
Two R1 and 2 HM1 independently targeted ES-cell clones were injected
into the inner cell mass of donor blastocysts. High-percentage chimeras
were generated and bred to Black Swiss mice (Taconic Farms, Germantown,
NY). ATF4+/ The neo cassette was not removed from the mutant mice. Although this
marker gene could affect expression of linked genes, a search of the
National Center for Biotechnology Information and Celera Genomics
(Rockville, MD) human and mouse genomic databases determined that the
closest gene is mannoside acetylglucosaminyltransferase 3 (Mgat3),
which is located 42 kb from the ATF4 gene. There is no other gene
within 100 kb of ATF4. Targeted inactivation of the Mgat3 gene was
previously shown to have no phenotypic effect.36 In
particular, Mgat3 Genotyping of ATF4-deficient mice
Hematologic analysis of embryonic and adult blood An incision was made in the extraembryonic membranes, with care taken to produce as little damage as possible to yolk sac blood vessels. The membranes were retracted but left attached to the placenta, and the uterine artery and vein were not severed. The embryo was dried with the tip of a Kimwipe (Kimberly-Clark, Dallas, TX). Blood was collected with a Microcaps micropipette (Drummond Scientific, Broomall, PA) from the carotid arteries of decapitated embryos. Blood smears were prepared by using the wedge technique followed by air drying and Wright-Giemsa staining. Reverse-phase high-performance liquid chromatography (HPLC) analysis of the globin chains was done on lysates of washed erythrocytes by using a Dynamax HPLC system (Rainin, Palo Alto, CA) with a Vydac C4 column as described previously.37 Individual globin chains were quantitated with Dynamax HPLC Method Manager software. In adult mice, blood was collected from the tail vein. Reticulocyte counts were determined by incubating blood in 100 ng/mL thiazole orange (Aldrich Chemical, Milwaukee, WI) for 15 minutes at 37°C and sorting on a fluorescence-activated cell-sorter machine (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ) and analysis with CellQuest software (Becton Dickinson). Complete blood counts were done with a Hemavet 1500R automated hematology system (CDC Technologies, Oxford, CT).In situ hybridization Embryos from the mating of wild-type Black Swiss mice were removed at 11.5 dpc and fixed in 4% paraformaldehyde overnight at 4°C. Fixed embryos were then dehydrated and embedded in paraffin. Six-micron sections were cut and either stained with hematoxylin and eosin for histologic analysis or used for RNA in situ hybridization. In situ probes consisted of -phosphorus 32-uridine triphosphate (UTP)-labeled full-length antisense murine ATF4 cDNA or the
corresponding labeled sense control. In situ hybridization was done as
described previously and was followed by counterstaining with
hematoxylin.32
Hematopoietic colony assays Single-cell suspensions from 15.5-dpc fetal livers were prepared in Iscoves modified medium supplemented with 2% fetal-calf serum (FCS). Total counts were determined by diluting cells in medium; nucleated cell counts were determined by diluting cells in 3% acetic acid with methylene blue. To quantitate colonies of erythroid colony-forming unit (CFU-Es), fetal-liver cells were plated in triplicate in 1 ml methylcellulose-based medium containing 150 U/mL recombinant erythropoietin (MethoCult M3334; Stem Cell Technologies, Vancouver, BC, Canada) at a density of 1.7 × 104 nucleated fetal-liver cells/35-mm Petri dish. Cells were incubated in a fully humidified atmosphere with 5% carbon dioxide (CO2) at 37°C. The number of CFU-E colonies was determined after 36 to 48 hours of culture. Colonies of erythroid burst-forming units (BFU-Es), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythrocyte-megakaryocyte-macrophage colony-forming units (CFU-GEMMs) were quantitated by plating 5.0 × 104 nucleated fetal-liver cells in 1 ml complete methylcellulose medium (MethoCult M3434). BFU-E colonies were counted at 7 days of culture and CFU-GM and CFU-GEMM colonies at 10 and 14 days, respectively. Assays of bone marrow progenitors in adult mice were done as described above with the following changes. Cells were collected by flushing the marrow cavities of the tibias and femurs with medium, and the cells were filtered through sterile nylon mesh. The number of nucleated cells was determined, and cells were plated at densities of 3.0 × 104 and 1.5 × 104 nucleated cells per plate, respectively, for CFU-E assays and assays of BFU-E, CFU-GM, and CFU-GEMM.Mouse embryonic fibroblast culture For culture of mouse embryonic fibroblasts (MEFs), 15.5-dpc embryos were obtained from the mating of ATF4 heterozygous mice. The embryo heads were processed for genotyping, and the liver, heart, lungs, and spleen were discarded. The remainder of the embryo was minced and incubated in 0.25% trypsin at 4°C for 12 hours and for 20 minutes at 37°C, with periodic disruption by pipetting. Remaining aggregates were discarded and the fibroblasts collected by centrifugation. Cells were maintained in Dulbecco modified Eagle medium supplemented with 15% FCS in 5% CO2 at 37°C. At passage 2, the MEFs were replated on 60-mm plates at a density of 2 × 104 cells/plate. Every 24 hours, duplicate plates for each embryo were trypsinized, and the number of MEFs per plate was determined.Statistical analysis Statistical analysis was done with InStat software (GraphPad Software, San Diego, CA). Significance was determined by using the Kruskal-Wallis nonparametric analysis of variance and the Dunn multiple comparison test. All errors shown represent the SEM.
Targeted inactivation of the ATF4 gene in mice The murine ATF4 gene was disrupted by homologous recombination in both R1 and HM1 ES cells. The targeting vector was designed to replace the entire ATF4 coding region after the first codon, as well as the polyadenylation signal, with a neomycin resistance cassette (Figure 1A).Electroporated ES cells were subjected to G418 and ganciclovir selection, and surviving clones were screened by PCR with a 3' primer external to the homology region. Correct targeting was confirmed in these ES cells by Southern blot analysis of genomic DNA digested with EcoRV and probing of the blot with the internal 3' probe indicated in Figure 1A. This resulted in the predicted 3.3-kb and 2.1-kb bands for the wild-type and homologous recombinant alleles, respectively (Figure 1B). HindIII digestion of DNA and probing of the blot with a fragment external to the 3' homology confirmed the correct 3' end of the homologous allele (data not shown). Similarly, the 5' end of the homologous recombinant allele was verified by double restriction-enzyme digestion with XbaI and SalI enzymes followed by Southern blot analysis using the 5' probe indicated in Figure 1A. Bands corresponding to wild-type (11.4-kb) and homologous recombinant (12.2-kb) alleles were observed as predicted (Figure 1C). Southern blotting analysis using the neomycin cassette probe detected only a single band, indicating that no random integration occurred in these clones (data not shown). To verify that the entire coding region of ATF4 was deleted, we conducted PCR amplification with primers within the ATF4 coding region. No PCR product was observed in the homozygous deletion mutant (data not shown). One in 15 clones resistant to G418 and ganciclovir was found to be correctly targeted. Euploidy was verified for 2 R1 and 2 HM1 targeted ES-cell clones, and these were injected into 3.5-dpc mouse blastocysts. High-percentage chimeras were generated and bred to Black Swiss mice. Heterozygous ATF4 mutant offspring were obtained from chimeras of both the R1 and the HM1 ES-cell line. Heterozygous mice were phenotypically normal. In particular, they had no significant changes in embryonic or adult hematopoiesis or eye formation. Heterozygous mice were intercrossed to obtain homozygous mice. These offspring were genotyped using PCR analysis, and findings were confirmed by Southern blot analysis. The expected Mendelian frequency of genotypes was observed at 17.5 dpc. Some neonatal deaths occurred; the ratio of wild-type to heterozygous to homozygous offspring at weaning was 1:2:0.4 (numbers of mice, 41:81:17). Homozygous pups generally died during the first hour after birth, although excess mortality occurred throughout the first 3 weeks of life. Most homozygous males were infertile. Among those that were fertile, the rate at which they impregnated females was greatly decreased, and they had a reduced period of fertility. Homozygous females were infertile despite having grossly normal ovaries and histologically normal ovarian follicles. When bred to wild-type males, homozygous females did form cervical mucus plugs indicating successful mating, although at a rate lower than normal. However, no homozygous female carried a litter to term and few supported pregnancies to 6.5 dpc. ATF4  -globin chains. From 12.5 to 16.5 dpc, the fetal liver is the
predominant site of erythropoiesis. The definitive (adult) erythrocytes
produced in the fetal liver undergo enucleation before release into the
circulation and express the adult chain. Finally, erythropoiesis
shifts to the bone marrow and spleen, where it remains in adults.
From 13.5 to 16.5 dpc, ATF4
ATF4  / embryos at 15.5 dpc, the percentage
of nucleated erythrocytes was increased 2.1 fold compared with that in
wild-type embryos (Table 1). The morphologic features of nucleated
erythrocytes from homozygous embryos were the same as those from
wild-type controls (Figure 3A). Because
cellular and nuclear volumes of primitive erythrocytes are closely
correlated with developmental age38 and presumably the
length of time the erythrocytes have been in circulation, this
similarity in morphologic characteristics suggests that these nucleated
erythrocytes represent primitive yolk sac-derived erythrocytes that
have persisted in circulation rather than ongoing yolk sac
hematopoiesis. HPLC analysis of lysates of peripheral blood obtained
from 15.5-dpc embryos was done to determine the level of embryonic ( ) and globin (h1 and  y) relative to adult
- and -globin chains. In the homozygous 15.5-dpc embryos, the
percentage of embryonic -globin chains was increased 2.1 fold and
the percentage of embryonic -globin chains was increased 2.2 fold
compared with values in their wild-type littermates (Table 1 and Figure
3B). Thus, the relative increase in the number of nucleated
erythrocytes in homozygous mutants resulted from persistence of
primitive yolk sac-derived erythrocytes rather than premature release
of nucleated definitive erythrocytes into circulation due to anemia,
that is, stress erythropoiesis.
The fetal liver is pale and hypoplastic in ATF4
To quantitate the decrease in fetal-liver size, the number of cells in
fetal livers was determined. The mean number of cells per liver in
ATF4 ATF4 is expressed at a high level in fetal livers of wild-type mice ATF4 is expressed ubiquitously in adult mice and in many cell lines.2,3,6 Reverse transcriptase-PCR amplification detected ATF4 expression in murine blastocysts and ES cells (data not shown). In situ hybridization of 6.5- to 8.5-dpc embryos detected uniform expression throughout the embryo, in agreement with previous findings.39If ATF4 is critical for fetal-liver hematopoiesis, one would expect the gene to be expressed in fetal livers of wild-type mice. In normal mice, the liver rudiment is first apparent at 9 dpc as an evagination of the gut into the septum transversum. Erythroblasts are first visible in the liver at 9 dpc, definitive hematopoiesis is detectable at 10 dpc, and the first long-term repopulating hematopoietic stem cells are observed at 11 dpc.40,41 This is associated with a rapid expansion of erythroid lineage cells in the fetal liver and a definitive adult pattern of globin expression. In this study, in situ hybridization detected ATF4 expression throughout 11.5-dpc embryos but at a much higher level in the liver than in other tissues (Figure 4C and D). A high level of expression was also present in the developing eye, in agreement with previous results.19 No signal above background level was detected with the sense-oriented control probe (data not shown). The high level of ATF4 expression in fetal livers of wild-type mice is consistent with an important role for this protein in fetal-liver hematopoiesis. Hematopoietic progenitor colonies in ATF4 /
embryos, we conducted in vitro progenitor assays. Compared with values
in wild-type embryos, no significant difference was found in the number
of colonies/105 nucleated fetal-liver cells, suggesting
that a similar fraction of the fetal liver in ATF4/
embryos and wild-type embryos is composed of hematopoietic cells. However, the number of CFU-E, BFU-E, and CFU-GM colonies per
ATF4/ fetal liver were reduced 2.2, 2.1, and 2.1 fold,
respectively, and the number of CFU-GEMM colonies was reduced 2.9 fold
compared with values in wild-type littermates (Figure
5A). The ratio of BFU-Es to CFU-Es was
normal in ATF4/ embryos, indicating that there was no
defect in the terminal differentiation of erythroid progenitors.
A striking decrease in colony size was observed for BFU-Es, CFU-GMs,
and CFU-GEMMs (Figure 5B). To quantitate this reduction, the number of
colonies, the number of cells per plate, and the number of cells per
colony were determined at 10 days of culture in methylcellulose medium
supplemented with growth factors that promote the growth of all myeloid
lineages. There was no significant difference in the total number of
colonies per plate. However, for fetal livers from
ATF4 Adult ATF4 / adults compared with controls but
no significant change in reticulocyte count or mean corpuscular
hemoglobin concentration (Table 2). Interestingly, leukocyte and platelet counts of ATF4/
mice were not significantly different from those of controls (Table 2
and data not shown).
The erythropoietic defect in ATF4 Primary MEFs from ATF4 / fibroblast
cultures were considerably less dense at each time point despite having
been plated at the same initial concentration as wild-type and
heterozygous cultures (Figure
6A). ATF4/ fibroblasts
divided at a greatly reduced rate, showing a mean doubling time of 45.2 hours, a 2.1-fold increase compared with the doubling time of 21.9 hours observed for both wild-type and heterozygous embryos (Figure 6B).
ATF4/ MEFs became contact inhibited at a density
similar to those of controls and did not reach the end of their
proliferative capacity sooner than those of wild-type controls (data
not shown). These results suggest that the homozygous cells did not
prematurely senesce and that ATF4/ cells had a
cell-autonomous defect in normal cellular proliferation.
ATF4 / embryos were smaller than
their wild-type and heterozygous littermates. From 13.5 dpc through
birth, the mean length of ATF4/ embryos was reduced 1.1 fold and the mean weight was reduced 1.3-fold compared with control
values. After birth, the weight of ATF4/ mice remained
lower than that of control mice. At 3 weeks of age, the difference in
weight was maximal: the weight of ATF4/ mice was
2.5-fold lower than that of controls. The weight of ATF4/ adult mice ultimately remained approximately
1.7-fold lower than that of controls.
Hair growth also appeared to be delayed in ATF4 Adult ATF4 homozygous mutants were severely microphthalmic, with no
recognizable lens, anterior chamber, iris, or vitreous body.
Development of the eye in ATF4 The phenotypic changes in ATF4
In studies in mice, we found that homozygous inactivation of the ATF4 gene results in severe fetal anemia. The hematocrit of 15.5-dpc mutant fetuses was 0.15, whereas that in controls was 0.35. The percentage of nucleated erythrocytes was increased 2 fold at this stage of development, and this increase was accompanied by an increase in embryonic globin chains, indicating that these cells represented persistence of primitive yolk sac-derived erythrocytes rather than premature release of nucleated definitive erythrocytes into the circulation. At 15.5 dpc, fetal livers in homozygous mutant fetuses were paler and much smaller than those in controls. Because most of the fetal liver at this stage is composed of erythroid elements, this finding also indicates the presence of a defect in fetal-liver hematopoiesis. In situ hybridization studies in wild-type mice showed that ATF4 is expressed at a high level in the fetal liver, a result consistent with an important role for ATF4 in fetal-liver hematopoiesis. In vitro methylcellulose colony assays of ATF4 ATF4 inactivation resulted in severe fetal anemia but relatively normal
steady-state erythropoiesis in adults. This phenotype has been
described in other mouse mutants, such as the "flexed," E2F4, and
signal transducer and activator of transcription (STAT) 5a STAT5a Although ATF4 The greater severity of anemia in embryos compared with adults was most
likely due to the low level of hematopoietic reserve capacity during
the fetal-liver stage of hematopoiesis. Adults have considerable
erythropoietic reserve capacity, and the rate of erythropoiesis can be
up-regulated 10 fold in response to stress such as hemorrhage or
anemia.49 The rate of fetal erythropoiesis is several-fold
higher than the steady-state erythropoietic rate in
adults.50,51 During the period of fetal-liver
hematopoiesis, the developing embryo has little hematopoietic reserve
capacity.50-52 Thus, anemias during this stage may reveal
factors required for maximal levels of hematopoiesis that are
unnecessary for normal levels of adult hematopoiesis. Adult mice with
such defects may be deficient in response to erythropoietic stress.
Interestingly, adult ATF4 Several other proteins, including XBP-1, Rb-1, and c-myb, are required
for normal fetal-liver hematopoiesis; and homozygous inactivation of
each results in pale, runted embryos with fetal-liver hypoplasia, a
relative increase in the number of nucleated erythrocytes, and a severe
anemia at 15.5 dpc caused by inhibition of definitive erythropoiesis.53-55 Like ATF4, these proteins are
expressed in many tissues but at the highest level in fetal liver.
Unlike inactivation of ATF4, homozygous inactivation of these proteins
results in embryonic death between 13.5 and 15.5 dpc. As was observed
in ATF4 We previously isolated ATF4 in a yeast 2-hybrid screen by using LCR-F1
as bait. A knockout mutation of LCR-F1 resulted in severe fetal anemia
in the C57BL/6J inbred mouse strain33 (unpublished data,
August 1999). In both LCR-F1 In addition to hematologic defects, ATF4-deficient embryos had defects
in lens formation, postnatal hair growth, and body size. ATF4 may play
a distinct role in each of the developmental pathways involved in these
features. However, it is most probable that a defect in proliferation
links all of the components of this phenotype, because the fetal liver,
embryonic lens, and hair follicle are all sites of rapid proliferation.
Consistent with this hypothesis, primary fibroblasts from
ATF4 No increase in apoptosis has been observed in the fetal livers of
ATF4 In summary, a knockout mutation of ATF4 resulted in severe fetal anemia. At 15.5 dpc, the hematocrit of ATF4 null fetuses was 0.15, whereas that in controls was 0.35. Fetal livers were pale and hypoplastic, and the number of hematopoietic progenitors of multiple lineages was decreased more than 2 fold. In addition to the reduction in progenitor numbers, the size of BFU-E, CFU-GM, and CFU-GEMM colonies was dramatically reduced. These results suggest that ATF4 is essential for the normal, high-level proliferation required for fetal-liver hematopoiesis.
We thank Jin-Xiang Ren for all ES-cell injections, Dr Vladimir Divoky for assistance with hematopoietic colony assays, Dr Thomas Ryan for assistance with hematologic assays, and Dr Peter Detloff and members of the Townes laboratory for critical review of the manuscript.
Submitted June 20, 2001; accepted September 24, 2001.
Supported in part by grant HL35559 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Tim M. Townes, Dept of Biochemistry, University of Alabama at Birmingham, 537 Kaul Genetics Bldg, 720 20th St S, Birmingham, AL 35294; e-mail: ttownes{at}uab.edu.
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Top
Abstract
Introduction
gt11
library screen by using the ATF binding site (GTGACGTACAG) as a
probe.
-aminobutyric acid type B
receptor.
for example, through activating or repressing expression
of genes involved in cell-cycle regulation. Importantly, the level of
ATF4 protein increases after growth stimulation of quiescent cells, with maximal levels occurring during late G1, and ATF4
affects cyclin A promoter activity in transient-transfection
assays.