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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3568-3577
RED CELLS
Analysis of ferrochelatase expression during hematopoietic
development of embryonic stem cells
Scott T. Magness,
Antonio Tugores, and
David A. Brenner
From the Departments of Medicine and Biochemistry and
Biophysics, Curriculum in Genetics and Molecular Biology, University of
North Carolina at Chapel Hill.
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Abstract |
Ferrochelatase, the last enzyme in the heme pathway, chelates
protoporphyrin IX and iron to form heme and is mutated in
protoporphyria. The ferrochelatase gene is expressed in all tissues at
low levels to provide heme for essential heme-containing proteins and
is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF-E2 sites, all of which bind their cognate
trans-acting factors in vitro. To investigate the role of these
elements during erythropoiesis, we introduced expression of the green
fluorescent protein (EGFP) transgenes driven by various ferrochelatase
promoter fragments into a single locus in mouse embryonic stem cells.
EGFP expression was monitored during hematopoietic differentiation in
vitro using flow cytometry. We show that a promoter fragment containing
the Sp1 sites, the NF-E2 and GATA elements, was sufficient to confer
developmental-specific expression of the EGFP transgene, with an
expression profile identical to that of the endogenous gene. In this
system the  0.275 kb NF-E2 cis-element is required for
erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between 0.375kb and 1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the
effect of an A-to-G polymorphism identified in the promoters of
patients with protoporphyria. There was no effect of the G haplotype on
transcriptional activity of the 1.1 kb transgene.
(Blood. 2000;95:3568-3577)
© 2000 by The American Society of Hematology.
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Introduction |
Heme is required at low levels in all cells for
respiratory cytochromes and other essential heme-containing proteins,
and it is required at high levels during erythropoiesis to provide heme
for hemoglobin. Ferrochelatase, the last enzyme in the heme biosynthetic pathway, chelates ferrous iron and protoporphyrin IX to
form heme. The human ferrochelatase gene is nuclear transcribed and,
after translation, is transported to its active site in the inner
mitochondrial membrane.1-4 The ferrochelatase protein has a
molecular weight of 40 to 42 kd in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis. Radiation
inactivation studies suggest that the ferrochelatase protein acts in
situ as an 82- to 84-kd dimer.5 Recently, crystallization
of human ferrochelatase confirmed a homodimer structure for the
enzyme.6
Investigating ferrochelatase gene regulation has primarily involved
assessing enzyme activities in human or mouse erythroleukemic cell
lines, identifying ferrochelatase gene transcripts in erythroid differentiated embryonic stem cells using reverse
transcription-polymerase chain reaction,7 and analyzing
cis-element function using transient transfection of reporter
genes into nonerythroid and erythroleukemic cells
lines.8-15 The ferrochelatase promoter contains 2 Sp1
cis-elements and upstream GATA and NF-E2
cis-elements,13,16 and each binds its cognate
trans-acting factors in vitro.13 Transient
transfection assays in nonerythroid and erythroid cells demonstrated
that the minimal promoter (0.150 kb), containing Sp1
binding sites, is sufficient to confer erythroid-preferential
expression, whereas the 0.375 kb GATA and NF-E2 elements in the
extended promoter failed to confer any additional increase in
erythroid-preferential expression.13
We hypothesized that appropriate use of these erythroid
cis-elements may require organized chromatin structure. Two
transgenic mouse lines, one containing the minimal promoter
(0.150 kb) and another containing the extended promoter
(4.0 kb), were generated. Unlike the transient transfection
studies, the transgenic mice studies demonstrated that
cis-elements 5' of the 0.150 kb Sp1 sites are
required for maximal erythroid-specific expression of the
ferrochelatase gene and furthermore implied that these
cis-elements require a structured chromatin environment for
appropriate tissue-specific expression.17 The 4.0 kb
ferrochelatase promoter fragment contains the 0.375 kb GATA and
NF-E2 cis-elements, but it also contains 3.8 kb of a sequence
that may contain additional functional cis-elements. Because
the studies in transgenic mice used only the minimal promoter (0.150 kb) or the extended promoter (4.0 kb), we were
unable to precisely identify the cis-elements required for
erythroid-preferential up-regulation of the ferrochelatase gene.
Protoporphyria is associated with a partial deficiency in
ferrochelatase18,19 and is typically transmitted as an
autosomal dominant disease with variable penetrance.20,21
Patients with protoporphyria consistently exhibit ferrochelatase
activities that are 15% to 30% of normal.21,22 They also
exhibit severe photosensitivity and sometimes hepatobiliary disease,
which may necessitate liver transplantation. Ferrochelatase mutations
identified in patients with protoporphyria are
heterogeneous23 and include missense and nonsense
mutations24-28 and, most commonly, mutations in splice
donor or acceptor sites that lead to aberrant splicing and exon
skipping.29-35
It has been proposed that the less than 50% ferrochelatase activities
described for protoporphyria arise from the combination of a null
allele and a "low-expressing" allele.36 Probable
promoter defects that could result in a low-expressing allele include
mutations in the ferrochelatase promoter and aberrant methylation of
the CpG island in the promoter. In this regard, an A-to-G polymorphism has been identified in the ferrochelatase promoter (251 bp from the start codon) that segregates with the protoporphyric
phenotype.36,37 The effect of the G haplotype on
ferrochelatase gene expression is unknown.
Based on our current understanding of transcription factor binding to
the NF-E2 and GATA sites at 0.375 kb of the ferrochelatase promoter, we predicted that use of the NF-E2 and GATA
cis-elements would be required during erythropoietic
up-regulation of the ferrochelatase gene, and because the A-to-G
polymorphism lies in a transcriptionally active region between the
0.150 kb Sp1 sites and the 0.375 kb GATA site, we thought
the G haplotype might play a role in decreasing expression of the
ferrochelatase gene.
To test these hypotheses, we used a gene-targeting method designed to
analyze single-copy transgenes integrated into a single locus, 5'
of the HPRT gene.38 We modified an HPRT gene-targeting vector to contain ferrochelatase promoter fragments driving expression of the green fluorescent protein (EGFP) and assessed transgene expression in live cells using fluorescence-activated flow cytometry. This method has advantages over traditional stable cell lines containing luciferase or  -galactosidase reporter genes in that EGFP
transgene expression can be analyzed in individual, living cell
populations devoid of multiple transgene copy numbers and integration sites.
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Materials and methods |
Reporter plasmid and targeting vector construction
The expression of green fluorescent protein (EGFP) reporter gene
vector, pEGFP, (Clontech, Palo Alto, CA) was modified by adding a
poly-cloning linker 5' of the EGFP cDNA that contained SalI and SacI restriction endonuclease sites. The EGFP
vector contains an enhanced version of the Aequorea victoria
protein39 with the poly A signal from SV40 and the neomycin
resistance gene driven by the SV40 promoter. Various regions of the
human ferrochelatase promoter, originally cloned into a luciferase
reporter gene construct,13,17 were digested with
SacI and SalI restriction endonucleases, and the
SalI/SacI fragments were subcloned into the modified
pEGFP vector.
NF-E2 and GATA mutations were introduced into the ferrochelatase
promoter using mutant oligonucleotides; the NF-E2 mutations are as
described.13 Mutations abolishing the GATA consensus site
were introduced into an oligonucleotide (mutations are underlined: TCCTGGAAAGGAAA). For the GATA/NF-E2 double
mutant, the same GATA mutations combined with the NF-E2 mutations were
introduced into an oligonucleotide (NF-E2 mutations are underlined:
TTTGCATCGTCA). Each mutant oligo was annealed to its
complementary sequence. Overhangs of the double-stranded oligos yielded
the restriction endonuclease sites HindIII and BamHI.
All mutant double-stranded oligonucleotides were ligated into the
HindIII and BamHI sites of the 0.375 kb EGFP.
To generate a reporter gene construct that contained the A-to-G
polymorphism, genomic DNA was isolated from a patient with protoporphyria who had only 1 allele of the ferrochelatase
gene40 with the G haplotype. A 1.1-kb fragment of the
ferrochelatase promoter was amplified using polymerase chain reaction
(PCR). The presence of the G haplotype (251) in this patient was
demonstrated by di-deoxy sequencing of the 1.1-kb PCR fragment. The PCR
product was digested with XbaI and SacII and then
cloned into the XbaI and SacII site of pBluescript SK+.
A clone that contained the G haplotype was digested with SalI
and SacI and cloned into the SalI and SacI
pEGFP reporter gene construct. The resultant pEGFP construct was
digested with EcoRI and SacI, and the promoter fragment was ligated into the EcoRI and SacI sites of the
HPRT/EGFP construct.
The HPRT targeting vector pMP8SKB38 (kindly provided by Drs
S. Bronson and O. Smithies) contains a polycloning site flanked 5' by DNA from the HPRT locus located upstream of the HPRT
promoter and flanked 3' by the promoter and exons 1, 2, and 3 of
the HPRT gene. To facilitate cloning, we modified the cloning site by
adding additional restriction endonuclease sites and the cDNA for EGFP by standard cloning methods. Additionally, the NotI and
SacI sites in the HPRT sequence were eliminated by digestion
with the appropriate restriction endonuclease, filling in of the
5' overhang with Klenow, and performance of a standard ligation
reaction. The mutant and wild-type human ferrochelatase promoter
regions from the pEGFP vectors (described earlier) were isolated using
EcoRI and SacI restriction endonuclease digests and
subcloned into the EcoRI/SacI site of the HPRT
targeting vector.
Cell culture/microscopy
The mouse embryonic stem (ES) cell line BK4, a subclone of E14TG2a,
was maintained in an undifferentiated state on embryonic fibroblast
feeder cells in Dulbecco's modified essential medium (DMEM) high
glucose, supplemented with 15% fetal bovine serum (ES qualified;
Gibco/BRL, Gaithersburg, MD), 0.1 mmol/L
-mercaptoethanol, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 1 mg/mL streptomycin. ES cells were grown at
37°C in a humidified atmosphere containing 5% CO2.
Before primary differentiation, the ES cells were weaned from the
feeder cells by passaging 2 to 3 times in DMEM-H, 15% fetal calf serum
(FCS; ES qualified; Gibco/BRL), 2 mmol/L glutamine, 100 U/mL
penicillin, 1 mg/mL streptomycin, 2U/mL LIF, and 125 µmol/L
-mercaptoethanol on tissue culture-treated plates coated with 0.1%
gelatin in phosphate-buffered saline (PBS). For primary differentiation,41,42 approximately
1.5 × 104 cells were plated in 6-cm Petri dishes
containing differentiation media (Iscove's-MDM with 10% FCS
(Hyclone, Logan, UT), 4.5 × 10 4 mol/L
monothioglycerol (MTG), 25 µg/mL ascorbic acid, 200 µg/mL iron-saturated transferrin, 2 mmol/L glutamine). Cells were grown in a
humidified atmosphere containing 5% CO2 at 37°C for 3 to 4 days. After 3 to 4 days in primary differentiation conditions, embryoid bodies were washed once in PBS and disrupted by trypsinization for 1 minute (using 0.5× concentration; 0.025% trypsin, 0.26 mmol/L EDTA). Approximately 1.0 to 3.0 × 105 cells
were plated in a secondary methylcellulose culture with IMDM containing
the above concentrations of glutamine, penicillin/streptomycin, ascorbic acid and MTG, 10% plasma-derived serum (Anatech, Tyler, TX),
5% protein-free hybridoma medium (PFHM II; Gibco/BRL), 1% methylcellulose, 2 U/mL erythropoietin (R&D Systems, Minneapolis, MN),
and 100 ng/mL stem cell factor (R&D Systems). Cells were grown in a
humidified atmosphere containing 5% CO2 at 37°C for an
additional 4 to 8 days.
To determine the half-life of EGFP, 5 to 8 × 105
pluripotent cells (from 2 different EGFP-expressing ES clones) were
plated into 6-well plates. Twenty-four hours later, 100 µg/mL
cyclohexamide43 was added at 2-hour intervals for 6 hours.
EGFP levels were then quantified by flow cytometry as later described.
An Olympus IX70 (Olympus, Melville, NY) was used for
bright-field and fluorescence microscopy. EGFP was detected using a
filter that allowed excitation at 488 nm, and porphyrins were detected using a filter that allowed excitation at 385 nm. Images were captured
using 200 ASA Kodak Elite slide film (Kodak, Rochester, NY). The film image was then digitized using a transparency scanner, and image overlays were generated using Adobe Photoshop
software (Adobe Systems, San Jose, CA). For bright-field, porphyrin,
and EGFP overlays, levels of red fluorescence above background levels were digitally selected and overlaid onto the bright-field image.
Stable integration of reporter gene constructs
Undifferentiated ES cells were isolated by trypsinization, and
approximately 2 × 107 cells were electroporated
with 15 µg of an EGFP/HPRT targeting vector linearized with
PvuI. The cells were suspended in 0.5 mL PBS with the DNA and
pulsed in a 4-mm gap electroporation cuvette with 350 V, 50 µF for 1 second (Bio-Rad, Hercules, CA). Twenty-four hours later,
the growth medium was replaced with HAT (final concentration of 0.1 mmol/L hypoxanthine, 0.4 µmol/L aminopterin, 16 µmol/L thymidine)
selection medium. HAT-resistant clones were isolated and expanded on
day 10 after initiation of selection. Individual clonal populations
were used in all experiments unless otherwise indicated.
Flow cytometry
A Becton Dickinson FACScan (Franklin Lakes, NJ) was
used to analyze ES cells for fluorescence intensities. Pluripotent ES cells, grown in the absence of feeders, were trypsinized, and washed
once in growth medium. Then 1 × 106 cells were
suspended in 1.0 mL PBS for analysis. For differentiated ES cells,
embryoid bodies (EB) were isolated by centrifugation and washed once
with PBS. After that, EBs were disrupted by brief trypsinization (using
0.25× trypsin solution for 1 minute), washed once in growth
medium, and finally suspended in 1.0 mL PBS for analysis. Each sample
was collected at 24-hour intervals. All cells were analyzed for EGFP
expression within 30 minutes of trypsinization.
Forward and side scatter plots were used to gate on the live population
of cells. The fluorescence of 50,000 to 80,000 cells was measured using
the FL1 (or fluorescein isothiocyanate) channel of the FACScan. EGFP
intensities were calculated from maximal fluorescence peak heights
using Cyclops software (Fort Collins, CO).
Northern blot and immunoblot analyses
Total RNA was isolated at 24-hour intervals during ES cell
differentiation using Trizol (Gibco/BRL, Gaithersburg, MD), and 15 µg
was separated on a 1.5% agarose/formaldehyde gel, transferred to nylon
membrane by capillary action in 20× SSC (3.0 mol/L NaCl, 0.3 mol/L sodium citrate), UV cross-linked in a Stratalinker (Stratagene, San Diego, CA) on automatic setting, and prehybridized in Rapid-hyb buffer (Amersham, Arlington Heights, IL). A random primed
32P-labeled mouse ferrochelatase, human EKLF, mouse ALAS-E,
or mouse -globin cDNA probe was applied to the membrane at
1 × 106 cpm/mL and incubated at 60°C for 4 hours. The membrane was washed once in 2× SSC/0.1% SDS at
21°C and twice at 55°C for 15 minutes. Then the wash buffer was
adjusted to 0.1× SSC/0.1% SDS and applied to membrane twice at
60° for 15 minutes. The membrane was then exposed to X-OMAT AR film
(Kodak) with intensifying screens for 14 hours (ferrochelatase) or
analyzed on a Storm 840 Phosphor Imager (Molecular Dynamics, Sunnyvale,
CA) for 8 hours (EKLF) or for 24 hours (ALAS-E and -globin).
To detect ferrochelatase protein, whole protein extracts were isolated
at 24-hour intervals in RIPA buffer (0.15 mol/L NaCl, 50 mmol/L
Tris-Cl, pH 7.2, 1% deoxycholic acid, 1% Triton-X 100, 0.1% SDS)
containing 10 µg/mL phenylmethylsulfonyl fluoride, and 15 µg whole
cell lysate was separated on a 12% SDS-polyacrylamide gel and
electrophoretically transferred to nitrocellulose. Equal loading of
protein was assessed by Ponceau S staining. The membrane was then
blocked with BLOTTO (5% wt/vol nonfat dried milk in water) for 15 minutes at room temperature. A 1:500 dilution (in BLOTTO) of
anti-recombinant human ferrochelatase polyclonal antibody44 was applied to the blot and allowed to incubate at room temperature for
4 hours. The blot was washed once with BLOTTO and twice in Tris-buffered saline containing 0.1% TWEEN-20 (TBS-TWEEN). A goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Santa
Cruz Biotechnologies, Santa Cruz, CA) was applied at a 1:1000 dilution
in BLOTTO and allowed to incubate for 1 hour at room temperature. The
membrane was washed once in TBS-TWEEN, once in BLOTTO, and twice in
TBS-TWEEN for 10 minutes, each time at room temperature. Western Blue
substrate (Promega, Madison, WI) was added, and the immunoblot was
allowed to develop for approximately 20 minutes.
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Results |
Single-copy, single-site integration of the ferrochelatase promoter
transgene
EGFP reporter gene-targeting constructs were introduced into the
mouse HPRT locus by electroporation to the mouse ES cell line, BK4.
This ES cell line is male-derived and is functionally deficient in the
X-linked HPRT gene resulting from a deletion of the HPRT promoter and
exons 1 and 2.45 On homologous recombination between the
endogenous HPRT gene and the exogenous HPRT targeting vector, the HPRT
mutation is corrected, and successfully targeted cells are able to grow
in HAT selection media. The HAT-resistant clones contain 1 copy of the
reporter gene located 5' of the HPRT gene (Figure
1A). Random integration of the targeting
vector does not reconstitute HPRT expression and the ability to grow in
HAT selective media.38,45 Various fragments of the human
ferrochelatase promoter were cloned into the HPRT targeting vector for
site-specific integration experiments (Figure 1B). The ferrochelatase
promoter fragments driving reporter gene expression contained
0.125 kb sequence with no functional elements; 0.150 kb
sequence containing 2 Sp1 elements; 0.375 kb sequence containing
the 2 erythroid elements, GATA and NF-E2 (with an overlapping AP-1
site); or 2 extended promoter fragments containing 1.1 kb
sequence or 4.0 kb sequence (Figure 1B).
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| Fig 1.
Targeting the mouse HPRT locus with the human
ferrochelatase promoter transgenes.
(A) To introduce ferrochelatase promoter genes 5' of the HPRT
locus, we designed HPRT targeting vectors that contained human
ferrochelatase promoter fragments driving expression of the EGFP cDNA
flanked 5' and 3', with HPRT sequences containing the HPRT
promoter, and exons 1, 2, and 3. After electroporation, a homologous
recombination event between the HPRT gene and the targeting vector
reconstitutes the HPRT gene function, which allows for selection in HAT
media, and introduces a single copy of the ferrochelatase promoter
transgene into the genomic DNA. (B) EGFP reporter genes contain various
fragments of the human ferrochelatase promoter.
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Up-regulated expression of the 4.0 EGFP reporter gene in
erythroid colonies
To determine whether expression of the extended promoter transgene
(4.0 kb) in the HPRT locus was responsive to erythroid cell
lineage trans-activation, we differentiated pluripotent ES cells in methylcellulose cultures to favor primitive and definitive erythroid cell formation.41,46 After 4 to 5 days of
secondary differentiation in methylcellulose, globinized
erythroid colonies became visible and continued to proliferate and
differentiate until day 8 (Figure 2A).
Although differentiation conditions favored hematopoietic development,
nonerythroid cells proliferated and surrounded the erythroid colonies
from days 7 to 8 of methylcellulose differentiation (Figure
2A).
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| Fig 2.
Transgene expression in erythroid colonies.
ES cells containing the targeted 4.0 kb transgene were
differentiated in primary culture for 5 days. Then, embryoid bodies
(EB) were disrupted to single cells and plated in a secondary
methylcellulose culture. Between 4 and 8 days of secondary culture,
colonies of erythroid cells were visible as foci of red cells
(globinized areas indicated with red arrows) with
nonglobinized cells (black arrows). The erythroid colonies were
surrounded by nonerythroid cells of unknown lineage. Original
magnification, × 200. (A) Erythroid colonies were observed using
either visible, (B) porphyrin-specific, or (C) EGFP-specific
wavelengths. (Yellow areas indicate emission filter bleed-through of
porphyrin fluorescence combined with green fluorescence) (D) To observe
areas of fluorescence in the erythroid colony, the porphyrin
fluorescence image was overlaid with the visible image, or (E) the EGFP
fluorescence image was overlaid with the visible image. (F) To identify
areas of both porphyrin and EGFP expression in the erythroid, the
images from porphyrin fluorescence and EGFP fluorescence were overlaid.
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To confirm early heme-pathway gene expression, erythroid colonies were
observed using fluorescence microscopy.  -Aminolevulinic acid synthase-erythroid form (ALAS-E) is the first and rate-limiting enzyme in the heme biosynthetic pathway. Its product, -ALA, is quickly converted to protoporphyrin. When erythroid colonies were observed using wavelengths that excite porphyrins, red fluorescence emanated exclusively from cells of the erythroid colony, indicating heme pathway gene up-regulation (Figure 2B).
To confirm that porphyrins were producing the red fluorescence in the
erythroid colonies, we isolated approximately 50 red-fluorescing erythroid colonies on day 4 to day 5 of secondary differentiation, extracted the pigments, and identified the porphyrins using
reverse-phase high-performance liquid chromatography
(HPLC).47,48 In the erythroid colonies there were high
levels of uroporphyrin, coproporphyrin, and protoporphyrin, as
indicated by an HPLC retention time and spectrofluorometric signatures
unique for each porphyrin (not shown). Levels of porphyrins in
nonerythroid cell extracts were below the level of detection using
this method.
To determine whether the ferrochelatase promoter EGFP-reporter gene was
expressed in cells of the erythroid colony, we viewed cells containing
the 4.0 kb promoter transgene with EGFP-specific wavelengths.
High levels of green fluorescence were observed in cells of the
erythroid colony, whereas no green fluorescence was found in
nonerythroid cells surrounding the erythroid colony (Figure 2C),
indicating that erythroid-preferential expression is controlled by
elements located within 4.0 kb of the ferrochelatase promoter.
Interestingly, there was a different temporal expression for early heme
pathway genes (indirectly monitored by porphyrin fluorescence) than for
the 4.0 kb ferrochelatase promoter transgene. Using fluorescence
microscopy, the fluorescence of individual erythroid colonies was
followed over a 5-day period. High levels of EGFP fluorescence were
observed from day 4 of primary differentiation through day 4 of
secondary differentiation. Porphyrin fluorescence was only observed
beginning on day 3 to 4 of secondary differentiation, which was
followed by globinization on day 5 to 6 of secondary differentiation. Bright-field and fluorescence images were overlaid to
demonstrate expression patterns of globins, early heme pathway genes,
and the ferrochelatase promoter transgene. Globinized primitive erythroid cells (Figure 2A, red arrows) were observed in the erythroid colony as were nonglobinized erythroid precursor cells (Figure 2A,
black arrows). To identify areas of early heme pathway gene expression
in cells of the erythroid colony, the porphyrin fluorescence image
(Figure 2B) was overlaid with the bright-field image (Figure 2D). To
identify areas of 4.0 kb transgene expression, the EGFP fluorescence image (Figure 2C) was overlaid with the bright-field image
(Figure 2E). To identify areas of early heme pathway expression and
4.0 kb EGFP expression, the porphyrin fluorescent image was overlaid with the EGFP fluorescent image (Figure 2F). Overlaid images
demonstrated different temporal expression of globins, early heme
pathway genes, and the ferrochelatase reporter gene (Figures 2D,
2E, and 2F).
Endogenous ferrochelatase gene and transgene expression are
up-regulated before the primitive erythroid cell stage
In primary cultures of 4.0 EGFP ES cells that contained early
hematopoietic lineages, there was a high level of green fluorescence that appeared to peak on day 3 to 4 of differentiation (Figures 3A, 3B). After day 4, the green
fluorescence was markedly decreased, and by day 6 to 7 it was below the
level of visual detection. Immunoblots for EGFP also indicated a
decrease in EGFP protein after day 4 of differentiation, corroborating
the fluorescence data (not shown). Flow cytometric analysis of live
cells confirmed the increase in transgene expression beginning on
day 2, peaking on day 4, and decreasing by day 5 of primary
differentiation (Figure 3C). Single-peak histograms were observed for
the gated population of live cells, indicating equivalent expression of
the transgene in each cell. The examination of early heme
pathway gene expression using fluorescence microscopy and HPLC
analysis of porphyrins indicated no detectable porphyrin production
at any stage of primary differentiation (not shown).
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| Fig 3.
Expression of the ferrochelatase promoter transgene
during primary in vitro differentiation.
ES cells containing the 4.0 kb transgene were differentiated in
a liquid culture (primary differentiation) for 5 days. Original
magnification, × 100. (A) Bright-field images and (B) green
fluorescent images were taken from day 0 (undifferentiated) through day
5 using identical exposure times. A transgene-negative, 4-day
differentiated EB was included as a negative control (TG-D4, A and B).
Arrows point to fluorescing cell clusters. (C) Quantitation of green
fluorescence was determined using flow cytometry. Each histogram
represents the fluorescent intensities of approximately 50 000 cells
determined at each 24-hour time point. Relative EGFP intensities are
indicated above each histogram.
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Fluorescence microscopy and flow cytometry data indicated that the
ferrochelatase promoter transgene was up-regulated early in
hematopoietic development, before the primitive erythroid-cell stage.
Therefore, we used primary differentiated embryoid bodies for
conducting expression analysis of the endogenous ferrochelatase gene
and the ferrochelatase promoter transgenes. Endogenous ferrochelatase mRNA expression was up-regulated beginning on day 2, peaking on day 4, and decreasing by day 5 (Figure 4A).
Erythroid Krüppel-like factor (EKLF), an erythroid-specific
transcription factor, was temporally expressed in a parallel fashion to
the ferrochelatase gene, indicating the erythropoietic potential of
cells in the embryoid bodies (Figure 4A).
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| Fig 4.
Endogenous ferrochelatase during primary in vitro
differentiation.
(A) (left) Total RNA was isolated from primary differentiated EBs over
a 5-day period. Northern blot analysis was conducted using 15 µg
total RNA and probed with a random-primed mouse ferrochelatase cDNA
(MFC). The membrane was stripped of the ferrochelatase probe and
reprobed with a random-primed human EKLF cDNA (EKLF). Amounts of RNA
loaded for each time point were assessed using ethidium bromide
staining of 18S and 28S ribosomal RNA (rRNA). (right) Graphical
representation of ferrochelatase mRNA levels depicted in Northern blot
(left). Values were normalized to rRNA levels using scanning
densitometry. (B) Endogenous ferrochelatase protein is demonstrated by
immunoblotting. (C) Ferrochelatase promoter transgene expression during
primary in vitro differentiation. Transgene expression was determined
at each 24-hour time point by disrupting the EBs with brief
trypsinization and quantifying EGFP expression in live cells using flow
cytometry. Black lines represent transgenes that have 0.150 kb
Sp1 elements and the 0.375 kb NF-E2 and GATA sites. Gray lines
represent the 0.150 kb transgene that contains only the Sp1
elements, the 0.125 kb transgene that contains no known
functional elements, and the transgene-negative cell line BK4.
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To analyze ferrochelatase protein expression during hematopoietic
development, we isolated whole cell extracts from undifferentiated and
2-, 3-, and 4-day differentiated embryoid bodies and assessed ferrochelatase protein levels by immunoblotting. There were equivalent levels of ferrochelatase protein in undifferentiated and 2-, 3-, and
4-day differentiated cells (Figure 4B).
cis-Elements located between 0.150 kb and
0.375 kb are required for erythroid preferential expression, but
maximal erythroid enhancement requires elements between 0.375 kb and
1.1 kb of the ferrochelatase promoter
To identify regions within the ferrochelatase promoter that
contributed to regulating ferrochelatase gene expression, we generated HPRT-targeted clonal cell lines containing a single copy of various regions of the ferrochelatase promoter-driving expression of EGFP (Figure 1B). Transgene expression was assessed over a 5-day period of
primary culture differentiation using flow cytometry. In pluripotent cells there were higher expression levels from the 0.375 kb, 1.1 kb, and 4.0 kb ferrochelatase promoter transgenes
compared to the 0.125 kb or 0.150 kb ferrochelatase
promoter transgenes (Figure 4C). Ferrochelatase promoter transgenes
containing the GATA and NF-E2 sites showed enhanced expression
beginning on day 2 of differentiation, peaking maximally on day 4, and
dropping to levels similar to those of pluripotent cells by day 5 (Figure 4C). Between day 0 and day 4 (maximal induction), there was a 1.9 ± 0.07-fold induction for the 1.1 kb transgene, a
2.4 ± 0.1-fold induction for the 4.0 kb transgene, and a
1.45 ± 0.35-fold induction for the 0.375 kb transgene.
Cells containing the 0.125 or 0.150 kb ferrochelatase
promoter transgenes showed no enhanced expression at any point of
differentiation. Maximal expression of ferrochelatase promoter
transgenes required additional sequences upstream of the GATA site
(from 0.375 kb to 1.1 kb).
The NF-E2 cis-element is required for erythroid-enhanced
expression, and the GATA cis-element functions as a
stage-specific repressor and enhancer
To elucidate the roles of the 0.375 kb NF-E2 and GATA
cis-elements, we mutated either the NF-E2 site, the GATA site,
or both and assessed expression of the wild-type and mutant transgenes integrated into the HPRT locus. Mutations within the NF-E2 site abolished binding activity of a protein corresponding to the migration pattern of NF-E2 but did not abolish binding of AP-1, as determined by
mobility shift assays.13 GATA binding was abolished by
either deleting the site ( GATA) or by introducing point mutations
(mGATA) (Figure 5A).
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| Fig 5.
Expression of transgenes that contain mutant GATA sites,
NF-E2 sites, or both in undifferentiated and 3-day differentiated
embryoid bodies.
(A) Summary of the constructs that contained mutations in either GATA,
NF-E2, or both (mGATA, mNF-E2, or mGATA/mNF-E2). The mutant
constructs contain point mutations that abolish the consensus site for
its transcription factor.  GATA contains a complete deletion of the
GATA cis-element. All mutations were introduced into the
0.375 kb promoter fragment. (B) Either undifferentiated (D0,
black bars) or 3-day differentiated (D3, hatched bars) ES cells were
disrupted to single cells by brief trypsinization and analyzed for EGFP
transgene expression using flow cytometry. The graph is representative
of 3 independent experiments.
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Transgene expression was determined using flow cytometry in pluripotent
or 3-day differentiated clonal populations of ES cell lines containing
either the NF-E2 mutation (mNF-E2), the GATA mutations (mGATA or
GATA), or both mutations (mNF-E2/mGATA). In pluripotent ES cells,
there was a 40% to 50% reduction in transgene expression in cell
lines containing the NF-E2 mutation compared to cells expressing the
0.375 kb wild-type promoter transgene (Figure 5B). In 3-day
differentiated cells, there was an 80% decrease in transgene
expression in the mutant NF-E2 transgenic cell line compared to
expression in the 0.375 kb wild-type promoter transgene cell
lines (Figure 5B).
In pluripotent ES cells containing either a deletion of the
0.375 kb GATA site or a mutated GATA site, there was a 20% to 25% increase in transgene expression compared to cells containing the
0.375 kb wild-type promoter (Figure 5B). In 3-day differentiated cells, there was an 18% decrease in transgene expression in the mutant
GATA cell line (mGATA) and an equivalent level of expression in the
cells containing the GATA deletion (GATA) compared to cells
containing the 0.375 kb wild-type promoter (Figure 5B).
When both the NF-E2 and the GATA sites were mutated, the transgene
expression was similar to the NF-E2 mutation alone. In the pluripotent
mGATA/mNF-E2 cell lines, transgene activity was decreased by 55%
compared to the 0.375 kb wild-type promoter transgene
expression. In 3-day differentiated cells that contained the double
NF-E2/GATA mutation, there was an 80% decrease in transgene expression compared to the 0.375 kb wild-type promoter
transgene. Furthermore, in pluripotent ES cells, the NF-E2 mutation
abrogated the increased expression resulting from the GATA mutation.
Transgene expression was determined using flow cytometry in pluripotent
or 3-day differentiated clonal populations of ES cell lines containing
either the NF-E2 mutation (mNF-E2), the GATA mutations (mGATA or
GATA), or both mutations (mNF-E2/mGATA). In pluripotent ES cells,
there was a 40% to 50% reduction in transgene expression
in cell lines that contained the NF-E2 mutation compared to cells
expressing the 0.375 kb wild-type promoter transgene (Figure
5B). In 3-day differentiated cells, there was an 80%
decrease in transgene expression in the mutant NF-E2 transgenic cell
line compared to expression in the 0.375 kb wild-type promoter
transgene cell lines (Figure 5B).
In pluripotent ES cells containing either a deletion of the
0.375 kb GATA site or a mutated GATA site, there was a
20% to 25% increase in transgene expression compared to cells
containing the 0.375 kb wild-type promoter (Figure 5B). In 3-day
differentiated cells there was no increase in transgene expression in
the mutant GATA cell line (mGATA), but there was a 30% increase in the
cells containing the GATA deletion (GATA) compared to cells
containing the 0.375 kb wild-type promoter (Figure 5B). When
both the NF-E2 and the GATA sites were mutated, the transgene
expression was similar to the NF-E2 mutation alone. In the pluripotent
mGATA/mNF-E2 cell lines, transgene activity was decreased by 55%
compared to the 0.375 kb wild-type promoter transgene
expression. In 3-day differentiated cells that contained the double
NF-E2/GATA mutation, there was an 80% decrease in transgene
expression compared to the 0.375 kb wild-type promoter
transgene. Furthermore, in pluripotent ES cells, the NF-E2 mutation
abrogated the increased expression resulting from the GATA mutation.
A-to-G polymorphism identified in the promoter of patients with
protoporphyria has no effect on transcription of the ferrochelatase
promoter transgene
It has been proposed that the "wild-type" allele of the
ferrochelatase gene is low expressing36 and contributes to
the less than 50% ferrochelatase protein levels and activities
observed in patients with protoporphyria. To determine whether an
A-to-G polymorphism identified in the promoter of patients with
protoporphyria36 contributed to a low-expressing allele, we
constructed an HPRT-targeting vector that contained the A haplotype or
the G haplotype in the context of the 1.1 kb ferrochelatase
promoter transgene (Figure 6A).
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| Fig 6.
The transcriptional effect of an A-to-G polymorphism
identified in the ferrochelatase promoter of patients with
protoporphyria.
(A) Summary of the HPRT targeting constructs used to assess the effect
of A or G polymorphism located at 251 bp from the start codon.
The G haplotype was introduced into the 1.1 kb reporter gene
construct by PCR amplification of this region from genomic DNA from a
patient with protoporphyria known to have the G polymorphism. The
1.1 kb fragment was then cloned into the EGFP reporter gene
vector (1.1 A to G). (B) Cells containing either the A
polymorphism or the G polymorphism were differentiated for 3 days in
primary culture. The embryoid bodies were disrupted by brief
trypsinization, and EGFP expression was determined in live cells using
flow cytometry. The EGFP intensities for the 0.125 (control
transgene), the 1.1 kb or the 1.1 A to G
transgenes were determined using maximal peak heights and are noted to
the right of each histogram.
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Using flow cytometry, we measured reporter gene expression in
pluripotent and 3-day differentiated cells containing a single copy of
the transgene integrated into the HPRT locus. Based on the
ferrochelatase activities observed in protoporphyria, we predicted that
transcription from a low-expressing promoter would range from 30% to
60% of a normal promoter. However, there was no significant difference
in transcriptional activity from a promoter transgene that contained
the G haplotype and a promoter transgene that contained the A haplotype
in either pluripotent (not shown) or 3-day differentiated cells (Figure
6B).
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Discussion |
The ability to use conventional stable cell lines to analyze and
compare functional elements in promoters that require organized chromatin for proper tissue-specific expression is confounded by the
inability to control chromosomal integration site and copy number of
transgenic reporter constructs. We have eliminated variable copy number
and integration site by using a gene-targeting method to introduce a
single copy of an EGFP reporter gene in a single locus at the 5'
end of the mouse HPRT gene. We have validated this method by showing
that ferrochelatase promoter transgene expression in the HPRT locus is
regulated in a tissue-specific manner and a time-course similar to the
endogenous gene. This technique has enabled us to identify that the
proximal NF-E2 element is required for enhanced expression during
hematopoiesis and that the GATA element functions as a stage-specific
repressor in pluripotent cells and an enhancer during early erythroid
development. Furthermore, we have demonstrated that undefined elements
between 0.375 kb and 1.1 kb are required for maximal
enhancement of ferrochelatase gene transcription during erythroid cell development.
EGFP has been shown to be very stable in Chinese hamster ovary (CHO)
cells.43 The implication of a highly stable EGFP reporter protein is that transgene induction rates may indicate an
accumulation of EFGP protein and not actual induction rates.
We addressed this concern by determining the half-life of EGFP
in ES cells. Contrary to the high stability of EGFP in CHO cells, our
findings indicated that in ES cells, the half-life of EGFP is between 3 and 4 hours (not shown; see "Materials and methods"). Therefore,
the induction rates of the ferrochelatase reporter genes in this study
most probably reflect actual induction rates.
Our data indicate that transcription of the human ferrochelatase
promoter transgene is an early event during hematopoietic differentiation of ES cells in culture that precedes both ALAS-E expression and globin expression. In primary differentiation
conditions, the ferrochelatase promoter transgene expression
(4.0 kb) increases on day 2, peaks on day 4, and decreases to
low basal levels by day 5 (Figures 3 and 4). Using Northern blot
analysis, we confirmed that the endogenous ferrochelatase gene followed
an expression pattern and a time course similar to that of the
transgene. No ALAS-E mRNA or -globin mRNA was observed in Northern
blot experiments. Fluorescence microscopy and HPLC analysis of
porphyrins demonstrated no porphyrin expression during any stage of
primary differentiation (not shown). Porphyrin expression was observed
just before the primitive erythroid cell stage (ie, globinization),
indicating late-stage transcription, translation, or both of
ALAS-E mRNA.
Although ferrochelatase mRNA levels increased 5-fold on day 3 of
primary differentiation, ferrochelatase protein levels remained equivalent to levels in undifferentiated cells (Figures 4A, 4B). A
hallmark of erythroid cell differentiation is selective expression and
stabilization of mRNA required by the cell after enucleation. Stabilization of  - and -globin mRNA requires cis-acting
elements in the 3'-UTR of both transcripts,49,50 and
expression of ALAS-E mRNA is controlled by a 5'-UTR stem loop
structure.51 The absence of increased ferrochelatase
protein expression during transcriptional induction may indicate a
translational control mechanism involving repressive stabilization of
ferrochelatase mRNA in noncommitted precursors with later expression in
committed erythroid cells.
We show that the NF-E2 cis-element is necessary for both the
basal expression in pluripotent cells and the up-regulated expression of the ferrochelatase gene during erythroid cell development. It is
noteworthy that AP-1 is still able to bind to the mutant NF-E2
cis-element,13 suggesting that AP-1 fails to
complement for the function of NF-E2 in this system. The failure of
AP-1 to compensate for NF-E2 function has also been observed in the PBGD promoter.52 Transcription factors capable of binding
the NF-E2 cis-element (besides Jun family factors) include
p45/Maf (NF-E2)53-55 and Bach/Maf dimers.56,57
The transcriptional enhancing mechanism of NF-E2 involves chromatin
remodeling in the human -globin and  -globin locus.58
Bach/Maf is highly expressed in fetal hematopoietic tissues before
NF-E257 and is also thought to enhance transcription
through chromatin rearrangement.57 Because ferrochelatase
up-regulation is an early hematopoietic event, we speculate that
enhanced transcription of the ferrochelatase gene may be initially
regulated through a Bach/Maf complex.
GATA-1 is known to be a positive regulator of erythroid gene
expression,59-61 and it is essential for erythroid
differentiation.62-64 Murine GATA-1 binds the 0.375
kb ferrochelatase GATA element in vitro13; therefore, we
predicted that the GATA site would be required for increased expression
during erythroid cell development. The results show that in pluripotent
ES cells, the GATA element functions as a transcriptional repressor,
and in 3-day differentiated cells the GATA element functions as a
transcriptional enhancer. The functional difference between the mGATA
transgene and the GATA transgene probably reflects the presence of
5'-flanking sequence in the mGATA construct, which is absent in
the GATA construct. One explanation for repression through the GATA
cis-element in pluripotent ES cells is that another
GATA-binding factor mediates this role. GATA-2 has previously been
shown to be a repressor.65 It is expressed initially in the
erythroid precursors, and later it is down-regulated as GATA-1
transcription increases.66,67 In addition, recent evidence
shows that GATA-1, GATA-2, and GATA-4 are able to mediate
transcriptional repression through a ternary complex with FOG factors
(Friend-of-GATA) and CtBP2.68,69
Although 0.375 kb of the ferrochelatase promoter is sufficient
for erythroid-enhanced expression, maximal expression during erythroid
cell development requires sequences between 0.375 kb and
1.1 kb. This upstream region has not been formally studied for
transcription factor binding; however, a computer database cis-element search has identified several putative
transcription factor-binding sites, GATA-1, CAC-binding
protein,70 and CACCC (EKLF),71 that may
contribute to up-regulation of the transgene in early erythroid cells
(Figure 7). We are currently investigating transcription factor binding to these cis-elements in vitro.
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| Fig 7.
Summary of putative cis-elements located between
0.375 kb and 1.1 kb of the human ferrochelatase promoter.
The TESS computer database was used to search for putative binding
sites for erythroid-specific or muscle-specific trans-acting
factors. The locations of these sites are indicated by circles (TESS
URL, http://www.cbil.upenn.edu/tess/).
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Phenotypic expression of "dominantly" transmitted protoporphyria
is thought to require inheritance of a null ferrochelatase allele and a
"low-expressed" ferrochelatase allele.36 The G haplotype segregates with the protoporphyric phenotype
and, in all but 1 reported case,37 segregates
with a low-expressing allele. We tested the effect of the G haplotype
on expression of the 1.1 kb ferrochelatase promoter transgene
using the gene-targeting method. There was no difference in
transcriptional activity between the transgene containing the G
haplotype and the transgene containing the A haplotype (Figure 6).
This result suggests there is another mutation located in the promoter
or elsewhere that contributes to a low-expressing allele. Hyper-methylation of CpG islands in some promoters causes a decrease in
gene expression and disease.72,73 It is conceivable that aberrant methylation of the ferrochelatase promoter could contribute to
decreased transcriptional activity from the imprinted allele. An
alternative possibility is that the presence of intron mutations, conferring cryptic splice sites, could result in mildly defective splicing, an unstable ferrochelatase mRNA, and lower levels of normal
ferrochelatase mRNA. Such mutations have been implicated in bipolar
disorder74 and Rabson-Mendenhall syndrome.75
Although we predicted high EGFP expression levels in erythroid
colonies, we unexpectedly observed equally intense EGFP expression in
rhythmically contracting foci of cells containing the 4.0 kb
ferrochelatase promoter transgene (Figure
8). When ES cells were differentiated under
conditions that favored cardiomyocyte formation,76,77
enhanced transgene expression correlated with the presence of
0.375 kb to 1.1 kb of the ferrochelatase promoter (Figure
8). Up-regulation of the ferrochelatase promoter transgene in
rhythmically contracting foci may indicate an increased demand for heme
in cardiomyocytes to produce myoglobin. Using the TESS computer
database search, we identified the putative cis-elements, myoD,
myogenin, and MEF2 in the region from 0.375 kb to 1.1 kb
of the ferrochelatase promoter that may contribute to increased expression of the promoter transgene in cardiomyocytes (Figure 7).
MyoD,78 myogenin, and MEF-279 are muscle
tissue-specific transcription factors belonging to the basic
helix-loop-helix family, and are all involved in developmental
up-regulation of muscle-specific genes.80,81
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| Fig 8.
Transgene expression in cardiomyocyte foci.
Areas of early cardiac cell lineages were visually identified at
approximately day 10 of secondary culture in methylcellulose in foci of
rhythmically contracting cells (A, C, E, G, |
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Abstract
Introduction