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RED CELLS
From the Department of Physiology, Monash University,
and the Rotary Bone Marrow Research Laboratories, Royal Melbourne
Hospital and Melbourne University, Melbourne, Australia; and the
Department of Hematology/Oncology, St Jude Children's Research
Hospital, Memphis, TN.
Erythroid Kruppel-like factor (EKLF) is a transcription factor of
the C2H2 zinc-finger class that is essential
for definitive erythropoiesis. We generated immortal erythroid cell
lines from EKLF The human Erythroid Kruppel-like factor (EKLF) is a transcription factor that was
identified by subtractive hybridization of DS-19 MEL and J774
monocyte-macrophage cell lines as part of a strategy to identify novel
erythroid-specific genes.2 The carboxy (C) terminus of
EKLF contains 3 C2H2-type zinc fingers similar
to those in the Drosophila gap gene Kruppel, and
the amino (N) terminus is rich in proline and acidic
residues.2 Modeling studies based on conserved amino acids
in the fingers of EKLF and Zif268 and the crystal structure of Zif268
bound to DNA suggest that EKLF binds to the consensus sequence
5'-NCNCNCCCN-3'.3 Sites of this nature are found in the
promoters of several erythroid-specific genes, including the adult
Gene targeting of embryonic stem cells to generate EKLF null
(EKLF To enable more complete analysis of the function of EKLF, we used the
J2 retrovirus that expresses 2 complimentary oncogenes, v-raf and v-myc,10 to immortalize
erythroid cell lines from EKLF In stably transduced cells, EKLF-ERTM remains dormant in the cytoplasm
until tamoxifen is added. Tamoxifen binds to the LBD of
ERTM, resulting in translocation of the hybrid protein to
the nucleus. Once in the nucleus, EKLF-ERTM can bind to its cognate
CACC sites and activate transcription of target genes. Because the LBD
of ERTM is specific to tamoxifen, estrogens present in
culture medium cannot induce translocation of EKLF-ERTM to the
nucleus.14 This situation provides a system in which the
transcriptional activity of EKLF can be tightly regulated, thus
allowing the role of EKLF in both Generation of EKLF EKLF+/ Immortalization of cell lines by using the J2 retrovirus
Individual colonies were picked from the methylcellulose at 5 days and
plated in 96-well plates in 100 µL 10% FCS/DMEM. Colonies that grew
robustly were expanded (1:3) to progressively larger volumes. When 5-mL
cultures reached a density of about 5 × 105 cells/mL,
aliquots were frozen and cultures were maintained by serial splitting
(1:3) every 2 to 3 days. Two EKLF Construction of retroviral vectors The primers AP68 5'-CGGGATCCGTGGACACCAGCCAGCCAT-3' and AP58 5'-CGGGATCCGAGGTGACGCTTCATGTGCAGA-3', which each contain a terminal BamHI restriction site (underlined), were used to generate a polymerase chain reaction (PCR) product from pSG5-EKLF.2 The PCR product that contained the entire open reading frame of murine EKLF was digested with BamHI and ligated into the BamHI site of pBabe Puro myc ERTM from which c-myc had been excised.14 The presence of an in-frame EKLF-ERTM fusion complementary DNA was confirmed by sequencing across the cloning site. The primers SEEK3 5'-CGGAATTCGTGGACACCAGCCAGCCAT-3' and SEEK4 5'-CGGAATTCTCAGATCGTGTTGGGGAAGC-3', which each contain a terminal EcoRI restriction site (underlined), were subsequently used to amplify the entire EKLF-ERTM open reading frame from pBabePuroEKLF-ERTM. This PCR product was digested with EcoRI and ligated into the EcoRI site of the retroviral vector MSCV-IRES-GFP13 to generate MSCV-EKLF-ERTM-IRES-GFP.Infection of EKLF  80°C. The transfected 293T cells were trypsinized and subjected to
fluorescence-activated cell-sorter scanning (FACS) analysis.
Nontransfected 293T cells were used to set the FACS gains and analysis
gates. Approximately 60% of 293T cells were green, which correlates
with a retroviral titer of about 1 × 106/mL according to
our previous experience.13 A formal retroviral titer was
not determined.
Hematopoietic cell lines were cocultured in retroviral supernatant (50%), DMEM, 10% FCS, and Polybrene (4 µg/mL) as described previously.16 After 24 hours, the cells were centrifuged (500g) and resuspended in a second aliquot of viral supernatant as described above. After an additional 72 hours, cells were harvested by centrifugation, washed 4 times in 5% FCS/PBS, and subjected to FACS. Nontransduced control A2 and B1 cell lines were used to set the FACS machine gains, compensations, and sorting gates. Cells were sorted according to side scatter, forward scatter, and GFP+ characteristics. Sorted GFP+ cells were cloned immediately by culture in 35-mm Petri dishes containing 1% methylcellulose (Life Technologies, Rockville, MD) in Iscove modified Dulbecco medium (IMDM), 10% FCS, and 1% penicillin/streptomycin. After clones had been in semisolid culture for 5 to 8 days, a Nikon E800 microscope and GFP-BP filter block were used to check them for GFP expression by observing green fluorescence after exposure to UV light. Individual GFP+ clones were picked from the methylcellulose and expanded in DMEM as described above. Southern blotting Genomic DNA was extracted from 40-mL cultures of all cell lines by using a standard method. DNA (20 µg) was digested with BamHI and resolved on a 0.7% Tris-borate-EDTA agarose gel. DNA was transferred to Hybond N+ (Amersham, Little Chalfont, United Kingdom) by alkali blotting, and the membrane was probed with a 250-base-pair BamHI/SacI EKLF fragment from pSG5-EKLF2 by using a standard protocol.RNAse protection analyses Total RNA was prepared17 and RNAse protection analyses were performed as described previously.6 Total RNA (2 µg) was hybridized simultaneously with murine -globin and
either human  -globin or human  -globin riboprobes, which were
generated as described previously.18 RNAse-protected mRNAs
were resolved on a 6% denaturing polyacrylamide gel, which was
subjected to autoradiography.
Western blotting Cultures of cell lines were harvested and lysed in 200 µL ice-cold lysis buffer (1% Triton X-100, 0.15 M sodium chloride, 0.01 M sodium phosphate (pH 7.2), 10% glycerol, 10 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 20 µg/mL aprotinin, and 2 µg/mL pepstatin). For Western blots, 5 µg of extracts were resolved on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Amersham). The membranes were probed with a rabbit anti-EKLF polyclonal antibody19 followed by a donkey antirabbit immunoglobulin (Ig)-horseradish peroxidase (HRP; NA934; Amersham). Hybridization was visualized by using the ECL Plus chemiluminescence system (Amersham). The Western blots were stripped by incubating in stripping buffer (100 mM-mercaptoethanol, 2% SDS, and 6.25 mM Tris HCl, pH 6.7) at 50°C for 30 minutes. They were reprobed with rat anti-GATA-1 IgG
(sc-265; Santa Cruz Biotechnology, Santa Cruz, CA) followed by sheep
antirat Ig HRP (Amersham).
Induction of nuclear transportation with 4-hydroxytamoxifen (tamoxifen) For the induction experiments, 20-mL cell cultures were set up in duplicate for each mutant line and the parent cell lines at a density of 1 × 105 cells/mL. Immediately before an experiment, 100 µL tamoxifen stock (1 mM; Sigma, St Louis, MO) in ethanol or 100% ethanol alone was diluted in 10 mL of culture medium. Then, 200 µL of this was added to one of each of the pairs of cultures to yield a final tamoxifen concentration of 100 nM. During the next 3 days, the number of viable cells in each culture was determined by counting the cells in 0.4% trypan blue with a hemocytometer. Four independent experiments were performed for each cell line. The cell counts in tamoxifen compared with ethanol were considered dependent variables for each experiment. Therefore, to test the null hypothesis that there was no significant difference between the cell counts for cultures grown in ethanol and those grown in tamoxifen, a paired t test was performed.Immunofluorescence Cells (1 × 105) were cytocentrifuged at 500g on glass slides and fixed in methanol and acetone (1:1). To detect-globin, slides were incubated with an
FITC-conjugated antibody raised against hemoglobin F as described
previously.6 To detect EKLF protein, slides were incubated
in rabbit polyclonal anti-EKLF (1:500) followed by antirabbit-biotin
(1:1000; Amersham) and streptavidin-Cy3 (1:1000; Amersham). Finally,
specimens were stained with 0.01% 4'6-diamidino-2-phenylindole (DAPI)
HCl in PBS for 10 minutes to identify all cell nuclei in the field.
Fluorescence was detected with a Nikon E-800 microscope. Photographs of
the same field were made after passage of emitted light through blue
(UV-2B) and red (B-1E) filter blocks. Images were scanned into
Photoshop software (Adobe, San Jose, CA). Images from control and
tamoxifen-treated samples were treated identically.
Establishment of EKLF -globin locus. Nonimmortalized cells of this
phenotype existed in the fetal livers of a cross between
EKLF+/ and YAC transgenic mice.6 Thus, we
decided to use an immortalization strategy that was reported to
preferentially transform erythroid fetal liver progenitors that
nevertheless retain some ability to differentiate in response to
erythropoietin.12
The J2 retrovirus has 2 complimentary oncogenes, a hybrid
v-raf-mil serine threonine kinase, and
v-myc.10 Fetal liver cells from
EKLF
Ninety-six consecutive colonies of this phenotype were picked from
cultures derived from both EKLF
Phenotype of EKLF / clones had erythroid morphologic
features on May-Grünwald-Giemsa staining of cytocentrifuge
preparations (Figure 1B). This finding was confirmed by positive
staining for hemoglobin with o-dianisidine (Figure 1C).
EKLF/ erythroid cell lines expressed large amounts of
human -globin as detected by direct immunofluorescence (Figure 1D),
whereas a control cell line, J2E,12 did not (Figure 1F).
The single EKLF+/ cell line displayed macrophage
morphologic characteristics on May-Grünwald-Giemsa staining (not
shown). This line became exclusively adherent after 6 weeks in culture
and could not be expanded further or rescued from frozen stocks. Thus,
we found that the absence of EKLF results in an enhanced ability of the
transforming genes raf-mil and v-myc to
immortalize erythroid progenitor cells. The J2 virus is capable of
immortalizing wild-type fetal liver cells, but it is very inefficient
unless additional mutations, such as loss of p53, are
present.15 Our results suggest that the absence of EKLF,
like the absence of p53, enhances erythroid cell immortalization by
v-myc and v-raf-mil.
Western blot analysis confirmed that the EKLF null cell lines B1 and A2
did not express EKLF but did express the
erythroid transcription factor GATA-1. Also, electromobility gel-shift
experiments using nuclear extracts from these lines and the
Reintroduction of EKLF To establish an in vivo assay for EKLF, we attempted to derive stable subclones of A2 and B1 by using an expression vector, pEF1-EKLF. The EF1 expression vector was previously used
successfully to express transcription factors in erythroid cell
lines.20 However, with this expression vector, we were
never able to derive stable sublines of A2 or B1 that expressed EKLF,
and we think that transduced cells underwent terminal differentiation
during selection for stable expression. Therefore, we employed an
efficient inducible expression system. We expressed EKLF as a fusion
protein with the LBD of the tamoxifen-inducible
ERTM.14 Expression was driven by the
retroviral promoter/enhancer in MSCV, which is efficient in
hematopoietic cells.21 Lastly, we chose to express GFP
from the same bicistronic mRNA as EKLF-ERTM by using the
encephalomyocarditis virus IRES to enable positive selection of
expressing cells.13
The retroviral vector, MSCV-EKLF-ERTM-IRES-GFP, was derived as
described above (Figure 2A). Amphotropic
infectious retroviral stocks were prepared, and A2 and B1 parent cell
lines were infected. After 72 hours in culture, GFP+
retrovirally transduced cells were sorted by FACS. Retroviral transduction was moderately efficient, with about 7% of cells showing
green fluorescence (Figure 2B). Sorted GFP+ cells were
immediately cloned in methylcellulose. Colonies were checked for green
fluorescence after exposure to UV light (Figure 2C) and picked for
expansion and analysis. Five subclones from the parental A2 line and 7 subclones (from 2 independent experiments) from the parental B1 cell
line were expanded for further analysis.
To check for clonal independence, Southern blotting was performed on
genomic DNA (Figure 3A). An 8.8-kb band present in all lanes
represents the endogenous targeted EKLF gene locus. All the transduced
A2 subclones contained integrated EKLF-ERTM and all had
independent sites of proviral integration, confirming their clonal
independence. Western blot analysis demonstrated that all the subclones
derived from the parent cell lines A2 and B1 (except A2.2) expressed
EKLF, although the levels of expression varied in comparison with the
levels of GATA-1 expression (Figure 3B and 3C). Clone A2.2 showed no
expression of EKLF even though Southern blotting demonstrated the
presence of the EKLF-ERTM transgene. This served as a useful negative
control cell line.
Tamoxifen-inducible nuclear expression Nuclear translocation of the hybrid EKLF-ERTM protein was achieved by adding 100 nM tamoxifen. To check that nuclear translocation was successful, about 1 × 105 cells were cytocentrifuged after 3 days of culture and fixed in methanol and acetate (1:1). The fixed cells were incubated with a rabbit anti-EKLF polyclonal antibody followed by antirabbit streptavidin-Cy3. The cells were costained with DAPI to identify all cell nuclei in the field. The blue (DAPI) and red (EKLF) images of the same cell field were photographed independently. Addition of tamoxifen resulted in efficient translocation of EKLF-ERTM protein to the nucleus of B1.4 cells (Figure 3F and 3G), whereas addition of ethanol vehicle did not (Figure 3D and 3E). Thus, until tamoxifen was added, EKLF was efficiently held as an inactive ERTM fusion protein in the cytoplasm of these cell lines grown in serum-containing medium.Reduced proliferation and enhanced differentiation Cell cultures of transduced A2 and B1 sublines were cultured for 3 days in either tamoxifen (100 nM) or ethanol (0.01%) as a vehicle control. Viable cells were counted daily for 3 days. The A2.2 subline and the A2 parent line did not show any significant change in growth between the ethanol-treated cultures and the tamoxifen-treated cultures, and they did not express EKLF. Conversely, the A2.1 and A2.3 cell lines showed a significant reduction in growth on addition of tamoxifen (P < .1 and P < .05, respectively), whereas the A2.4 and A2.6 cell lines demonstrated a small but nonsignificant reduction in cell growth on addition of tamoxifen (Figure 4B). Thus, there was a correlation between EKLF expression levels and a block in cell proliferation in these independent sublines.
After 3 days in tamoxifen, the cell pellets of lines A2.1, A2.3, A2.4, and A2.6 all had a red appearance consistent with an increase in hemoglobin content (Figure 4A). The pellet sizes were also much smaller, particularly in the A2.1 and A2.3 sublines that expressed higher levels of EKLF. On the other hand, lines A2.2 and A2 were pale after tamoxifen treatment, like the ethanol-treated cultures. All the B1 sublines tested (B1.8, B1.9, and B1.10) had high levels of EKLF expression and behaved like the A2.3 line. They all displayed a significant slowing in cell growth (Figure 4C) on activation of EKLF and the cell pellets were markedly red compared with those in the ethanol-treated control cultures (data not shown). Role of EKLF in globin gene expression Previous studies suggested that EKLF may play a role in the-globin to -globin gene switch.6,7,22 Therefore, we
wanted to determine whether activation of EKLF in the null cell lines resulted in a change in human globin gene expression. The results of
RNAse protection assays for human , human , and murine -globin stable mRNA transcripts are shown in Figure
5A and 5B. As expected, no change in
-globin expression was observed in the parent EKLF null cell lines,
A2 and B1, after tamoxifen treatment. However, a marked increase in
human -globin expression was detected in the A2.3, A2.4, and
B1.2-B1.6 cell lines after tamoxifen was added. There was no
significant increase in -globin in 2 sublines of A2 (A2.1 and A2.6)
that expressed low levels of EKLF. Again, there was a consistent
correlation between EKLF expression and tamoxifen-induced -globin
gene expression in the independent sublines. The levels of -globin
transcripts in all the sublines of A2 and B1 remained high after
addition of tamoxifen and were similar to those of the endogenous mouse
-globin transcripts in each line. This most likely reflects the
remarkable stability of -globin mRNA rather than indicating a lack
of a link between human -globin gene activation and -globin gene
silencing in these cell lines. Thus, additional experiments examining
transient -globin transcripts must be done to confirm whether these
lines will be valuable tools for a structural and functional analysis
of the role of EKLF in human hemoglobin switching.
The principal aim of these experiments was to develop a cell-based
assay for the role of EKLF in erythropoiesis and globin gene
regulation. To achieve this, we used the J2 retrovirus, which encodes
the complimentary oncogenes v-myc and v-mil-raf,
to immortalize fetal liver progenitor cells from EKLF Studies of several transcription factors and some protein kinases have
been aided by linking them to the LBD of steroid hormone receptors.
Most commonly, the LBD of the human estrogen receptor (hER) is used.
The resultant fusion proteins are inactive in the absence of their
specific ligand because they are excluded from the nucleus by a variety
of cytoplasmic inhibitor proteins, including heat shock protein (Hsp)
70 and Hsp90. Addition of ligand to the culture medium results in
dissociation of the fusion protein from inhibitory complexes and
translocation to the nucleus. Although hER has been used successfully
for this purpose, the system has had considerable technical
complications. The most troublesome problem is activation of hER by
estrogens in FCS. To overcome this problem, Littlewood et
al14 used the LBD of ERTM, which is unable to
bind estrogen but is able to bind the synthetic steroid,
4-hydroxytamoxifen. They fused the LBD of ERTM to the
C-terminal end of c-Myc and showed that
Myc-induced proliferation and apoptosis in fibroblasts
depended entirely on the presence of 4-hydroxytamoxifen and that
addition of estrogen to the culture medium had no
effect.14 Similarly, we successfully fused the LBD of ERTM to the C-terminal end of EKLF and
demonstrated that addition of 4-hydroxytamoxifen to the culture medium
resulted in translocation of the hybrid EKLF-ERTM protein to the
nucleus. There was minimal, if any, activation of the sensitive EKLF
target gene, human EKLF is a negative regulator of cellular proliferation Our ability to control the transcriptional activity of EKLF in this study revealed an unexpected role for it in cellular proliferation. The greatly enhanced frequency of derivation of immortal clones from EKLF/ compared with EKLF+/
fetal liver cells using the J2 retrovirus suggested that EKLF discourages immortalization. In other words, the absence of EKLF resulted in a markedly increased rate of transformation by the cooperating oncogenes, v-mil-raf and v-myc. This
result is similar to that of Metz et al,15 who showed that
absence of p53 resulted in an increased frequency of immortalization of
erythroid cells by v-mil-raf and v-myc. Thus,
like p53, EKLF could be considered a suppressor of immortalization.
Superficially, these results appear to contradict those of Spadaccini
et al,8 who inactivated EKLF in the
myc-raf-transformed erythroid cell line J2 by using an
antisense oligonucleotide approach. Although these authors showed
effects on globin gene expression and hemoglobinization, they found no
effects on proliferation. In view of our results and those of Metz et
al15 showing that wild-type fetal liver progenitors are
difficult to immortalize with the J2 virus,15 we
suggest that the J2E cell line probably underwent additional undetermined mutational events during its initial derivation. These may
preclude it from slowing in growth rate in response to a reduction
(albeit incomplete) in EKLF levels. Our results that begin from an EKLF
null background offer a cleaner system in which to address the role of
EKLF in proliferation.
The activation of EKLF-ERTM with tamoxifen resulted in a slowing of
proliferation consistent with the role of EKLF-ERTM as an antiproliferation factor (Figure 4). Other C2H2
zinc-finger proteins, such as the Wilms tumor 1 (WT-1) protein, have an
antiproliferative effect. WT-1 has a proline-rich N-terminus and 4 C-terminus zinc fingers and it binds to GC-rich promoter sites similar
to those bound by EKLF.23 WT-1 is lost in many cases of
inherited Wilms tumor,24 and enforced reexpression of WT-1
in a cell line derived from a Wilms tumor (RM1) resulted in cessation
of proliferation.25 If EKLF is also a tumor suppressor,
then erythroleukemia might be expected to occur in mice lacking EKLF.
Unfortunately, EKLF EKLF induces terminal differentiation and hemoglobinization Reintroduction of EKLF into EKLF/ erythroid cell
lines resulted in marked hemoglobinization, as demonstrated by the
finding of small red cell pellets (Figure 4A). Thus, EKLF may
coordinate expression of other genes involved in terminal
differentiation and hemoglobinization of erythroblasts. We previously
showed that correction of the globin chain imbalance in
EKLF/ fetal liver erythroblasts fails to substantially
improve their intrinsic defect,9 thereby suggesting that
other EKLF target genes must be important. Also, Spadaccini et
al8 introduced an antisense EKLF construct into the
erythroid cell line J2E and reported that hemoglobinization was
impaired. They showed that expression of 2 rate-limiting enzymes in the
heme biosynthetic pathway, aminolevulinic acid synthetase (ALAS) and
ferrochelatase, were expressed at reduced levels when EKLF was
inhibited. Interestingly, the erythroid-specific promoter of ALAS has
an EKLF binding site that is functionally critical.27
Future studies with our cell lines could determine the nature of
other potential EKLF target genes involved in terminal differentiation
and hemoglobinization.
EKLF and globin gene regulation Activation of EKLF resulted in a dramatic activation of the human-globin gene in the cell lines we used, confirming the critical role
of EKLF in -globin gene expression. The cell lines also expressed
high levels of -globin, but the levels of processed -globin RNA
transcripts did not decrease after activation of EKLF. In view of the
suggested role of EKLF as a mediator of competition between the
-globin promoter and -globin promoter for the
LCR,6,7 these results were initially surprising. They may
simply reflect the long half-life of processed -globin transcripts.
Alternatively, EKLF may not play a direct role in -globin gene
silencing. Future dynamic studies of the rate of generation of new
-globin transcripts, using such techniques as "nuclear run-ons"
or in situ hybridization with probes specific for prespliced nuclear
RNA species28 will resolve this issue.
We appreciate the expert cell-culture advice from Peta Tillbrook and Peter Klinken and the comments from Stuart Orkin.
Submitted January 10, 2000; accepted October 26, 2000.
Supported by an Australian NH & MRC grant (981010), grant PO1 HL53749-03 from the National Institutes of Health, Cancer Center Support CORE Grant P30 CA 21765, the American Lebanese Syrian Associated Charities (ALSAC), and the Assisi Foundation of Memphis. E.C. was supported by an Australian Postgraduate Research Fellowship, and A.P. by a Wellcome Senior Research Fellowship.
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: Andrew Perkins, Department of Physiology, Monash University, Wellington Road, Vic 3186, Australia.
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  T. Sengupta, K. Chen, E. Milot, and J. J. Bieker Acetylation of EKLF Is Essential for Epigenetic Modification and Transcriptional Activation of the {beta}-Globin Locus Mol. Cell. Biol., October 15, 2008; 28(20): 6160 - 6170. [Abstract] [Full Text] [PDF]
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 S. A. Eaton, A. P. W. Funnell, N. Sue, H. Nicholas, R. C. M. Pearson, and M. Crossley A Network of Kruppel-like Factors (Klfs): Klf8 IS REPRESSED BY Klf3 AND ACTIVATED BY Klf1 IN VIVO J. Biol. Chem., October 3, 2008; 283(40): 26937 - 26947. [Abstract] [Full Text] [PDF]
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 F. Bouilloux, G. Juban, N. Cohet, D. Buet, B. Guyot, W. Vainchenker, F. Louache, and F. Morle EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation Blood, August 1, 2008; 112(3): 576 - 584. [Abstract] [Full Text] [PDF]
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N. Sue, B. H. A. Jack, S. A. Eaton, R. C. M. Pearson, A. P. W. Funnell, J. Turner, R. Czolij, G. Denyer, S. Bao, J. C. Molero-Navajas, et al. Targeted Disruption of the Basic Kruppel-Like Factor Gene (Klf3) Reveals a Role in Adipogenesis Mol. Cell. Biol., June 15, 2008; 28(12): 3967 - 3978. [Abstract] [Full Text] [PDF]
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 J. K. Alder, R. W. Georgantas III, R. L. Hildreth, I. M. Kaplan, S. Morisot, X. Yu, M. McDevitt, and C. I. Civin Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo J. Immunol., April 15, 2008; 180(8): 5645 - 5652. [Abstract] [Full Text] [PDF]
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T. S. Kendrick, C. J. Payne, M. R. Epis, J. R. Schneider, P. J. Leedman, S. P. Klinken, and E. Ingley Erythroid defects in TR{alpha}-/- mice Blood, March 15, 2008; 111(6): 3245 - 3248. [Abstract] [Full Text] [PDF]
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P. Frontelo, D. Manwani, M. Galdass, H. Karsunky, F. Lohmann, P. G. Gallagher, and J. J. Bieker Novel role for EKLF in megakaryocyte lineage commitment Blood, December 1, 2007; 110(12): 3871 - 3880. [Abstract] [Full Text] [PDF]
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P. Basu, T. K. Lung, W. Lemsaddek, T. G. Sargent, D. C. Williams Jr, M. Basu, L. C. Redmond, J. B Lingrel, J. L. Haar, and J. A. Lloyd EKLF and KLF2 have compensatory roles in embryonic {beta}-globin gene expression and primitive erythropoiesis Blood, November 1, 2007; 110(9): 3417 - 3425. [Abstract] [Full Text] [PDF]
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A. P. W. Funnell, C. A. Maloney, L. J. Thompson, J. Keys, M. Tallack, A. C. Perkins, and M. Crossley Erythroid Kruppel-Like Factor Directly Activates the Basic Kruppel-Like Factor Gene in Erythroid Cells Mol. Cell. Biol., April 1, 2007; 27(7): 2777 - 2790. [Abstract] [Full Text] [PDF]
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D. Hodge, E. Coghill, J. Keys, T. Maguire, B. Hartmann, A. McDowall, M. Weiss, S. Grimmond, and A. Perkins A global role for EKLF in definitive and primitive erythropoiesis Blood, April 15, 2006; 107(8): 3359 - 3370. [Abstract] [Full Text] [PDF]
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 H. Im, J. A. Grass, K. D. Johnson, S.-I. Kim, M. E. Boyer, A. N. Imbalzano, J. J. Bieker, and E. H. Bresnick Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs within a broad chromosomal region PNAS, November 22, 2005; 102(47): 17065 - 17070. [Abstract] [Full Text] [PDF]
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F. Laub, L. Lei, H. Sumiyoshi, D. Kajimura, C. Dragomir, S. Smaldone, A. C. Puche, T. J. Petros, C. Mason, L. F. Parada, et al. Transcription Factor KLF7 Is Important for Neuronal Morphogenesis in Selected Regions of the Nervous System Mol. Cell. Biol., July 1, 2005; 25(13): 5699 - 5711. [Abstract] [Full Text] [PDF]
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X. Chen and J. J. Bieker Stage-Specific Repression by the EKLF Transcriptional Activator Mol. Cell. Biol., December 1, 2004; 24(23): 10416 - 10424. [Abstract] [Full Text] [PDF]
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S. Smaldone, F. Laub, C. Else, C. Dragomir, and F. Ramirez Identification of MoKA, a Novel F-Box Protein That Modulates Kruppel-Like Transcription Factor 7 Activity Mol. Cell. Biol., February 1, 2004; 24(3): 1058 - 1069. [Abstract] [Full Text] [PDF]
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H. Qi, D. J. Aguiar, S. M. Williams, A. La Pean, W. Pan, and C. M. Verfaillie Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells PNAS, March 18, 2003; 100(6): 3305 - 3310. [Abstract] [Full Text] [PDF]
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 J. M. Naciff, M. L. Jump, S. M. Torontali, G. J. Carr, J. P. Tiesman, G. J. Overmann, and G. P. Daston Gene Expression Profile Induced by 17{alpha}-Ethynyl Estradiol, Bisphenol A, and Genistein in the Developing Female Reproductive System of the Rat Toxicol. Sci., July 1, 2002; 68(1): 184 - 199. [Abstract] [Full Text] [PDF]
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R. C. Brown, S. Pattison, J. van Ree, E. Coghill, A. Perkins, S. M. Jane, and J. M. Cunningham Distinct Domains of Erythroid Kruppel-Like Factor Modulate Chromatin Remodeling and Transactivation at the Endogenous {beta}-Globin Gene Promoter Mol. Cell. Biol., January 1, 2002; 22(1): 161 - 170. [Abstract] [Full Text] [PDF]
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J. J. Bieker Kruppel-like Factors: Three Fingers in Many Pies J. Biol. Chem., September 7, 2001; 276(37): 34355 - 34358. [Full Text] [PDF]
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Top
Abstract
Introduction
, A
, and 
2-J2 retroviral
producer cells
indicates EtOH; 
,
tamoxifen.
