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First published online 30 April 2008
doi: 10.1242/dev.018200
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Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, Box 1020, 1 Gustave Levy Place, New York, NY 10029, USA.
* Author for correspondence (e-mail: james.bieker{at}mssm.edu)
Accepted 3 April 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Megakaryocytic-erythroid progenitor, Lineage fate decision, ES cell in vitro differentiation, Eklf (Klf1)
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
In lower vertebrates and mammals alike, signaling by Bmp4, a member of the
Tgfβ-like group of growth factors that signal via the Smad protein family
of intracellular effectors, is essential for the establishment of
hematopoiesis shortly after gastrulation
(Gupta et al., 2006;
Maeno et al., 1996;
Winnier et al., 1995). In the
mouse embryo, Bmp4 induces a mesodermal cell population within the primitive
streak at embryonic day (E) 7.0 to give rise to a common progenitor for
hematopoietic and endothelial lineages termed the hemangioblast. This
progenitor subsequently migrates into the extraembryonic yolk sac where it
differentiates into the hematopoietic, endothelial and vascular smooth muscle
cell lineages that will form the cardiovascular system of the embryo
(Huber et al., 2004).
Of particular importance to the understanding of the mechanisms that
underlie these early events of hematopoietic commitment has been the
application of embryonic stem (ES) cell in vitro differentiation technology
(Keller, 2005). With regard to
yolk sac hematopoiesis, ES cell differentiation studies have shown that Bmp4
signaling is required throughout embryoid body (EB) development, from the
initial establishment of hematopoietic fate from mesoderm
(Park et al., 2004) to its
subsequent progression to erythroid commitment
(Adelman et al., 2002). In
addition to Bmp4 signaling, members of the GATA-motif-binding family of
zinc-finger transcription factors play key regulatory roles in hematopoietic
development. Gata2 expression occurs early in hematopoiesis and is crucial for
the proliferation and expansion of hematopoietic stem cells and progenitors
(Tsai et al., 1994), whereas
Gata1 is activated subsequently and is essential for the
establishment of erythroid commitment and differentiation
(Pevny et al., 1991;
Weiss et al., 1994).
Importantly, Gata2 is a direct Bmp4 target gene
(Lugus et al., 2007), and
Smad-dependent Bmp4 signaling is necessary and sufficient to induce the
expression of Gata1 alongside that of erythroid Krüppel-like factor
(Eklf; Klf1) (Adelman et al.,
2002), another key regulator of erythropoiesis.
Eklf was originally identified as an erythroid-specific transcription
factor that functions as an essential regulator of β-like globin
switching in red cells (Miller and Bieker,
1993; Nuez et al.,
1995; Perkins et al.,
1995). However, in contrast to this well-defined role, Eklf must
fulfill additional functions during hematopoiesis as it is expressed much
earlier, prior to erythroid differentiation. In the developing mouse embryo,
Eklf message is first detected in situ in the extraembryonic mesoderm
of the yolk sac as early as at the neural plate stage, by E7.5
(Southwood et al., 1996),
which coincides with the presence of primitive erythroid progenitors
(Palis et al., 1999).
Similarly, during the differentiation of mouse ES cells in vitro, Eklf
expression is activated prior to terminal erythroid differentiation
(Adelman et al., 2002). These
observations raise the possibility that Eklf plays a role prior to erythroid
commitment and lead us to ask where exactly Eklf fits into the transcription
factor hierarchy that establishes the hematopoietic program.
In order to address these issues, we have developed novel reporter gene and RNAi-based loss-of-function assays for use during ES cell in vitro differentiation, as well as a transgenic mouse model to characterize the onset of Eklf expression during hematopoiesis. We show that Eklf is activated in a progenitor population of erythroid-megakaryocytic potential prior to erythroid commitment and delineate the transcriptional mechanisms that govern its differential expression in the two compartments. Based on our findings, a model integrating Bmp4- and Gata factor-mediated transcriptional control of hematopoiesis is presented.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) into the
EcoRI site of the plox-plasmid
(Kyba et al., 2002). GFP
(pEGFP-C1, Clontech) was inserted downstream of Peklf, replacing
the Eklf with the GFP translation start codon (ATG), placing GFP
upstream of the plox-plasmid NotI site, followed by poly(A). The
Peklf-intron-GFP construct was generated by inserting murine Eklf
exon 1 and intron 1 into Peklf-GFP upstream of GFP, mutating Eklf
exon 1 ATG to GTA to make the GFP start codon the first ATG present in the
resulting spliced mRNA. All mutations and deletions were introduced using
QuikChange (Stratagene); primer sequences are available upon request.
The plox-GFP-Intron-miR plasmid was generated by inserting the miR-30
backbone (SalI to PmeI) of pSM2 (Open Biosystems) into the
β-globin intron of pSG5 (Stratagene) at MfeI. The resulting
Intron-miR sequence was placed between the GFP and poly(A) of
pEGFP-C1, subsequently inserting GFP-Intron-miR-poly(A) into the
plox-plasmid. shRNAs sequences were generated by PCR as described previously
(Paddison et al., 2004) and
cloned into the miR-30 backbone (XhoI to EcoRI) of
plox-GFP-Intron-miR. shRNA sequences against NM_008541 (shRNA-1 is HP_248638;
shRNA-2 is HP_257681) were obtained from RNAiCodex
(http://codex.cshl.edu/scripts/newmain.pl).
The non-silencing shRNA-control was purchased from Open Biosystems
(RHS1703).
ES cell culture and in vitro differentiation
Ainv18 mouse ES cells (Kyba et al.,
2002) were maintained and differentiated according to established
protocols (Choi et al., 2005;
Kennedy and Keller, 2003) and
have been described previously (Manwani et
al., 2007; Zafonte et al.,
2007). Each ES cell clone was generated by targeting the
respective plox construct to the Ainv18 homing site by co-electroporation with
a Cre recombinase expression plasmid and subsequent antibiotic selection.
Site-specific integration of the plox construct was checked by PCR on genomic
DNA. Retention of hematopoietic differentiation capacity as measured by Flk1
expression levels on EB day 4 was verified for each clone. All reporter assay
experiments were performed in the absence of doxycycline (dox) (Sigma); shRNA
expression was induced by adding of 1 µg/ml dox.
Flow cytometry and colony assays
Single-cell suspensions of murine fetal livers or EBs were prepared and
stained using the following antibodies: CD71-PE, Ter119-APC (eBiosciences),
Flk1-PE, Ter119-PE, CD41-APC, Gr1-APC, Mac1-APC, CD42d-APC (BD Biosciences).
Flow cytometry data were analyzed using FlowJo (TreeStar). Sorted cells were
cultured in MethoCult M3234 (StemCell Technologies) supplemented with
cytokines according to manufacturer's instructions. Cytospins were fixed with
May-Grünwald Solution (Fluka) and stained with Giemsa Solution (Sigma),
and pictures were taken using an Axioplan2 microscope (Zeiss) with a
100x or 63x oil-immersion objective.
Phylogenetic sequence comparison and regulatory site prediction
Genomic sequences of mammalian Eklf or Gata1 loci were
obtained from the ENSEMBL Genome Browser between February and August, 2005.
Each alignment and the subsequent identification of conserved blocks of
sequence homology and putative transcription factor binding sites were
performed according to Chapman et al.
(Chapman et al., 2004). Smad
binding motif consensus sequences: 5'-AGAC (and complement
5'-GTCT) (Shi et al.,
1998; Zawel et al.,
1998); 5'-GTCTT (Lee et
al., 2004); 5'-CAGC
(Korchynskyi and ten Dijke,
2002); 5'-GCTG
(Benchabane and Wrana, 2003);
5'-TGGAGC (Hata et al.,
2000); 5'-TGGACC (Oren
et al., 2005). Sp1/E2F/Smad binding motif: 5'-CCGCCC
(Chen et al., 2002;
Frederick et al., 2004;
Yagi et al., 2002).
Chromatin immunoprecipitation (ChIP) assay
ChIP experiments were performed as previously described for G1E-ER-Gata1
cells (Im et al., 2005) or EBs
(Lugus et al., 2007).
G1E-ER-Gata1 cells were treated with 0.1 µM 17β-estradiol (Sigma) for
14 hours to induce ER-Gata1 nuclear translocation prior to cross-linking.
Anti-Gata2 and anti-Gata1 rabbit polyclonal antibodies were a gift from Emery
H. Bresnick (University of Wisconsin Medical School, Madison, WI). Normal
rabbit IgG (Upstate) was used as a negative control. Primers were designed
using Primer Express software (Applied Biosystems). Primer specificity was
verified by showing that each primer pair generated a single amplicon and
dissociation curve; primer sequences are available upon request.
Gene expression analysis by qRT-PCR
Purified total RNA (1 µg) was reverse-transcribed using the ImProm-II RT
Kit (Promega). Eklf, Gata1, Gata2, GFP, globin βH1, Smad1,
Smad5, Gapdh mRNA or 18S rRNA expression was detected using pre-made
TaqMan Gene Expression Assays (Applied Biosystems) according to the
manufacturer's protocol. Gene expression levels were calculated using a
relative standard curve made from serially diluted cDNA samples, followed by
normalization to 18S rRNA or Gapdh mRNA. For each clone and time
point, three independent RNA samples derived from three independent EB
differentiations performed in parallel were used to calculate the arithmetic
mean (average) and s.d. P-values were computed using Student's
t-test.
Protein expression analysis by western blot
Primary antibodies: Eklf (Southwood et
al., 1996), Gata1 (N-6, Santa Cruz Biotechnology), Gata2 (H-116,
Santa Cruz Biotechnology), Hsp90 (H-114, Santa Cruz Biotechnology), GFP (JL-8,
Clontech). Secondary antibodies: goat anti-rabbit IgG(H+L)-HRP (Southern
Biotechnologies); goat anti-mouse IgG(H+L)-HRP and goat anti-rat IgG(H+L)-HRP
(Pierce).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
which had been shown to direct erythroid-specific transgene expression in
developing mouse embryos (Xue et al.,
2004). However, to avoid the pitfalls usually associated with the
random integration of a transgene, such as position-effect variegation, we
took advantage of a site-specific integration mechanism featured in the
previously engineered Ainv18 ES cell line
(Kyba et al., 2002). Using
Cre-mediated recombination, we targeted a single copy of the Peklf-GFP
construct in a uni-directional fashion to a loxP homing site in a genomic
locus of open chromatin conformation and selected transgenic ES cell clones
based on antibiotic resistance (Fig.
1A). After establishing that the tet operon (tetOP)
contained in Ainv18 ES cells does not interfere with the transcriptional
control conferred by Peklf (see Fig. S1 in the supplementary material), we
created a Peklf-GFP `reporter mouse' by injecting ES cells of one Peklf-GFP
Ainv18 clone into mouse embryos at the blastocyst stage and breeding chimeric
animals to homozygosity.
Next we investigated the pattern of Eklf expression in vivo by analyzing
Peklf-GFP transgene activity during fetal liver hematopoiesis, as it is the
site of blood cell production between E11 and E16 in the developing mouse
embryo. Flow cytometric analysis of fetal livers from Peklf-GFP embryos
revealed that 
36% of all cells were GFP+ at E13.5
(Fig. 1B). To correlate GFP
expression with the status of erythroid lineage commitment in fetal liver
cells, we determined the transferrin receptor (CD71; Tfrc - Mouse Genome
Informatics) versus Ter119 (Ly76) cell-surface marker expression profile of
fetal liver cells as an indicator of hematopoietic differentiation, as
previously described (Zhang et al.,
2003). At E13.5, we found 61% of all fetal liver cells in the
committed erythroid compartment, as indicated by their
CD71hi/Ter119- to CD71lo/Ter119+
expression profile (Fig. 1C,
Table 1). Most GFP+
cells were part of this population as they also expressed the Ter119 marker
(34%, Fig. 1D) and were
thus identified as erythroid cells. By contrast, 12% of fetal liver cells
were characterized as progenitors by their low to medium CD71 levels in
combination with a lack of Ter119 expression
(CD71lo/med/Ter119-,
Fig. 1C,
Table 1). Interestingly, a
small fraction of GFP+ cells (2-3% of total,
Fig. 1D; 17% of progenitors,
Table 1) were found in this
progenitor compartment (CD71lo/med/Ter119-) of the
murine fetal liver.
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The sorted GFP+/Ter119- population gave rise to a substantial enrichment of erythroid progenitor-derived colonies (colony-forming unit erythroid, CFU-E; burst-forming unit erythroid, BFU-E) in culture (Fig. 1E,I). Most interestingly, it was also enriched for megakaryocytic progenitor-derived colonies, which were present in significant numbers (Fig. 1F,I), but not for granulocytic (Fig. 1G,I) or macrophage colonies (that are readily detectable in assays seeded with GFP-/Ter119- cells, Fig. 1H). Our data clearly indicate that the Eklf promoter (Peklf) activates transcription in progenitor cells well before the onset of erythroid differentiation. More specifically, Peklf-GFP expression preferentially marks progenitors that will give rise to erythroid or megakaryocytic colonies.
According to current models of hematopoiesis, the erythroid and
megakaryocytic lineages are derived from a common, bipotential progenitor
(megakaryocytic-erythroid progenitor, MEP), which in turn stems from a
progenitor common to all myeloid cells
(Adolfsson et al., 2005;
Forsberg et al., 2006).
Therefore, our findings strongly suggest that Eklf is expressed at least as
early as in a progenitor population of erythroid-megakaryocytic potential.
Eklf expression is initiated prior to erythroid commitment in differentiating EBs
To better define the onset and pattern of Eklf expression during
hematopoiesis in the context of stage- and lineage-specific cell-surface
markers and genes, we next employed the differentiation of the Peklf-GFP
Ainv18 ES cell clone to EBs as an in vitro assay of hematopoietic development.
Throughout an 8-day ES cell differentiation time-course, GFP
transcription under control of Peklf closely mirrored that of the endogenous
Eklf gene with regard to onset and levels of expression
(Fig. 2A). Expression of Eklf
and GFP was initiated at or near day 4 during the stage of EB development that
is characterized by the presence of mesoderm-derived hematopoietic progenitor
cells as indicated by a peak in Gata2 expression
(Fig. 2A) and high levels of
the receptor tyrosine kinase Flk1 (Kdr - Mouse Genome Informatics)
(Chung et al., 2002)
(Table 2). Interestingly, the
activation of Eklf (and GFP) expression occurred
simultaneously to that of Gata1 at day 4, and all three were expressed in
virtually the same pattern thereafter (Fig.
2A). Following the initial activation of expression, Eklf and GFP
levels began to rise rapidly around day 5 of EB development. By day 6, Eklf
and GFP expression peaked, whereas the levels of the progenitor markers Gata2
and Flk1 decreased, which coincided with the appearance of committed erythroid
cells in EBs as indicated by the expression of the red cell-restricted
cell-surface marker Ter119 (Table
2) and embryonic β-like globin (globin βH1; Hbb-bh1 -
Mouse Genome Informatics) (Fig.
2A). Subsequently, mature myeloid cells emerged by day 8 of EB
development (see Fig. S2 in the supplementary material). At that point, GFP
expression among these lineage-committed cells was restricted to erythroid
cells and did not occur in cells of the granulocyte-macrophage or
megakaryocytic lineages (see Fig. S2 in the supplementary material).
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Therefore, the expression of Eklf (GFP) during the hematopoietic differentiation of EBs can be divided into two phases: the onset of expression in a progenitor population between days 4 and 6 of EB development, followed by erythroid-restricted expression after day 6.
Comparative phylogenetic sequence analysis of the Eklf locus
To elucidate the mechanism regulating Eklf expression in a progenitor
population, we sought to identify genomic regulatory regions that control
Eklf transcription at this early stage in hematopoiesis, in contrast
to previous studies, which had been focused on the regulation of Eklf
expression in differentiating erythroid cells.
In order to predict putative transcription factor binding sites based on
cross-species sequence conservation
(Chapman et al., 2004), we
generated a comparative, multi-sequence alignment of the Eklf locus
from different mammalian species. This approach has been demonstrated to be
superior in identifying functional cis-regulatory elements to pairwise
alignments, which had previously been performed for the Eklf locus
(Chen et al., 1998).
Fig. 3A shows a five-species
alignment across the entire genomic Eklf locus between the two
neighboring genes. Importantly, aside from exons, only two domains of
non-coding sequence display a significant level of conservation between all
five species across the 17 kb of the alignment: a stretch of 1000 bp with
three peaks of conservation directly upstream of the Eklf
transcription start site, and a shorter peak region within the first intron.
The former corresponds to the 950 bp region used in the Peklf-GFP construct
described above, which harbors an upstream enhancer, two erythroid
hypersensitive sites (EHS1 and EHS2) (Chen
et al., 1998) and a proximal promoter
(Crossley et al., 1994). The
latter consists of a previously unreported sequence element just upstream of
the splice branch site preceding the second exon, which we designated as a
putative intronic enhancer.
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); and a Gata factor site next to a Cp1 (Nfya - Mouse Genome
Informatics) binding site within the proximal promoter
(Crossley et al., 1994). In
addition, we found two previously unreported WGATAR motifs in the putative
intronic enhancer in an arrangement similar to that of the upstream enhancer,
albeit with a lesser degree of homology as only one of the intronic GATA
motifs is conserved between all seven species. Most importantly, however, all three regulatory regions of the Eklf gene display a similar layout, in which Smad sites are clustered around one or two Gata factor binding motifs. The upstream enhancer contains four Smad sites and a putative Sp1/E2F/Smad site surrounding the GEG motif, whereas the proximal promoter contains one Smad site next to the Gata/Cp1 sites. Similarly, the five Smad sites in the intronic enhancer are arranged around the one perfectly conserved Gata site.
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Gata factor and Smad binding motifs regulate EKLF expression
To examine the contribution of the identified cis-regulatory elements and
individual transcription factor binding sites to the control of Eklf
transcription during hematopoiesis, we expanded the ES cell in vitro
differentiation reporter assay by generating a set of new GFP transgene
reporter constructs that incorporated insertions, deletions or point mutations
into Eklf cis-regulatory elements based on the alignment in
Fig. 3. In total, we created
four new Ainv18 ES cell clones as shown in
Fig. 4A.
The introduction of a point mutation in each of the two Gata binding sites of the upstream Eklf enhancer (Peklf-2xG/A-GFP) completely abolished GFP transgene transcription between days 4 and 7 of EB development (Fig. 4B). By contrast, a small but significant rise in GFP transgene expression levels occurred between days 4 and 5.5 when a point mutation was introduced into the Gata site at the proximal Eklf promoter (Peklf-G/A-GFP; Fig. 4C). Only subsequently, upon erythroid commitment after day 5.5, did this Gata site mutation result in a failure to increase GFP transgene levels, whereas mRNA levels of the endogenous Eklf gene continued to grow.
Next we tested the potential contribution of the newly identified Smad
binding motifs found within the three Eklf cis-regulatory regions to
the control of GFP transgene expression. As the onset of Eklf expression at
day 4 of EB differentiation requires functional Gata binding sites within the
upstream Eklf enhancer, which also contains Smad binding motifs, we
hypothesized that Smad-mediated control of Eklf transcription was
most likely to occur at the upstream enhancer. To test this, we deleted the
two Smad binding motifs in the upstream enhancer that directly surround the
GEG motif (Peklf-2x
SBM-GFP), without altering the GEG motif itself so
as to avoid interfering with Gata factor binding. The deletion of the two Smad
binding sites abolished the onset of GFP transgene transcription between days
4 and 5.5 of EB development (Fig.
4D). Thereafter however, transgene expression was activated as GFP
levels began to rise between days 5.5 and 7. Importantly, the GFP expression
pattern resulting from the deletion of Smad sites in the upstream enhancer
differed from the pattern produced by the mutation of GATA motifs in the
upstream enhancer, arguing that the deletion of Smad sites in the upstream
enhancer does not simply impact Gata binding in this region.
As Smads and Gata factors act on the upstream enhancer, we reasoned that a similar mode of transcriptional control might occur at the intronic Eklf enhancer, given that both regulatory regions display a similar layout of Gata sites surrounded by Smad binding motifs. Inclusion of the intronic Eklf enhancer (Peklf-intron-GFP) increased the maximal level of GFP expression about threefold as compared with levels recorded with Peklf-GFP alone, without altering the overall pattern of transgene expression (Fig. 4E). Furthermore, the intronic Eklf enhancer extended the rise of transgene expression until day 8 of EB development, in contrast to expression of the endogenous Eklf, which plateaus at day 7. This argues that although the newly identified, highly conserved region within the first intron of Eklf is not required for transgene expression, it indeed acts as an enhancer of Eklf transcription.
In summary, our EB reporter assay results in the context of a transgene integrated at a homing site suggest that Gata factor and Smad binding at the Eklf upstream enhancer are required for the onset of Eklf transcription in a progenitor population. By contrast, upon erythroid commitment, Gata factor-mediated control of transcription at the upstream enhancer and the proximal promoter is sufficient for the maintenance of Eklf expression. In addition, the highly conserved region within the first Eklf intron that was identified through the phylogenetic alignment acts as an enhancer of Eklf transcription throughout hematopoiesis.
Gata2 binds to the Eklf locus in undifferentiated G1E-ER-Gata1 cells and at the progenitor stage of hematopoietic EB differentiation
Having established that Gata factor binding to the upstream Eklf
enhancer is required for the onset of transgene expression in EBs at day 4, we
next asked which Gata factor regulates Eklf expression at the progenitor
stage. Gata2 is an obvious candidate in this regard, as its functional role in
hematopoietic progenitors is well documented and we find Eklf
transcription initiated in EBs at a time when high levels of Gata2, but not of
Gata1, are present. To test this, we performed quantitative chromatin
immunoprecipitation (ChIP) assays in G1E-ER-Gata1 cells and in differentiating
EBs.
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;
Weiss et al., 1997;
Welch et al., 2004), which
coincides with a rise in Eklf expression levels (see Fig. S4 in the
supplementary material) (see Im et al.,
2005). In undifferentiated G1E-ER-Gata1 cells, Gata2 occupied the Eklf upstream enhancer (Fig. 5A). Gata2 occupancy levels at the upstream enhancer were twofold enriched compared with those at the proximal promoter and the intronic enhancer, which did not display any significant Gata2 occupancy as compared with a non-conserved GATA motif in the 3'UTR of the murine Eklf locus that served as a negative control. Upon differentiation of G1E-ER-Gata1 cells, Gata2 occupancy levels at the Eklf upstream enhancer dropped, which, in turn, coincided with an increase of ER-Gata1 fusion protein binding to the Eklf upstream enhancer and to the proximal promoter (Fig. 5A). This switch in Gata factor occupancy argues that ER-Gata1 replaces Gata2 at the upstream enhancer while also binding to the proximal promoter during the induced erythroid differentiation of G1E-ER-Gata1 cells, which correlates with an increase in Eklf expression.
To assess Gata factor-mediated control of Eklf expression under conditions
that more closely resemble the endogenous state than does a cell line, we next
examined Gata occupancy patterns at the Eklf locus during
hematopoiesis in differentiating EBs. At day 4 of EB development, an
enrichment of Gata2 occupancy was detected at the upstream enhancer and the
proximal promoter of the Eklf locus, and to a lesser degree at the
intronic enhancer (Fig. 5B).
Therefore, the onset of Eklf expression at the progenitor stage coincides with
Gata2 occupancy at all three cis-regulatory sites of the Eklf gene.
[Similar to Lugus et al., we detected an overall background signal with the
anti-Gata2 ChIP in EBs that was higher than in G1E-ER-Gata1 cells, most likely
owing to the cellular heterogeneity of EBs
(Lugus et al., 2007).] At day
6 of EB differentiation, Gata2 occupancy levels decreased. By contrast, Gata1
binding occurred at all three conserved Eklf cis-regulatory regions,
especially at the upstream enhancer (Fig.
5B). Thus, a Gata switch at the Eklf locus dependent on
the progression of hematopoietic development is observed in EBs. This in vivo
occupancy profile of Gata factors suggests a mechanism of transcriptional
control by which Gata2 binding mediates the initiation of Eklf expression
during hematopoiesis prior to erythroid commitment, whereas Gata1 replaces
Gata2 at the Eklf locus during erythroid differentiation, which
results in an increase of Eklf expression.
Importantly, a Gata factor switch upon erythroid commitment, similar to the
one observed at the Eklf locus between days 4 and 6 of EB
differentiation, was also detected at a cis-regulatory region of the
Gata1 gene (Fig. 5B),
analogous to results previously described in G1E-ER-Gata1 cells
(Pal et al., 2004) and
reproduced here (Fig. 5A). This
regulatory region, termed hypersensitive site 1 (HS1) within the hematopoietic
enhancer (HE) of the Gata1 (G1) gene (G1HE/HS1, see Fig. S3 in the
supplementary material), is known to be required for Gata1 expression in a
progenitor population (Vyas et al.,
1999). Thus, the initial onset of Eklf and Gata1
transcription does not only occur at the same time during the progenitor stage
in EBs, but is also regulated in a similar fashion, dependent on Gata2 binding
to specific enhancer elements.
The knockdown of Smad5 in EBs results in reduced Eklf and Gata1 expression
As Bmp4 signaling via the Smad pathway is necessary and sufficient to
induce Eklf and Gata1 expression at day 4 of EB development
(Adelman et al., 2002), we
hypothesized that the onset of Eklf expression in a progenitor population
prior to erythroid commitment is regulated directly by Smad proteins, in light
of the fact that Smad binding sites in the Eklf upstream enhancer are
required for GFP transgene expression between days 4 and 5.5 of EB
development. The most likely Bmp4 effector in this regard is Smad5, as it has
been implicated in promoting the establishment of erythroid fate during
hematopoiesis (Fuchs et al.,
2002; Liu et al.,
2003; McReynolds et al.,
2007). By contrast, Smad1 acts earlier in hematopoiesis, at the
hemangioblast stage (Zafonte et al.,
2007).
As no anti-Smad5 antibodies functional in ChIP assays have been reported to
date, we developed a novel loss-of-function assay for the Ainv18 ES cell line
that allowed us to specifically knockdown Smad5 levels in an inducible,
RNAi-mediated manner during EB development so as to test the impact of Smad5
activity on Eklf expression. Utilizing the tet operon of the Ainv18
ES cell line (Kyba et al.,
2002), we coupled the transcription of a specific, short hairpin
RNA (shRNA) within a microRNA (miR) backbone to that of a GFP cDNA in
a doxycycline (dox)-dependent fashion (Fig.
6A). We generated three different expression cassettes, each of
which was stably inserted into the plox homing site of the Ainv18 ES cell line
via Cre-mediated recombination. Two of these encoded shRNAs targeting a unique
sequence within the Smad5 mRNA (shRNA-1 and shRNA-2), whereas a third
encoded a no-target control shRNA. Fig.
6B shows the tight transcriptional control imparted by the
tet operon in response to dox treatment as exemplified by GFP
expression levels in the case of the shRNA-1 Ainv18 ES cell clone.
As we hypothesized that Smad5-mediated transcriptional control occurs at
the onset of Eklf expression prior to erythroid commitment, we
induced the knockdown of Smad5 mRNA at day 4 of EB development by
treating the shRNA-1 Ainv18 ES cell clone with dox for 24 hours. To quantify
the maximal RNAi effect, we sorted EBs at day 5 into dox-treated,
GFP-expressing cells (+dox/GFP+) or untreated, GFP-negative cells
(-dox/GFP-), and isolated RNA for qRT-PCR analysis. In the
shRNA-1-expressing population (+dox/GFP+), the Smad5 mRNA
level was knocked down to 40% of the level seen in the non-expressing
population (-dox/GFP-), demonstrating that the transgenic RNAi
assay is functional (Fig. 6C).
More importantly, the dox-induced knockdown of Smad5 coincided with a
reduction of Eklf and Gata1 mRNA to 25% and 30%,
respectively, of the levels observed in untreated cells
(-dox/GFP-), suggesting that Smad5 regulates both genes.
Given the strong knockdown effect observed in the shRNA-1 Ainv18 clone upon
dox treatment, we reasoned that a cell-sorting step based on GFP expression
should not be required to detect the substantial drop in Smad5, Eklf and Gata1
levels. Instead, we used unsorted, whole EBs for a more extended
loss-of-function analysis. Analogous to the effect described above, a 24 hour
dox treatment of developing EBs from day 4 to 5 resulted in a 50% reduction of
Smad5 mRNA levels as compared with EBs not treated with dox
(Fig. 6D). Moreover, the
knockdown of Smad5 correlated once again with a significant drop in
Eklf and Gata1 levels, to 65% and 70% of those
observed in untreated EBs, respectively. Importantly, shRNA-1 targets
Smad5 mRNA specifically, as the levels of Smad1 mRNA were
unperturbed, despite the high degree of sequence homology between the two
mRNAs. In addition, Gata2 mRNA levels were not affected by the dox
treatment, demonstrating that the negative effect observed on Eklf
and Gata1 transcription between EB day 4 and 5 is in fact a direct
and specific consequence of the reduction of Smad5 mRNA levels.
To further corroborate this point, we next included the shRNA-2 and the shRNA-control clones in our experiments. A western blot analysis of whole-cell lysates made from EBs on day 6 of differentiation after 48 hours of dox treatment revealed that Eklf and Gata1 protein levels are strongly reduced in cells carrying either shRNA-1 or shRNA-2, but not in cells carrying the shRNA-control (Fig. 6E). By contrast, Gata2 protein levels did not display any significant variability between dox-treated and untreated cells in any of the three different clones. Importantly, the observed RNAi effect is due to the specific knockdown of Smad5, as two independent shRNAs complementary to different regions of the target mRNA produced the same result, in contrast to the no-target control shRNA that did not alter the expression of any of the genes tested. We conclude that Smad5 protein is required at day 4 of EB development for the correct onset of Eklf and Gata1 transcription.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), we propose a two-tiered, stage- and lineage-dependent model
of Eklf regulation during hematopoiesis, as described below.
Model of stage- and lineage-dependent activation of Eklf
Early in hematopoiesis, Gata2 and Smad5 activate Eklf in a
cooperative fashion. This integration of the Bmp4 signaling pathway with the
Gata2 target gene network is achieved at the upstream enhancer of
Eklf, where functional Gata and Smad binding sites are found in close
proximity to one another. It results in the low-level expression of
Eklf. Upon erythroid lineage commitment, Gata1 replaces Gata2 and
binds to the Eklf upstream enhancer and proximal promoter, regulating
Eklf expression at high levels throughout erythroid differentiation
in a Smad-independent manner. At this stage, Gata1 nucleates an Scl (Tal1 -
Mouse Genome Informatics)-containing protein complex occupying the GEG motif
at the upstream enhancer (Rodriguez et
al., 2005), similar to the GATA/E-box-bound complexes regulating
the erythroid-specific expression of other Gata1 target genes
(Anguita et al., 2004). In
parallel, Gata1 binding occurs at the Eklf proximal promoter next to
a CCAAT-box, possibly in a complex with Cp1 or a C/EBP family member
(Crossley et al., 1994;
Gordon et al., 2005), while
occupancy changes from Gata2 to Gata1 at the intronic enhancer as well. Such
`Gata-switches' at the same GATA motif have also been shown to occur during
the regulation of Gata1 (Pal et al.,
2004) (see also below), 
-globin
(Anguita et al., 2004), Scl
(Lugus et al., 2007), Gata2
(Grass et al., 2003;
Grass et al., 2006;
Kobayashi-Osaki et al., 2005)
and Kit expression (Jing et al.,
2008), resembling the two-tiered regulatory mechanism described
here for Eklf. Thus, the layout of transcription factor binding sites
contained in the Eklf cis-regulatory regions enables a response of
dual specificity that is dependent on the stage of hematopoiesis, which in
turn results in a transcriptional output of varying degree in a
lineage-specific manner.
Gata-Smad cooperation
Parallels to the Gata2-Smad5 cooperation in our model of Eklf
activation can be found in the regulation of cardiac-specific genes during
embryonic heart development (reviewed by
Peterkin et al., 2005).
Similar to the architecture observed in the cis-regulatory regions of
Eklf, neighboring Smad and Gata factor binding sites are found in
enhancers of Nkx2.5, which is the earliest known marker of
cardiogenesis, and the presence of both types of consensus motif is required
for conveying transcriptional control in response to BMP signaling
(Brown et al., 2004;
Lee et al., 2004).
Given the requirement of Bmp4 signaling for the establishment and progression of (yolk sac) hematopoiesis as well as the prominence of Gata2-controlled gene expression in hematopoietic progenitors, we propose that the functional cooperation of (BMP-activated) Smads and hematopoietic Gata factors underlies target gene regulation in hematopoietic development and lineage fate decisions, as exemplified here for the case of Eklf.
Model of Gata1 activation at the progenitor stage
In support of the above notion, we show that the onset of Gata1 expression
is dependent on Smad5 as well. We identify a cluster of conserved Smad binding
motifs next to a known Gata site in the HE/HS1 region of the Gata1
locus, which we find to be occupied by Gata2 at the progenitor stage during EB
differentiation. This Gata1 enhancer region is required for transgene
expression in both the erythroid and megakaryocytic lineage
(Vyas et al., 1999). Thus, as
the regulation of Gata1 expression is stage- and lineage-dependent, we
speculate that the onset of Gata1 transcription in a progenitor
population of erythroid-megakaryocytic potential is mediated via Gata2-Smad5
cooperation at the G1HE/HS1 element, followed by Gata2 replacement and Gata1
autoregulation upon erythroid commitment in a Smad-independent manner.
These models of Eklf and Gata1 activation argue that a
layout featuring GATA motifs in combination with Smad binding sites denotes a
cis-regulatory element that is utilized at the progenitor stage and could
explain the block in erythroid differentiation that is observed upon
perturbation or lack of Smad5 expression during hematopoiesis
(Fuchs et al., 2002;
Liu et al., 2003;
McReynolds et al., 2007) or in
response to stress erythropoiesis
(Porayette and Paulson,
2008).
A novel role for Eklf in erythroid lineage commitment
The addition of Eklf to the growing list of Bmp4-regulated genes
illustrates that the Bmp4 signaling pathway and Smad activity are required
throughout hematopoiesis, as opposed to being necessary only for the initial
establishment of hematopoietic fate from mesoderm during development
(Lugus et al., 2007;
Pimanda et al., 2007;
Zafonte et al., 2007). As Gata
factors regulate a multitude of genes central to the hematopoietic program,
the delineation of mechanisms that integrate the cross-talk between Bmp4
signaling and the Gata factor-controlled gene network is essential to an
understanding of hematopoiesis (Loose et
al., 2007). Of particular interest in this regard (and for stem
cell biology in general) are the mechanisms underlying lineage fate decisions
and the accompanying progression from a highly proliferative progenitor state
to that of lineage commitment and differentiation.
According to models of lineage fate decisions from a (bipotential)
progenitor cell, commitment to one specific lineage over another is
established through a cross-antagonistic mechanism of opposing transcription
factors (Cantor and Orkin,
2002). Quantitatively, the uncommitted progenitor state is
characterized by the co-expression of such antagonistic transcription factors
at a low level. However, this priming state is disrupted following a rise in
the transcription levels and thereby dominance of one regulatory factor over
the other, which subsequently leads to lineage commitment and ultimately
lineage differentiation (Huang et al.,
2007; Roeder and Glauche,
2006).
Here, we describe how the stage- and lineage-dependent integration of Gata2
activity and Bmp4/Smad5 signaling versus Gata1-anchored complex binding to the
same Eklf cis-regulatory element translates into such a two-tiered
transcriptional profile between the megakaryocytic-erythroid progenitor and
differentiating erythroid cells. As the expression of Eklf prior to erythroid
commitment has recently been corroborated
(Bottardi et al., 2006;
Frontelo et al., 2007),
studies conducted in parallel in our laboratory show that Eklf indeed plays a
directive role in erythroid versus megakaryocytic development in accordance
with the antagonistic model described above
(Frontelo et al., 2007;
Siatecka et al., 2007). Thus,
a new role for Eklf as a regulator of lineage fate decisions during
hematopoiesis is defined, the misregulation of which could potentially
underlie disease mechanisms.
A new `toolbox' for the ES cell in vitro differentiation system
Expanding on the gain-of-function assay previously described for the Ainv18
ES cell line (Kyba et al.,
2002; Manwani et al.,
2007; Willey et al.,
2006), we provide novel transgenic reporter and loss-of-function
assays for use in ES cells and EBs. In combination with in vivo binding
studies performed during the in vitro differentiation of ES cells, we are able
to delineate the mechanisms of Eklf transcriptional regulation at a
higher resolution than had previously been achieved, either in transgenic
mouse models of Eklf expression (Anderson
et al., 2000; Xue et al.,
2004) or in gene ablation studies of Gata factors
(Pevny et al., 1991;
Tsai et al., 1994) and Smad5
(Liu et al., 2003). Together
with the rapidly expanding number of protocols available for the
differentiation of ES cells in culture, these assays provide a powerful
`toolbox' for the study of genetic interactions that govern early mouse
development and lineage decision processes, similar to techniques established
for the fish and frog model systems.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/12/2071/DC1
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ACKNOWLEDGMENTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Adelman, C. A., Chattopadhyay, S. and Bieker, J. J.
(2002). The BMP/BMPR/Smad pathway directs expression of the
erythroid-specific EKLF and GATA1 transcription factors during embryoid body
differentiation in serum-free media. Development
129,539
-549.[Medline]
Adolfsson, J., Mansson, R., Buza-Vidas, N., Hultquist, A.,
Liuba, K., Jensen, C. T., Bryder, D., Yang, L., Borge, O. J., Thoren, L. A. et
al. (2005). Identification of Flt3+ lympho-myeloid stem cells
lacking erythro-megakaryocytic potential a revised road map for adult blood
lineage commitment. Cell
121,295
-306.[CrossRef][Medline]
Anderson, K. P., Crable, S. C. and Lingrel, J. B.
(1998). Multiple proteins binding to a GATA-E box-GATA motif
regulate the erythroid Kruppel-like factor (EKLF) gene. J. Biol.
Chem. 273,14347
-14354.
Anderson, K. P., Crable, S. C. and Lingrel, J. B.
(2000). The GATA-E box-GATA motif in the EKLF promoter is
required for in vivo expression. Blood
95,1652
-1655.
Anguita, E., Hughes, J., Heyworth, C., Blobel, G. A., Wood, W.
G. and Higgs, D. R. (2004). Globin gene activation during
haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2.
EMBO J. 23,2841
-2852.[CrossRef][Medline]
Benchabane, H. and Wrana, J. L. (2003). GATA-
and Smad1-dependent enhancers in the Smad7 gene differentially interpret bone
morphogenetic protein concentrations. Mol. Cell. Biol.
23,6646
-6661.
Bottardi, S., Ross, J., Pierre-Charles, N., Blank, V. and Milot,
E. (2006). Lineage-specific activators affect beta-globin
locus chromatin in multipotent hematopoietic progenitors. EMBO
J. 25,3586
-3595.[CrossRef][Medline]
Brown, C. O., 3rd, Chi, X., Garcia-Gras, E., Shirai, M., Feng,
X. H. and Schwartz, R. J. (2004). The cardiac determination
factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a
novel upstream enhancer. J. Biol. Chem.
279,10659
-10669.
Cantor, A. B. and Orkin, S. H. (2002).
Transcriptional regulation of erythropoiesis: an affair involving multiple
partners. Oncogene 21,3368
-3376.[CrossRef][Medline]
Chapman, M. A., Donaldson, I. J., Gilbert, J., Grafham, D.,
Rogers, J., Green, A. R. and Gottgens, B. (2004). Analysis of
multiple genomic sequence alignments: a web resource, online tools, and
lessons learned from analysis of mammalian SCL loci. Genome
Res. 14,313
-318.
Chen, C. R., Kang, Y., Siegel, P. M. and Massague, J.
(2002). E2F4/5 and p107 as Smad cofactors linking the TGFbeta
receptor to c-myc repression. Cell
110, 19-32.[CrossRef][Medline]
Chen, X., Reitman, M. and Bieker, J. J. (1998).
Chromatin structure and transcriptional control elements of the erythroid
Kruppel-like factor (EKLF) gene. J. Biol. Chem.
273,25031
-25040.
Choi, K., Chung, Y. S. and Zhang, W. J. (2005).
Hematopoietic and endothelial development of mouse embryonic stem cells in
culture. Methods Mol. Med.
105,359
-368.[Medline]
Chung, Y. S., Zhang, W. J., Arentson, E., Kingsley, P. D.,
Palis, J. and Choi, K. (2002). Lineage analysis of the
hemangioblast as defined by FLK1 and SCL expression.
Development 129,5511
-5520.
Crossley, M., Tsang, A. P., Bieker, J. J. and Orkin, S. H.
(1994). Regulation of the erythroid Kruppel-like factor (EKLF)
gene promoter by the erythroid transcription factor GATA-1. J.
Biol. Chem. 269,15440
-15444.
Forsberg, E. C., Serwold, T., Kogan, S., Weissman, I. L. and
Passegue, E. (2006). New evidence supporting
megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic
progenitors. Cell 126,415
-426.[CrossRef][Medline]
Frederick, J. P., Liberati, N. T., Waddell, D. S., Shi, Y. and
Wang, X. F. (2004). Transforming growth factor beta-mediated
transcriptional repression of c-myc is dependent on direct binding of Smad3 to
a novel repressive Smad binding element. Mol. Cell.
Biol. 24,2546
-2559.
Frontelo, P., Manwani, D., Galdass, M., Karsunky, H., Lohmann,
F., Gallagher, P. G. and Bieker, J. J. (2007). Novel role for
EKLF in megakaryocyte lineage commitment. Blood
110,3871
-3880.
Fuchs, O., Simakova, O., Klener, P., Cmejlova, J., Zivny, J.,
Zavadil, J. and Stopka, T. (2002). Inhibition of Smad5 in
human hematopoietic progenitors blocks erythroid differentiation induced by
BMP4. Blood Cells Mol. Dis.
28,221
-233.[CrossRef][Medline]
Gordon, C. T., Fox, V. J., Najdovska, S. and Perkins, A. C.
(2005). C/EBPdelta and C/EBPgamma bind the CCAAT-box in the human
beta-globin promoter and modulate the activity of the CACC-box binding
protein, EKLF. Biochim. Biophys. Acta
1729,74
-80.[Medline]
Grass, J. A., Boyer, M. E., Pal, S., Wu, J., Weiss, M. J. and
Bresnick, E. H. (2003). GATA-1-dependent transcriptional
repression of GATA-2 via disruption of positive autoregulation and domain-wide
chromatin remodeling. Proc. Natl. Acad. Sci. USA
100,8811
-8816.
Grass, J. A., Jing, H., Kim, S. I., Martowicz, M. L., Pal, S.,
Blobel, G. A. and Bresnick, E. H. (2006). Distinct functions
of dispersed GATA factor complexes at an endogenous gene locus.
Mol. Cell. Biol. 26,7056
-7067.
Gupta, S., Zhu, H., Zon, L. I. and Evans, T.
(2006). BMP signaling restricts hemato-vascular development from
lateral mesoderm during somitogenesis. Development
133,2177
-2187.
Hata, A., Seoane, J., Lagna, G., Montalvo, E.,
Hemmati-Brivanlou, A. and Massague, J. (2000). OAZ uses
distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf
signaling pathways. Cell
100,229
-240.[CrossRef][Medline]
Huang, S., Guo, Y. P., May, G. and Enver, T.
(2007). Bifurcation dynamics in lineage-commitment in bipotent
progenitor cells. Dev. Biol.
305,695
-713.[CrossRef][Medline]
Huber, T. L., Kouskoff, V., Fehling, H. J., Palis, J. and
Keller, G. (2004). Haemangioblast commitment is initiated in
the primitive streak of the mouse embryo. Nature
432,625
-630.[CrossRef][Medline]
Im, H., Grass, J. A., Johnson, K. D., Kim, S.-I., Boyer, M. E.,
Imbalzano, A. N., Bieker, J. J. and Bresnick, E. H. (2005).
Chromatin domain activation via GATA-1 utilization of a small subset of
dispersed GATA motifs within a broad chromosomal region. Proc.
Natl. Acad. Sci. USA 102,17065
-17070.
Ishida, W., Hamamoto, T., Kusanagi, K., Yagi, K., Kawabata, M.,
Takehara, K., Sampath, T. K., Kato, M. and Miyazono, K.
(2000). Smad6 is a Smad1/5-induced smad inhibitor.
Characterization of bone morphogenetic protein-responsive element in the mouse
Smad6 promoter. J. Biol. Chem.
275,6075
-6079.
Jing, H., Vakoc, C. R., Ying, L., Mandat, S., Wang, H., Zheng,
X. and Blobel, G. A. (2008). Exchange of GATA factors
mediates transitions in looped chromatin organization at a developmentally
regulated gene locus. Mol. Cell
29,232
-242.[CrossRef][Medline]
Keller, G. (2005). Embryonic stem cell
differentiation: emergence of a new era in biology and medicine.
Genes Dev. 19,1129
-1155.
Kennedy, M. and Keller, G. M. (2003).
Hematopoietic commitment of ES cells in culture. Methods
Enzymol. 365,39
-59.[Medline]
Kobayashi-Osaki, M., Ohneda, O., Suzuki, N., Minegishi, N.,
Yokomizo, T., Takahashi, S., Lim, K. C., Engel, J. D. and Yamamoto, M.
(2005). GATA motifs regulate early hematopoietic lineage-specific
expression of the Gata2 gene. Mol. Cell. Biol.
25,7005
-7020.
Korchynskyi, O. and ten Dijke, P. (2002).
Identification and functional characterization of distinct critically
important bone morphogenetic protein-specific response elements in the Id1
promoter. J. Biol. Chem.
277,4883
-4891.
Kyba, M., Perlingeiro, R. C. and Daley, G. Q.
(2002). HoxB4 confers definitive lymphoid-myeloid engraftment
potential on embryonic stem cell and yolk sac hematopoietic progenitors.
Cell 109,29
-37.[CrossRef][Medline]
Lee, K. H., Evans, S., Ruan, T. Y. and Lassar, A. B.
(2004). SMAD-mediated modulation of YY1 activity regulates the
BMP response and cardiac-specific expression of a GATA4/5/6-dependent chick
Nkx2.5 enhancer. Development
131,4709
-4723.
Liu, B., Sun, Y., Jiang, F., Zhang, S., Wu, Y., Lan, Y., Yang,
X. and Mao, N. (2003). Disruption of Smad5 gene leads to
enhanced proliferation of high-proliferative potential precursors during
embryonic hematopoiesis. Blood
101,124
-133.
Loose, M., Swiers, G. and Patient, R. (2007).
Transcriptional networks regulating hematopoietic cell fate decisions.
Curr. Opin. Hematol. 14,307
-314.[Medline]
Lugus, J. J., Chung, Y. S., Mills, J. C., Kim, S. I., Grass, J.
A., Kyba, M., Doherty, J. M., Bresnick, E. H. and Choi, K.
(2007). GATA2 functions at multiple steps in hemangioblast
development and differentiation. Development
134,393
-405.
Maeno, M., Mead, P. E., Kelley, C., Xu, R. H., Kung, H. F.,
Suzuki, A., Ueno, N. and Zon, L. I. (1996). The role of BMP-4
and GATA-2 in the induction and differentiation of hematopoietic mesoderm in
Xenopus laevis. Blood
88,1965
-1972.
Manwani, D., Galdass, M. and Bieker, J. J.
(2007). Altered regulation of beta-like globin genes by a
redesigned erythroid transcription factor. Exp.
Hematol. 35,39
-47.[CrossRef][Medline]
McReynolds, L. J., Gupta, S., Figueroa, M. E., Mullins, M. C.
and Evans, T. (2007). Smad1 and Smad5 differentially regulate
embryonic hematopoiesis. Blood
110,3881
-3890.
Miller, I. J. and Bieker, J. J. (1993). A
novel, erythroid cell-specific murine transcription factor that binds to the
CACCC element and is related to the Kruppel family of nuclear proteins.
Mol. Cell. Biol. 13,2776
-2786.
Nuez, B., Michalovich, D., Bygrave, A., Ploemacher, R. and
Grosveld, F. (1995). Defective haematopoiesis in fetal liver
resulting from inactivation of the EKLF gene. Nature
375,316
-318.[CrossRef][Medline]
Oren, T., Torregroza, I. and Evans, T. (2005).
An Oct-1 binding site mediates activation of the gata2 promoter by BMP
signaling. Nucleic Acids Res.
33,4357
-4367.
Orkin, S. H. and Zon, L. I. (2008).
Hematopoiesis: an evolving paradigm for stem cell biology.
Cell 132,631
-644.[CrossRef][Medline]
Paddison, P. J., Cleary, M., Silva, J. M., Chang, K., Sheth, N.,
Sachidanandam, R. and Hannon, G. J. (2004). Cloning of short
hairpin RNAs for gene knockdown in mammalian cells. Nat.
Methods 1,163
-167.[CrossRef][Medline]
Pal, S., Cantor, A. B., Johnson, K. D., Moran, T. B., Boyer, M.
E., Orkin, S. H. and Bresnick, E. H. (2004).
Coregulator-dependent facilitation of chromatin occupancy by GATA-1.
Proc. Natl. Acad. Sci. USA
101,980
-985.
Palis, J., Robertson, S., Kennedy, M., Wall, C. and Keller,
G. (1999). Development of erythroid and myeloid progenitors
in the yolk sac and embryo proper of the mouse.
Development 126,5073
-5084.[Abstract]
Park, C., Afrikanova, I., Chung, Y. S., Zhang, W. J., Arentson,
E., Fong, G., Rosendahl, A. and Choi, K. (2004). A
hierarchical order of factors in the generation of FLK1- and SCL-expressing
hematopoietic and endothelial progenitors from embryonic stem cells.
Development 131,2749
-2762.
Perkins, A. C., Sharpe, A. H. and Orkin, S. H.
(1995). Lethal beta-thalassaemia in mice lacking the erythroid
CACCC-transcription factor EKLF. Nature
375,318
-322.[CrossRef][Medline]
Peterkin, T., Gibson, A., Loose, M. and Patient, R.
(2005). The roles of GATA-4, -5 and -6 in vertebrate heart
development. Semin. Cell Dev. Biol.
16, 83-94.[CrossRef][Medline]
Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S.
F., D'Agati, V., Orkin, S. H. and Costantini, F. (1991).
Erythroid differentiation in chimaeric mice blocked by a targeted mutation in
the gene for transcription factor GATA-1. Nature
349,257
-260.[CrossRef][Medline]
Pimanda, J. E., Donaldson, I. J., de Bruijn, M. F., Kinston, S.,
Knezevic, K., Huckle, L., Piltz, S., Landry, J. R., Green, A. R., Tannahill,
D. et al. (2007). The SCL transcriptional network and BMP
signaling pathway interact to regulate RUNX1 activity. Proc. Natl.
Acad. Sci. USA 104,840
-845.
Porayette, P. and Paulson, R. F. (2008).
BMP4/Smad5 dependent stress erythropoiesis is required for the expansion of
erythroid progenitors during fetal development. Dev.
Biol. (in press).
Rodriguez, P., Bonte, E., Krijgsveld, J., Kolodziej, K. E.,
Guyot, B., Heck, A. J., Vyas, P., de Boer, E., Grosveld, F. and Strouboulis,
J. (2005). GATA-1 forms distinct activating and repressive
complexes in erythroid cells. EMBO J.
24,2354
-2366.[CrossRef][Medline]
Roeder, I. and Glauche, I. (2006). Towards an
understanding of lineage specification in hematopoietic stem cells: a
mathematical model for the interaction of transcription factors GATA-1 and
PU.1. J. Theor. Biol.
241,852
-865.[Medline]
Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague, J. and
Pavletich, N. P. (1998). Crystal structure of a Smad MH1
domain bound to DNA: insights on DNA binding in TGF-beta signaling.
Cell 94,585
-594.[CrossRef][Medline]
Siatecka, M., Xue, L. and Bieker, J. J. (2007).
Sumoylation of EKLF promotes transcriptional repression and is involved in
inhibition of megakaryopoiesis. Mol. Cell. Biol.
27,8547
-8560.
Southwood, C. M., Downs, K. M. and Bieker, J. J.
(1996). Erythroid Kruppel-like factor exhibits an early and
sequentially localized pattern of expression during mammalian erythroid
ontogeny. Dev. Dyn. 206,248
-259.[CrossRef][Medline]
Tsai, F.-Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J.,
Rosenblatt, M., Alt, F. W. and Orkin, S. H. (1994). An early
haematopoietic defect in mice lacking the transcription factor GATA-2.
Nature 371,221
.[CrossRef][Medline]
Vyas, P., McDevitt, M. A., Cantor, A. B., Katz, S. G., Fujiwara,
Y. and Orkin, S. H. (1999). Different sequence requirements
for expression in erythroid and megakaryocytic cells within a regulatory
element upstream of the GATA-1 gene. Development
126,2799
-2811.[Abstract]
Weiss, M. J., Keller, G. and Orkin, S. H.
(1994). Novel insights into erythroid development revealed
through in vitro differentiation of GATA-1 embryonic stem cells.
Genes Dev. 8,1184
-1197.
Weiss, M. J., Yu, C. and Orkin, S. H. (1997).
Erythroid-cell-specific properties of transcription factor GATA-1 revealed by
phenotypic rescue of a gene-targeted cell line. Mol. Cell.
Biol. 17,1642
-1651.[Abstract]
Welch, J. J., Watts, J. A., Vakoc, C. R., Yao, Y., Wang, H.,
Hardison, R. C., Blobel, G. A., Chodosh, L. A. and Weiss, M. J.
(2004). Global regulation of erythroid gene expression by
transcription factor GATA-1. Blood
104,3136
-3147.
Willey, S., Ayuso-Sacido, A., Zhang, H., Fraser, S. T., Sahr, K.
E., Adlam, M. J., Kyba, M., Daley, G. Q., Keller, G. and Baron, M. H.
(2006). Acceleration of mesoderm development and expansion of
hematopoietic progenitors in differentiating ES cells by the mouse Mix-like
homeodomain transcription factor. Blood
107,3122
-3130.
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L.
(1995). Bone morphogenetic protein-4 is required for mesoderm
formation and patterning in the mouse. Genes Dev.
9,2105
-2116.
Xue, L., Chen, X., Chang, Y. and Bieker, J. J.
(2004). Regulatory elements of the EKLF gene that direct
erythroid cell-specific expression during mammalian development.
Blood 103,4078
-4083.
Yagi, K., Furuhashi, M., Aoki, H., Goto, D., Kuwano, H.,
Sugamura, K., Miyazono, K. and Kato, M. (2002). c-myc is a
downstream target of the Smad pathway. J. Biol. Chem.
277,854
-861.
Zafonte, B. T., Liu, S., Lynch-Kattman, M., Torregroza, I.,
Benvenuto, L., Kennedy, M., Keller, G. and Evans, T. (2007).
Smad1 expands the hemangioblast population within a limited developmental
window. Blood 109,516
-523.
Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W.,
Vogelstein, B. and Kern, S. E. (1998). Human Smad3 and Smad4
are sequence-specific transcription activators. Mol.
Cell 1,611
-617.[CrossRef][Medline]
Zhang, J., Socolovsky, M., Gross, A. W. and Lodish, H. F.
(2003). Role of Ras signaling in erythroid differentiation of
mouse fetal liver cells: functional analysis by a flow cytometry-based novel
culture system. Blood
102,3938
-3946.
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