(Received for publication, October 3, 1996, and in revised form, December 19, 1996)
From the University of Cambridge, Department of Haematology,
Medical Research Council Centre, Hills Road, Cambridge CB2 2QH,
United Kingdom and the The Walter and Eliza Hall
Institute of Medical Research, Melbourne,
Victoria 3050, Australia
The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. The molecular basis for expression of SCL in stem cells and its subsequent modulation during lineage commitment is of fundamental importance for understanding how early "decisions" are made during hematopoiesis.
We now compare the activity of SCL promoters 1a and 1b in erythroid cells and in CD34 positive primitive myeloid cells. SCL mRNA expression in CD34 positive myeloid cells did not require GATA-1. Promoter 1a activity was weak or absent in CD34 positive myeloid cells and appeared to correlate with the presence or absence of low levels of GATA-1. However, promoter 1b, which was silent in committed erythroid cells, was strongly active in transient assays using CD34 positive myeloid cells, and functioned in a GATA-independent manner. Interestingly, RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells. These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. Moreover these observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Stable transfection experiments showed that both core promoters were silent following integration in erythroid or CD34 positive myeloid cells. Our data therefore indicate that additional regulatory elements were necessary for both SCL promoters to overcome chromatin-mediated repression.
One of the principal issues facing modern developmental biology concerns the molecular mechanisms whereby a multipotent stem cell gives rise to a variety of phenotypically distinct differentiated progeny. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for studying this process. Since lineage commitment and differentiation involve alterations in patterns of gene expression, the function and regulation of lineage-restricted transcription factors are of central importance for the behavior and subsequent fate of hematopoietic stem cells (1, 2).
The SCL/tal-1 gene (hereafter termed SCL) encodes a lineage-restricted basic helix-loop-helix transcription factor with a pivotal role in the regulation of hematopoiesis. Mice lacking SCL protein die by embryonic day 10 and exhibit a complete absence of all hematopoietic cells (3-6). Previous antisense experiments have suggested that SCL may perform different functions in distinct hematopoietic cell types. Introduction of antisense constructs into a multipotent cell line resulted in reduced proliferation and self-renewal (7), whereas similar experiments inhibited erythroid differentiation of a committed erythroid cell line (8). In addition, loss of SCL function increased apoptosis of a T cell line following serum starvation (9). However, despite its implicit involvement in several physiological processes, target genes for SCL have not yet been identified.
SCL is expressed predominantly in hematopoietic cells, although SCL mRNA and/or protein have also been detected in adult and developing brain together with endothelial cells (10-13). Within the hematopoietic system SCL is expressed in committed erythroid, mast and megakaryocytic cells as well as in interleukin-3 dependent cell lines (10, 12-16). Erythroid differentiation of committed erythroid cell lines is accompanied by up-regulation of SCL mRNA although protein levels actually fall (10, 17). SCL is also expressed in multipotent progenitors prior to lineage commitment. The use of growth factors to induce erythroid differentiation of a multipotent progenitor cell line was accompanied by up-regulation of SCL mRNA (but not protein), whereas induced granulocyte/monocyte differentiation resulted in extinction of SCL expression (18). Indeed, down-regulation of SCL expression not only accompanies, but may actually be required for normal myeloid differentiation, since over-expression of exogenous SCL impaired macrophage differentiation of M1 cells (19).
The mechanisms that regulate this complex pattern of expression are almost completely unknown. Studies of Drosophila embryogenesis have shown that complex patterns of gene expression are frequently specified by autonomous regulatory elements directing expression to distinct cell types (20, 21). Similar mechanisms may operate in vertebrate stem cell systems. However, direct experimental data are lacking and several aspects of Drosophila embryogenesis are very different from post-zygotic vertebrate development. We have therefore chosen to analyze the molecular mechanisms responsible for SCL expression in stem cells and its subsequent modulation during lineage commitment. Such studies should also shed light on the fundamental question of lineage determination during hematopoiesis. Moreover, by studying in detail the regulation of this one gene, we will gain insight into the molecular mechanisms necessary to direct exogenous gene expression in stem cells as well as in specific differentiated hematopoietic cell types.
In view of the powerful experimental tools available for studying
hematopoiesis in the mouse, we have focused on the murine SCL gene. The structure of the murine gene is very similar
to that of the human gene (22-24). Transcription of both genes is driven by two promoters in alternate 5 exons. SCL promoter
1a directs lineage-restricted expression in erythroid cells and is regulated by GATA-1 (8, 25, 26), but the molecular basis for
SCL expression in hematopoietic stem cells or in other
specific cell types is not known. Several lines of evidence suggest
that GATA-1 and GATA-2 regulate both overlapping and unique sets of target genes (27). In addition, a number of observations have implied
that SCL may be regulated by GATA-2 in primitive
hematopoietic cells, a role that is subsequently assumed by GATA-1
following commitment to the erythroid lineage. SCL is almost
invariably co-expressed with GATA-1 and/or GATA-2
in hematopoietic cells (10, 15, 18) and a critical GATA motif in the
SCL promoter has been shown to bind GATA-2 (8). In addition,
the ratio of GATA-2 to GATA-1 mRNA is high in
multipotent cells but reversed during erythoid differentiation (18).
Finally, proerythroblasts from GATA-1 null mice express
SCL at normal levels but GATA-2 mRNA levels
were markedly up-regulated (27).
Taken together these data suggest that GATA-1 and GATA-2 may act through the same regulatory elements to direct SCL expression in distinct human cell types. To address this issue, we have compared the mechanisms responsible for SCL expression in erythroid cells with those operating in primitive myeloid cells which express the stem cell antigen, CD34. Our results show that very different mechanisms are responsible for transcriptional activity of the SCL promoter in CD34 primitive myeloid cells compared with erythroid cells. Moreover, in the CD34 positive cells, transcriptional activity of the SCL promoter region was independent of GATA motifs. This suggests that GATA-2 does not direct SCL promoter activity in primitive hematopoietic cells, an observation with important implications for the hierarchy of transcription factors operating in hematopoietic stem cells.
The murine CD34 positive myeloid cell lines M1 and 416B, the murine T-cell line BW 5147, and the murine erythroid cell line J2E have been described previously (25, 28-30). Cell lines M1, 416B (kindly provided by Dr. T. Enver, LRF Center, Institute of Cancer Research, London, United Kingdom), and J2E (kindly provided by Dr. P. Klinken, Biochemistry Department, Royal Perth Hospital, Western Australia) were grown in RPMI 1640 plus 10% fetal calf serum and the BW 5147 and F4N cell lines in Dulbecco's modified Eagle's medium plus 10% fetal calf serum.
PlasmidsSCL promoter sequences in all
constructs are from a genomic clone isolated from a Balb/C genomic
library (24) and have been subcloned into the pGL-2 basic luciferase
reporter vector (Promega) as described previously (25). The numeric
nomenclature of the different constructs refers in all cases to the 5
end of exon 1a (GenBank accession number U05130[GenBank]; position 551). pEF-BOS lacZ contains the lacZ gene under the control of the
pEF-promoter (31) and the pA3RSVluc positive control luciferase plasmid
contains the enhancer and promoter of the 3
-long terminal repeat of
Rous sarcoma virus and were kindly provided by Dr. K. Chatterjee
(Department of Medicine, Cambridge University, United Kingdom). The
construction of pT7 SCL 1b is described under
"Ribonuclease Protection Assay." PEF-BOS GATA-1 has been described
(25) and pCDNA 3 expression vector was purchased from
Invitrogen.
Site-directed mutants were
created using the Kunkel method (32) or the protocol described by Wong
and Komaromy (33). The following oligonucleotides were used as primers
in the mutagenesis reaction: 37 GATA,
5
-CGTGTGCGCCGCCGAATTCAGGAGCCGCCTCGGG-3
;
69 GATA,
5
-GGCCCGCCCGCCCCCATTCAGCGCCTCGGCCATT-3
;
63 SP1,
5
-CCGAGATAAGGAGCCCGCCTCGGGCCCGAATTCCCCCGATAAGCGCCTCGGCC-3
;
101 AP1,
5
-GGCCATTATGGGCCAAATGAATTCTTTTAATTTGGCAATTTC-3
; +242 MAZ,
5
-AGTCTCCCCGGCTGCAGGGGCAGCGGAGGG-3
; +259 ETS,
5
-GCGCAAGGGGAAAAGGGGGAGCTCGGAAGAGAGTCTCCCC-3
. All mutations
were subsequently confirmed by nucleotide sequence analysis.
Furthermore, gel shift assays were used to demonstrate that the
mutations did not introduce new protein-binding sites.
DNA methylation analysis has been described in detail (25).
Transient Transfection of Cell LinesTransient transfection were performed as described (25) with the difference that during the electropulse voltages of 260 V (for the J2E cell line) and 240 V (for the M1 and 416B cell lines) were applied.
-Galactosidase
and luciferase assays were carried out as described (25). A linear
relationship between light units and amount of luciferase reporter
plasmid was confirmed in all cell lines. In the transfection
experiments 10 µg of pEF-BOS lacZ vector and 15 µg of
the appropriate luciferase reporter construct were used. In the
co-transfection experiments, 15 µg of the pEF-BOS GATA-1 expression
vector (25) or 15 µg of pCDNA 3 empty expression vector
(Invitrogen) were used together with 10 µg of pEF-BOS lacZ vector and 15 µg of the
2000 1a luciferase reporter construct. For
each pulse the relative light units were normalized for transfection efficiency against
-galactosidase values obtained from the same sample. The relative light units values presented are the mean of at
least four independent experiments and the same pattern was also
obtained using a different DNA preparation.
3 × 107 F4N or M1 cells were electroporated (960 microfarads, 250 V) with 3 µg of linearized phosphoglycerate kinase gene promoter puromycin poly(A) vector (kindly provided by Dr. S. Cory, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) and a 10-fold molar excess of the linearized luciferase plasmid. After 24 h, electroporated cells were transferred into selective medium containing 2 µg/ml (F4N) or 10 µg/ml (M1) puromycin (Sigma). Cells electroporated with SCL reporter constructs were maintained as three independent pools.
Puromycin resistant pools were derived 2-4 weeks following electroporation and luciferase assays were performed using extracts derived from 3 × 105 cells for each assay point as described above. For each experiment, assays were performed in duplicate on each pool and a positive control (a pool of cells transfected with pA3RSVluc) and a negative control (a pool of cells transfected with pGL-2 basic) were included in each experiment. This experiment was repeated on three separate occasions for each SCL construct. Results were expressed as fold elevation over the negative control. In addition, this relative luciferase activity was normalized for luciferase DNA content by Southern blot analysis of BamHI and HindIII digested DNA. Filters were probed with a nonrepetitive sequence from the mouse vav gene locus1 to normalize DNA loading and with the HindIII/BamHI fragment of the luciferase gene from pGL-2 basic. Relative luciferase values were also corrected by normalizing for luciferase DNA content of test pools compared with the control pool carrying pGL-2 basic.
Electrophoretic Mobility Shift AssaysConditions for the band shift assays were as described (25). Oligonucleotides used in the band shift assays are depicted in Table II. The N6 anti-GATA-1 antibody (34) was kindly provided by Dr. G. Partington, Randall Institute, London, United Kingdom.
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Poly(A)+ RNA was isolated from cell lines and blotted as described previously (10). Filters were sequentially hybridized to the following probes: murine SCL, a 1.8-kb2 XbaI cDNA fragment (35); murine GATA-1, full-length cDNA (36); murine GATA-2, 0.7 kb cDNA fragment kindly provided by Prof. S. H. Orkin (Howard Huges Medical Institute Boston, MA); murine GATA-3, full-length cDNA kindly provided by Prof. H. Clevers (Utrecht University, Utrecht, The Netherlands); murine CD34 cDNA kindly provided by Dr. T. Enver (Institute for Cancer Research, London, United Kingdom); and human GAPDH (37).
RNase Protection AssayTotal cellular RNA was prepared
using the RNAzol method according to the manufacturer's protocol
(Bio/Gene, Bolnhurst, United Kingdom). Genomic murine SCL
fragments encompassing nucleotides 760-1156 (24) were inserted in
antisense orientation into the pT7 vector (38) to generate pT7
SCL 1b. Radiolabeled antisense transcripts were generated by
transcription in vitro using T7 RNA polymerase (Promega).
Ribonuclease protection assays were performed using the Ambion RPA II
kit (Ambion, Austin, TX) and 40 µg of total RNA for each protection.
The amount and integrity of each RNA preparation was confirmed using
the pTRI--actin mouse plasmid (Ambion) which contains a
250-bp mouse
-actin gene fragment in antisense
orientation and gives rise to a 304-nucleotide transcript. The size of
the corresponding protected fragments was determined by running a
sequencing reaction alongside the protected fragments.
SCL mRNA is expressed in
committed erythroid, mast, and megakaryocytic cells as well as in
multipotent stem cells (10, 14-16, 18). Since it is difficult to
obtain sufficient numbers of normal stem cells for biochemical
experiments, we have chosen to study two primitive myeloid cell lines.
Both of these cell lines expressed mRNA for the hematopoietic
"stem cell" antigen CD34 (Fig. 1). 416B and M1 cells
also expressed SCL mRNA, as did two erythroid lines (F4N
and J2E). The two erythroid lines expressed high levels of both
GATA-1 and GATA-2 mRNA. By contrast,
GATA-1 mRNA was undetectable in M1 cells and only very
low levels were observed in 416B cells. These results are in accord
with previous data (14, 16, 39) and suggest that SCL
mRNA expression in primitive myeloid cells may not require
detectable GATA-1 expression.
SCL Promoter 1a Activity in Erythroid and CD34 Positive Primitive Myeloid Cells
We have previously demonstrated that SCL promoter 1a is active in a murine erythroleukemia cell line but not in a murine T cell line (25). Since murine erythroleukemia cell lines cannot be equated with normal erythroid cells, it was important to confirm the generality of our results using an alternative erythroid line, which had been generated in a different manner. For this purpose it was decided to study the J2E cell line, which was derived in vitro by infection of fetal liver with a retrovirus containing myc and raf oncogenes (30). J2E cells are growth factor independent but undergo terminal differentiation in response to erythropoietin.
Luciferase reporter constructs containing SCL promoter 1a
were introduced into J2E and a murine T cell line (BW 5147) in
transient assays. A -galactosidase control plasmid was also included
to control for variation in DNA uptake, and luciferase values were normalized against the corresponding
-galactosidase values. A construct, containing approximately 2 kb of exon 1a upstream sequence, was active in J2E cells (34-fold stimulation over background) but not
in BW 5147 (Fig. 2A). Deletion of sequences
between
2000 and
187 did not decrease promoter 1a activity but
further deletion to
55 resulted in significant loss of promoter
activity. The pattern obtained using the promoter 1a deletion
constructs in J2E cells was therefore similar to that previously seen
in murine erythroleukemia cells (25). We therefore conclude that
sequences between
187 and +26 were sufficient for lineage-restricted
activity of promoter 1a in both murine erythroleukemia cells (F4N) and J2E cells.
The same constructs were then used to perform transient assays in 416B
and M1 cells. Luciferase constructs containing approximately 2 kb of
sequence upstream of promoter 1a were weakly active in both 416B and M1
cells (Fig. 2A). Deletion of sequences between 2000 and
187 upstream of promoter 1a had no effect. By contrast, further
removal of sequences between
187 and
55 significantly reduced
promoter activity. The pattern of promoter activity obtained with the
various constructs in M1 and 416B cells was similar to the pattern
observed in erythroid cells. However, the overall activity of promoter
1a was much weaker in the primitive myeloid cell lines than in J2E
cells.
SCL promoter
1b with 128-bp upstream sequence has previously been shown to be
inactive in both murine erythroleukemia cells (F4N) and T cells (25).
Since murine erythroleukemia cells contain activated ETS family members
and since a potential ETS binding motif is present upstream of promoter
1b, it was especially important to confirm the inactivity of promoter
1b in a different erythroid cell line. As shown in Fig. 2B,
a construct containing both promoters together with 2 kb of DNA
upstream of promoter 1a, was active in J2E cells but not in BW 5147 cells. The activity of this construct (2000 1a1b, Fig. 2B)
was similar to that observed with an analogous construct containing
promoter 1a alone (
2000 1a, Fig. 2A). Moreover, the
activity of
2000 1a1b in J2E cells was abolished by removal of part
or all of promoter 1a, and a construct containing promoter 1b alone
(with 128 bp of upstream sequence) was inactive. This pattern of
activity mirrored that previously observed in F4N cells (25).
Very different results were obtained in M1 and 416B cells (Fig.
2B). In both cell lines, 2000 1a1b exhibited much higher luciferase levels than
2000 1a (40-95-fold over background compared with 5-12-fold over background). Moreover, deletion of part or all of
promoter 1a did not reduce the activity. Indeed the +209 1b construct,
which only contained 128 bp upstream of exon 1b, was still fully
active. Deletion of promoter 1a removed the GATA sites that are
situated upstream of exon 1a. Since neither +26 1b nor +209 1b
constructs contain any additional GATA motifs, these data demonstrate
that the activity of promoter 1b in myeloid cells was independent of
GATA motifs.
The transient transfection assays described above suggest that promoter 1a is active in erythroid cells and promoter 1b in primitive myeloid cells. However, such experiments do not take into account the additional effects of distant regulatory elements, methylation status, or chromatin structure. It was therefore important to study the activity of the endogenous SCL promoter region in erythroid and primitive myeloid cells.
The SCL promoter locus is CpG-rich in both human and murine
SCL genes (22-24). Furthermore, in cell lines (but not in
primary cells) the methylation status of CpG-rich promoters correlates with their transcriptional activity (40, 41). It was therefore important to see whether differential promoter usage in distinct cell
types reflected cell-specific differences in promoter methylation. We
therefore analyzed methylation of the SCL promoter region in J2E, BW 5147, 416B, and M1 cells. As shown in Fig. 3,
both SCL promoters are contained within a 0.8-kb
Sau3A fragment. Genomic DNA extracted from the four cell
lines was digested with Sau3A alone or together with the
methylation-sensitive enzyme HpaII and hybridized to the
indicated probe. As anticipated, digestion with Sau3A alone
generated a 0.8-kb fragment in all cell lines (Fig. 3). Addition of
HpaII resulted in complete digestion of the 0.8-kb fragment
in all SCL expressing cell lines (J2E, M1, and 416B), but
not in BW 5147 cells which did not express SCL mRNA.
These data demonstrate that the endogenous SCL promoter was
predominantly unmethylated in both erythroid and primitive myeloid cell
lines, an observation that fully accords with the transcriptional
activity of the SCL promoter region in transient reporter
assays. Moreover, within the resolution of this study, no difference
was observed in the methylation pattern of the SCL promoter
region in erythroid cells compared with primitive myeloid cells.
Reverse transcriptase-polymerase chain reaction has been used
previously to assess endogenous transcription of exon 1a and 1b in
different cell types (15, 24). However, the generation of a polymerase
chain reaction product using a primer specific for exon 1b could
represent either activity of promoter 1b or read-through transcripts
initiating from promoter 1a. RNase protection assays were therefore
used to determine the endogenous activity of the two SCL
promoters in erythroid, CD34 positive myeloid and T cells. A
radiolabeled probe of 449 nucleotides was generated containing part of
exon 1b as well as 117 nucleotides of intronic sequence between exon 1a
and exon 1b (Fig. 4). This probe was annealed with total
RNA extracted from F4N, J2E, 416B, M1, and BW 5147 cells. A murine
-actin antisense transcript which was predicted to
protect a 250-nucleotide fragment was also annealed with the same RNA
preparations as a control for equal loading. SCL transcripts
initiating from promoter 1b were predicted to give rise to a protected
fragment of 280 nucleotides, whereas read-through transcripts that had
initiated in exon 1a would protect a larger 397-nucleotide fragment. As
shown in Fig. 4, a 280-nucleotide fragment was protected in all
erythroid and CD34 positive primitive myeloid lines but not in the T
cell line. In addition, a 397-nucleotide band was detected in both
erythroid lines but not in the T cell or in either primitive myeloid
cell line, although prolonged exposure of the autoradiograph gave rise
to a faint band in the 416B lane (data not shown). It is probably no
coincidence that 416B cells, which express low levels of
GATA-1 mRNA, also exhibited low but detectable activity
of promoter 1a in transient reporter assays together with a low level
of exon 1a transcripts by RNase protection.
Taken together, these data demonstrate that the endogenous promoter 1b was active in both erythroid and primitive myeloid cell lines but that endogenous promoter 1a activity was only readily detectable in the erythroid cell lines. These results accord with the transient transfection data for promoter 1a. However, promoter 1b was strongly active following transient transfection into primitive myeloid cells, but was silent in erythroid cells. These results have two significant implications. First, they suggest that promoter 1b required additional sequences and/or integration in chromatin for its activity in erythroid cells. Second, they demonstrate that the activity of promoter 1b was achieved by very different mechanisms in erythroid and CD34 positive primitive myeloid cells.
Both Core Promoters Were Silent Following Integration in ChromatinIn view of the possibility that promoter 1b may be chromatin dependent and/or require additional regulatory elements in erythroid cells, a series of stable transfection experiments were performed. The core promoter 1a and promoter 1b luciferase constructs were electroporated into M1 and F4N cells. Three independent pools were generated for each SCL reporter construct in each cell type. Luciferase activity of each pool was analyzed on three separate occasions along with luciferase activity of positive and negative control pools.
As shown in Table I neither promoter 1a nor promoter 1b were active after integration in chromatin. By contrast the positive control vector produced high levels of luciferase activity. These results demonstrate that integration in chromatin was not sufficient for promoter 1b activity in erythroid cells. Indeed, integration in chromatin suppressed activity of both promoter 1a in erythroid cells and also promoter 1b in primitive myeloid cells. Our data therefore suggest that additional regulatory elements were necessary for both SCL core promoters to overcome the repressive effect of chromatin.
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The results presented above demonstrate that SCL
promoter 1a and SCL promoter 1b exhibited endogenous
activity in J2E cells. However, only promoter 1a was independently
active in transient assays and it was therefore readily amenable to
further analysis. Previous results from this laboratory (25) have shown
that the 37 GATA site was essential for promoter 1a activity in
murine erythroleukemia cells. By contrast, both the
63 Sp1 and
69
GATA sites were dispensable. In J2E cells, mutation of the
37 GATA site dramatically impaired activity of a
2000 1a promoter construct (Fig. 5A). This result is consistent with
previous studies and underlines the importance of the
37 GATA site
for SCL transcriptional activity (8, 25, 26). No significant
reduction of promoter activity was observed when the
101 AP-1 site
was mutated which accords with our previous findings using F4N cells
(25). However, mutation of the
69 GATA and the
63 Sp1 consensus
binding sites reduced promoter 1a activity in J2E cells from 37-fold
stimulation over background to 19- and 9-fold stimulation, respectively
(Fig. 5A). In F4N cells mutation of the
69 GATA site
produced a modest fall in activity but mutation of the Sp1 motif had
little effect. The contrasting results obtained with Sp1 mutation in
F4N and J2E cells may reflect differences in the stage of erythroid
differentiation or in the complement of genetic changes present in the
two cell lines.
Band shift assays were performed to characterize the proteins in J2E
extracts that bound to the GATA and Sp1 sites. A single strong complex
bound the 37 GATA site (Fig. 6, lane 2).
This was competed by cold probe (lane 3) but not by an
oligonucleotide with a mutated
37 GATA site (lane 4) or an
unrelated oligonucleotide (lane 5). The complex was shown to
contain GATA-1 since a monoclonal antibody to GATA-1 produced a clear
supershift (lane 6).
Two major complexes in J2E extracts bound an oligonucleotide containing
both 63 Sp1 and
69 GATA sites (Fig. 6, filled arrows). Binding of both complexes was completed by cold probe (lane
9) but not by an unrelated oligonucleotide (lane 10).
The upper complex bound to the
63 Sp1 concensus site and contained a
member of the Sp1 family of transcription factors. Complex formation
was not competed by an oligonucleotide in which the Sp1 site was
mutated (lane 11) but was competed by an Sp1 consensus
oligonucleotide (data not shown). Moreover, the complex co-migrated
with recombinant Sp1 protein (data not shown). The lower complex
contained GATA-1: binding was competed by two different
oligonucleotides each containing consensus GATA motifs (lanes
12 and 13), but not by an unrelated oligonucleotide
(lane 10) or by an oligonucleotide in which the
69 GATA
site was mutated (lane 15). Furthermore, the N6 GATA-1 monoclonal antibody supershifted this complex (compare lanes
14 and 15). Since the GATA-1 specific supershift
co-migrated with the upper complex, the supershift was performed in the
presence of cold oligonucleotide, containing a mutated GATA-1 site but an intact Sp1 site, to compete the upper Sp1 complex.
A more rapidly migrating complex was evident in the presence of the N6 antibody (lanes 6 and 14). This complex was also found in the absence of antibody or competitor oligonucleotide in lane 8 and, after prolonged exposure of the autoradiograph, in lane 2. This complex has been observed previously (36, 42). Its nature was not specifically addressed in our experiments, but it is likely to contain either a smaller proteolytic product of GATA-1 or the product of an alternative AUG start codon, as recently reported by Calligaris and colleagues (43). Both products will have lost the binding site for N6 and will therefore not supershift in our band shift assay.
The low/absent activity of promoter 1a in 416B and M1 cells correlated
with the low/absent expression of GATA-1 mRNA. However, both 416B and M1 cells expressed GATA-2 (Fig. 1) and it has
been suggested that GATA-2 may be able to substitute for GATA-1 in directing SCL expression in primitive hematopoietic
progenitors (18, 27). Moreover, recombinant GATA-2 protein has been
reported to bind to the SCL promoter (8). Inactivity of
promoter 1a in 416B and M1 cells could therefore reflect either absence
of GATA-2 binding to the SCL promoter or failure of bound
GATA-2 to transactivate the SCL promoter. Band shift
analysis using oligonucleotides containing the 37 or the
69 GATA
site revealed no specific binding to either GATA site in M1 cells (data
not shown), thus suggesting that endogenous GATA-2 does not bind to
promoter 1a GATA sites in M1 cells. Furthermore, co-transfection of
GATA-1 with SCL
2000 1a produced a 2-3-fold increase in
luciferase activity (Fig. 7). These findings suggested
that GATA-1 expression in M1 cells is sufficient to allow activity of
SCL promoter 1a. It is therefore tempting to speculate that
the low/absent SCL promoter 1a activity in 416B and M1 cells
is, at least in part, due to low/absent GATA-1 expression in these cell
lines.
SCL Promoter 1b Activity in CD34 Positive Primitive Myeloid Cells Required MAZ and ETS Motifs
The experiments described above have demonstrated that the activity of promoter 1b was achieved by very different mechanisms in erythroid and CD34 positive primitive myeloid cells. To dissect these mechanisms we have begun to characterize the transcription factors that regulate promoter 1b.
Previous sequence comparison of the human and murine promoter 1b has
revealed a highly conserved region upstream of exon 1b (25).
Furthermore, a MAZ-binding site at +242 and overlapping tandem ETS
motifs at +264 were necessary for full transcriptional activity of
constructs containing both promoter 1a and promoter 1b in F4N cells
(25). Since promoter 1b was independently active in the primitive
myeloid cell lines, mutations of the MAZ and ETS sites were introduced
into the +209 1b construct. As shown in Fig. 5B, both
mutations markedly reduced activity of promoter 1b in 416B cells and M1
cells. This effect is unlikely to represent the introduction of novel
protein-binding sites since nuclear extracts from J2E, 416B, or M1
cells did not give rise to complexes after incubation with labeled
mutant oligonucleotide (Fig. 8A, lanes 13, 14, and 22).
Band shift analysis was then performed to characterize the proteins binding to the MAZ and ETS motifs. Using an oligonucleotide containing the MAZ site as a probe, specific binding of three major complexes was observed (Fig. 8A). The same three complexes were seen in J2E, 416B, and M1 cells (lanes 2, 7, and 16). Three lines of evidence suggest that either MAZ or a MAZ related protein are present in these complexes. First, all complexes were competed with an excess of an oligonucleotide containing the known ME1a1 MAZ-binding site (44) from the myc promoter (lanes 6, 11, and 20). Second, all three complexes required an intact MAZ site since an oligonucleotide containing a mutated MAZ site could not compete for binding of any of the complexes (lanes 4, 9, and 18). Third, the three complexes co-migrated with complexes obtained using a known MAZ-binding site (ME1a1) as a probe (data not shown).
Band shift assays were also performed to study the proteins binding to the ETS motif. The oligonucleotide used as a probe for this experiment contained three overlapping TTCC motifs. Using J2E, 416B, and M1 nuclear extracts, three main complexes were detected in all three cell lines (Fig. 8B, lanes 2, 6, and 10). Addition of excess cold probe abolished binding of all complexes (lanes 3, 7, and 11), whereas an unrelated oligonucleotide did not compete for binding (lanes 5, 9, and 13). An oligonucleotide in which two of the three ETS binding motifs were mutated still competed for binding of the upper complex (lanes 4, 8, and 12) but not for binding of the lower two complexes. During these experiments it was noted that the addition of an excess of unlabeled oligonucleotide seemed to enhance the binding of the middle complex (asterisk) in all cell lines. We did not further investigate this finding, but one possible explanation would be the presence of an inhibitor which reduces binding of the complex but which is sequestered by the addition of excess cold oligonucleotide. These results suggest that the lower two complexes bound to the TTCCTT sequence that was mutated in the mETS oligonucleotide (Table II), but that the upper complex bound elsewhere within the oligonucleotide.
Taken together these data demonstrate that the MAZ and ETS sites were important for full activity of promoter 1b in primitive myeloid cells. However, a similar pattern of complexes was observed in J2E cells in which transient reporter assays showed promoter 1b to be silent. These observations suggest that the MAZ and ETS sites are necessary but not sufficient for promoter 1b activity, and that additional lineage-restricted transcription factors may be needed for promoter 1b activity in primitive myeloid cells. Nevertheless, our data do not exclude the possibility that transcription factors binding to the MAZ and ETS sites may undergo lineage-restricted post-translational modifications which are not detectable by band shift analysis, or may represent comigrating but functionally distinct transcription factor family members.
The SCL gene is essential for the development of all hematopoietic lineages and is expressed in hematopoietic stem cells as well as committed erythroid, mast, and megakaryocytic cells. Although GATA-1 has been shown to be important for transcriptional regulation of SCL in erythroid cells, the molecular basis for SCL expression in other hematopoietic cell types is unknown. In this paper we have shown that the molecular basis for SCL expression is very different in CD34 positive primitive myeloid cells compared with committed erythroid cells.
Transient reporter assays have previously been used to show that SCL promoter 1a was active in murine erythroleukemia cells but not in murine T cells and that core promoter 1b was inactive in both cell types (25). These studies, together with work from other laboratories (8, 26), have also demonstrated a critical role for GATA-1 in regulating the activity of SCL promoter 1a. Murine erythroleukemia cells cannot be equated with normal erythroid progenitors, not least because the former express ETS family members as a result of retroviral insertions. It was therefore important to confirm the pattern of SCL promoter activity in a different class of erythroid cells. Whereas murine erythroleukemia cell lines are generated by infection of mice with Friend virus, the J2E cell line was generated in vitro by infection of fetal liver cells with a replication incompetent virus carrying raf and myc. Analysis of SCL promoter contructs in J2E cells gave rise to the same pattern of activity (active promoter 1a and silent promoter 1b) as previously found in murine erythroleukemia cells. In addition, GATA sites within promoter 1a bound GATA-1 and were critical for promoter 1a function.
Very different results were obtained when SCL promoter
activity was studied in CD34 positive primitive myeloid cell lines. In
both 416B and M1 cells promoter 1a activity was weak and no specific
transcription factor binding could be detected at the 37 and
69
GATA sites upstream of promoter 1a. However, the most striking
observation was the presence of strong core promoter 1b activity in
both 416B and M1 cells. A series of 5
deletion constructs was used to
show that removal of promoter 1a and the upstream GATA sites did not
reduce activity of promoter 1b. Since the constructs with promoter 1b
alone did not contain additional GATA sites, these data demonstrate
that promoter 1b was functioning in a GATA-independent manner.
GATA-1 plays a central role in the regulation of SCL
promoter 1a in erythroid cells. GATA-1 binds to the functionally
important 37 and
69 GATA motifs and also transactivates promoter 1a
in heterologous cells (8, 25, 26). However, levels of GATA-1 are low in
primitive progenitors (18, 45, 46) and as, shown here,
GATA-1 mRNA may be undetectable in primitive myeloid
cells expressing SCL. Furthermore, in mice lacking GATA-1
protein, erythroid differentiation was blocked at the level of the
proerythroblast, but GATA-1 target genes including SCL were
still expressed at relatively normal levels along with markedly raised
levels of GATA-2 (27). It has therefore been suggested that GATA-2 and GATA-1 regulate both overlapping and unique sets of genes (47). According to this hypothesis, SCL would be regulated by
GATA-2 in primitive hematopoietic progenitors, a role that is taken
over by GATA-1 during erythroid differentiation. Several additional observations are consistent with this scenario: (i) the ratio of GATA-2
to GATA-1 is high in multipotent progenitors and reversed during
erythroid differentiation (18, 45, 46); (ii) SCL and GATA-2 both
regulate self-renewal in multipotent progenitor cell lines (7, 48);
(iii) both SCL and GATA-2 are required for the
survival and/or differentiation of pluripotent stem cells during
development (3-6); (iv) GATA-2 binds to a functional GATA motif in the
SCL promoter (8); (v) GATA responsive promoters can
frequently be transactivated by different GATA proteins (49-52).
Taken together these previous data suggested that GATA-2 regulates SCL in primitive hematopoietic progenitors, and that this is likely to be achieved by GATA-2 interacting with the same regulatory elements that are bound by GATA-1 in erythroid cells. Our results argue against this model and demonstrate that the SCL promoter is active in a GATA-independent manner in CD34 positive primitive myeloid cells. Moreover, these data raise the possibility that initial activation of SCL transcription in pluripotent hematopoietic stem cells may be GATA independent, a speculation that accords with the recent finding that lack of SCL abolishes hematopoiesis even more effectively than lack of GATA-2 (5, 6, 53). However, our results do not address the possibility that GATA-2 may regulate SCL distal regulatory elements in primitive hematopoietic cells.
Transient reporter assays provide valuable information about the transcription factor environment within a cell. However, they do not reflect constraints imposed by chromatin structure or by distant regulatory elements, absent from the reporter constructs. An RNase protection assay was therefore established to investigate the activity of the endogenous SCL promoter in erythroid and primitive myeloid cells. Both promoter 1a and promoter 1b were active in murine erythroleukemia cells and J2E cells. This is consistent with previous reverse transcriptase-polymerase chain reaction data which revealed the presence of transcripts including exon 1a in erythroid cells (15, 24). However, a very different pattern was observed in the two CD34 positive primitive myeloid cell lines, both of which exhibited clear activity of endogenous promoter 1b but weak/absent promoter 1a activity. These results demonstrate differential usage of promoter 1a in different SCL-expressing cell types. Moreover, they suggest that promoter 1b may be the critical site of transcription initiation in stem cells that exhibit low or absent GATA-1 expression.
Our observations also suggest that the mechanisms responsible for promoter 1b activity are fundamentally different in committed erythroid cells compared with CD34 positive progenitor cells. RNase protection assays demonstrated that the endogenous SCL promoter 1b was active in both erythroid and primitive myeloid cells. However, in transient reporter assays the core promoter 1b was strongly active in primitive myeloid cell lines, but silent in erythroid cells. This observation implies that in erythroid, but not in primitive myeloid cells, the core promoter 1b required integration in chromatin and/or additional sequences for its activity.
To distinguish these possibilities stable transfections were performed. These demonstrated that integration in chromatin was not sufficient to allow activity of promoter 1b in erythroid cells. In fact both core promoters were silent following integration in erythroid or myeloid cells. Thus, although some hematopoietic promoters are active after integration into chromatin (54, 55), this is clearly not the case for either SCL promoter. Instead our results strongly suggest that additional regulatory elements are necessary for both SCL promoters to overcome chromatin-mediated suppression.
It will now be important to dissect the different mechanisms responsible for endogenous promoter 1b activity in the two cell types, not least because it may be possible to construct promoter 1b variants that target expression to hematopoietic stem cells. Mutagenesis of promoter 1b has previously demonstrated that the +242 MAZ and +264 ETS motifs were important for full activity of a reporter construct containing both promoter 1a and 1b in erythroid cells. However, it was not clear whether these effects were mediated by promoter 1a or promoter 1b (25). We have now shown that the MAZ and ETS motifs were critical for activity of the core promoter 1b in primitive myeloid cells. Nevertheless, band shift analysis revealed the same pattern of complexes binding to the MAZ and ETS motifs in erythroid and primitive myeloid cells. The simplest interpretation of these results would be that MAZ and ETS sites are necessary but not sufficient for promoter 1b activity in primitive myeloid cells. Alternatively, subtle qualitative differences may exist between the comigrating complexes in erythroid and primitive myeloid cells. Thus, closely related transcription factors or post-translational modification may give rise to lineage-restricted functional differences that are not readily detectable by band shift analysis. Distinguishing between these possibilities may allow the construction of promoter 1b variants that, together with distal regulatory elements, will direct expression specifically to hematopoietic stem cells.
We acknowledge the computing expertise of James Gilbert, the excellent technical assistance of Koula Kosmopoulos, and Lorraine Ives for typing the manuscript. We also are grateful for the reagents kindly provided by S. Orkin, S. Cory, P. Klinken, T. Enver, K. Chatterjee, G. Partington, and H. Clevers.