(Received for publication, September 15, 1994; and in revised form, January 19, 1995)
From the
Using J2E cells and the murine -globin
promoter as a model, we have performed the first direct analyses of
erythropoietin (EPO)-activated transcription from defined templates.
The -346 to +26
promoter was shown to
comprise a target for maximal activation. This included a positive role
for a -346 to -107-base pair (bp) domain in J2E cells, but
not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element
within this domain showed that this effect did not require GATA-1
binding. In contrast, a critical role for GATA-1 at a -60-bp
(G)GATAG element was defined by mutagenesis (GGgTAG and TGATAG),
complementation with a synthetic TGATAA element, and the demonstrated
specific binding of GATA-1. Proximal CCAAT(-75) and
CACCC(-90) elements also were shown to contribute to
transcriptional activation in J2E cells, yet exerted quantitatively
distinct effects in the F-MEL system. Based on these results, minimal
[TGATAA]
-TATA and TGATAA-CACCC-TATA promoters
were constructed and assayed in each system. Remarkably, the
[TGATAA]
-TATA promoter, but not the
TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E
cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by
Me
SO in F-MEL cells. These findings suggest that mechanisms
of EPO-induced transcription in J2E cells involve GATA-1 and differ
from chemically activated mechanisms studied previously in F-MEL cells.
Globin induction in J2E cells was not associated with effects of EPO on
levels or nuclear translocation of GATA-1. However, hemoglobinization
was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding
consistent with induction mechanisms that may involve modulated
serine/threonine phosphorylation.
Erythrocyte production in marrow, spleen, and fetal liver
depends upon the exposure of pro-erythroblasts to the glycoprotein
hormone erythropoietin (EPO). ()EPO is known to exert two
general effects on late BFU-e and CFU-e. As a mitogen, EPO promotes the
rapid expansion of CFU-e while inhibiting
apoptosis(1, 2) ; and as a differentiation factor, EPO
is required for the subsequent expression of late erythroid genes
including
- and
-globins(3) , the anion transporter
band 3, and the cytoskeletal protein 4.1(4) . Through the
cloning of the EPO receptor and the reconstitution of its mitogenic
function in heterologous factor-dependent hematopoietic cell lines,
significant progress recently has been made in identifying factors
involved in proliferative signaling(5, 6) . This
includes the recent identification of Jak2 as an essential
receptor-associated protein tyrosine kinase (7) and the
demonstrated association of p85/phosphatidylinositol 3-kinase (8) and Shc/Grb2/mSos-1 (9) with EPO receptor
complexes. In contrast, comparably little is understood concerning
mechanisms of EPO-induced late erythroid gene expression. This is due,
in part, to limitations of model systems. Programs of late erythroid
differentiation can be induced and have been widely studied in Friend
virus-transformed murine erythroleukemia (F-MEL) cells (10) and
human erythroleukemia K562 cells(11) . However, induction
requires exposure to polar planar chemicals (Me
SO,
hexamethylene-bisacetamide), butyrate, or hemin and is not activated by
EPO. Among cell lines which are EPO-responsive, the primary response
typically is proliferative(12, 13) , even among
erythroid lines(14, 15, 16) . Notable
exceptions are provided by SKT6, B6SUtA, and J2E cell lines. SKT6 cells
were derived from splenocytes of mice infected with the anemia-inducing
Friend leukemia virus complex and undergo efficient hemoglobinization
in response to EPO(17) . A requirement for a transient decrease
in c-myb expression during EPO-induced differentiation in SKT6
cells has been defined(18) , and tyrosine phosphorylation
events associated with induced differentiation have been
described(19) . B6SUtA cells were derived from an
interleukin-3-dependent line(20) , and upon exposure to EPO
(3-6 days) at least certain subclones express
-,
-,
-, and
-globin transcripts at ratios consistent with
to
switching(21) . J2E
cells were established via infection of murine fetal hepatocytes in
vitro with the v-raf/v-myc-encoding virus,
J2(22) , and respond to EPO with a rapid accumulation of
-,
-, and
-globin
transcripts, hemoglobinization (30-40% of cells), enucleation,
and differentiation to reticulocytes(23) . Transient decreases
in c-myb transcript levels in J2E cells likewise have been
associated with EPO-induced differentiation.
Using J2E cells as a
model, in the present study we have tested whether EPO-induced
expression of the murine -globin gene involves
direct transcriptional activation at the proximal promoter, and if so,
what cis-elements and trans-factors mediate
induction. These analyses comprise the first study of regulated
transcription from defined erythroid promoter constructs in an
EPO-responsive cell line and were facilitated through our recent
development of a novel transferrin/poly-L-lysine conjugate for
receptor-mediated transfection(24) . The choice of the murine
-globin promoter as a model was based on the rapid
increase in
transcript levels in J2E cells
following EPO exposure(22, 23) . Also, regulatory
elements of this promoter have been well studied in F-MEL cells, thus
allowing for direct comparisons of
promoter
construct activities in an alternate, albeit chemically inducible,
erythroid system.
Within the murine -globin
promoter, a -106 to +26-bp region has been shown in F-MEL
cells to be necessary and sufficient for regulated
transcription(25) , and for enhancement by hypersensitive site
2 (HS2) of the murine
locus control region (LCR)(26) . In
this proximal promoter (and in the majority of
-like globin
promoters (27) ) TATA(-30), CCAAT(-75), and
CACCC(-90) elements occur and contribute to
promoter activity in erythroid and non-erythroid
cells(25, 28) . Decreased activity in F-MEL cells of
promoters mutated at the CACCC element correlates
with loss of binding of both Sp1 and a 44-kDa protein (29) with
binding specificity of the recently cloned erythroid Krüppel-like factor, EKLF(30) .
Me
SO induction in F-MEL cells increases dimethyl sulfate
protection both at the CACCC element (26) and at an imperfect
direct repeat element (
DRE; position -53 to -32 bp)
which contributes to
promoter inducibility in the
F-MEL system(31, 32) . A positive role for a
nonconsensus GATA-1 element, (G)GATAG at -60 bp, also has been
implicated in the expression of a -346 to +26
promoter in F-MEL cells(33) . GATA-1 is an erythroid
member of a Cys-2/Cys-2 zinc finger family of transcriptional
activators, is required for red cell development in chimeric mice, and
binds to cis-elements within essentially all erythroid genes
studies to date (see (34) for review).
The upstream region
of the murine -globin promoter also influences
transcriptional induction. In F-MEL cells an inhibitory domain at
-100 to -250 bp represses transcription 6- and 4-fold
following induction with hexamethylene-bisacetamide(33) . This
repression involves a conserved element at -165 (BB1, which binds
a ubiquitous factor in uninduced cells) and co-localized elements for
GATA-1(-215) and NF1(-250).
Using wild type, mutated,
and synthetic murine -globin promoter constructs, we
presently demonstrate: (i) that the proximal -106 to +26
promoter is sufficient for activation by EPO in J2E
cells; (ii) that maximal transcriptional activation by EPO requires the
-346 to -107-bp promoter domain, as well as intact proximal
CACCC(-90), CCAAT(-75), and GGATAG(-60) elements;
(iii) that mechanisms of EPO-induced transcription in J2E cells from
the distal
promoter, the proximal
promoter, and synthetic promoters comprised of TGATAA and CACCC
elements differ significantly from Me
SO-dependent
mechanisms in F-MEL cells; and (iv) that GATA-1 (or a direct co-factor)
may play a central role in mediating EPO-induced expression of the
-globin gene and possibly additional late erythroid
genes. Levels and cytosolic versus nuclear distributions of
GATA-1 remained constant during EPO-induced differentiation. However,
J2E cells were shown to differentiate in response to okadaic acid and
cAMP-elevating agents, suggesting that serine and/or threonine
phosphorylation events may mediate induction.
Figure 1:
The murine wild type
-globin proximal promoter and derived mutant
constructs. cis-Elements of the murine
-globin promoter are diagrammed together with point
mutations prepared for present transcriptional analyses. (See
``Materials and Methods'' for construction of reporter
plasmids with mutations 1-5.)
COS cells were transfected using Lipofectin reagent (Life
Technologies, Inc.) as described previously(7) .
Chloramphenicol acetyltransferase assays were performed using
[H]chloramphenicol and butyryl-CoA (40) with the following modifications: freeze/thaw extracts
were heated for 10 min at 60 °C; butyryl-CoA concentrations were
increased to 0.3 µg/µl; incubations with substrates were
extended to 16 h; and following TMPD/xylenes extractions, samples were
back-extracted with an equal volume of 0.25 M Tris, pH 7.5.
Figure 2:
Distal sequences of the murine
-globin promoter increase EPO-induced transcription
in J2E cells. A, for comparison and as a control, activities
of p
106, p
340, and p
G215 promoter/CAT reporter
constructs initially were assessed in F-MEL cells (uninduced, solid
bars; Me
SO-induced, fill patterns).
Activities are standardized versus the activity of the
p
106 promoter in uninduced F-MEL cells (1.0 unit = 360
cpm). B, activities of the p
106, p
340, and
p
G215 promoters in J2E cells (uninduced, solid bars;
EPO-induced, fill patterns). Activities are standardized versus the activity of the p
106 promoter in uninduced J2E
cells (1.0 unit = 28 cpm). t test values: p
340 versus p
106, p = 0.07; p
340 versus p
G215, p = 0.40. Results are
representative of
three independent
experiments.
Footprinting
of the above -346 to +426 promoter
domain using extracts from J2E cells demonstrated specific factor
binding to a naturally occurring AGATAA site (consensus GATA-1 element)
at -215 (not shown). Based on this observation, this potential
activating element was mutated within the -346
promoter and the resulting construct (p
215-CAT) was
assayed for transcriptional activity. However, no effect of this
mutation was detectable in either J2E or F-MEL cells, indicating that
alternate distal elements mediate activation in J2E cells of the
-346
promoter.
In contrast, analyses of
the proximal -106 to +26-bp promoter domain indicated an
essential role for GATA-1 at a -60-bp (G)GATAG element. First,
mutation of this element to GGgTAG (p106
G60-CAT)
resulted in a dramatic loss of EPO-induced activity in J2E cells,
whereas mutation to a consensus TGATAG element
(p
106+G60-CAT) increased activity approximately 1.9-fold (Fig. 3A). Second, a synthetic consensus TGATAA element
cloned immediately 5` to the -106 promoter was shown to increase
promoter activity approximately 5-fold and to functionally complement
the -60 GGgTAG mutant (Fig. 3B). Third, in
non-erythroid COS cells, mutation of this -60 (G)GATAG element
had little or no effect on
106 promoter activity, indicating that
activation at this element is erythroid-specific (Table 1).
Finally, while GATA-1 binding at the -60 element was not detected
by footprinting, or by gel mobility shift using a -72 to
-48-bp promoter fragment, sequence-specific binding of GATA-1
(from J2E cell extracts) to a
P-labeled -106 to
+26 promoter was demonstrated via its co-immunoprecipitation with
GATA-1/N-6 complexes (Fig. 3C). This -60 (G)GATAG
element does not overlap with any previously identified
-globin element (including the adjacent
DRE at
-53 to -32)(31, 32) , and corresponds in
sequence to an element defined by polymerase chain reaction binding
site selection and transactivation assays in NIH 3T3 cells as a
functional GATA-1 element(47) . In F-MEL cells a requirement
for this -60 GGATAG element recently has been demonstrated within
a -346
promoter construct (33) and is
supported further by activity data in Me
SO-induced F-MEL
cells for these five presently studied constructs (Table 2).
Figure 3:
Requirement for the -60 GGATAG
element in EPO-induced transcription at the -106 promoter. A, effects on transcriptional activity of
mutating the -60 GGATAG element within the -106
promoter were assayed in EPO-induced J2E cells:
p
106
G60, GGgTAG mutant; p
106+G60, TGATAG mutant. B, placement of a synthetic TGATAA element for GATA-1 5` to
the above -106 promoter increased activity approximately 5-fold,
and functionally complemented the GGgTAG mutant (pGATA106
G60 versus pGATA106). Activities are standardized versus the activity of p
106-CAT in EPO-induced J2E cells. C, binding of GATA-1 to the -60 GGATAG element of the
wild type -106
promoter. Specific binding of GATA-1 to the
wild type (WT) versus a control -60 GGgTAG
mutant promoter (
G) was assayed by co-immunoprecipitation
of
P-end labeled promoter fragments with mAb N-6 and
GATA-1 from nuclear extracts of J2E cells. Specificity of binding was
assessed further by competition with TGATAA versus AP-1
oligonucleotides. Values represent averages minus background (i.e.
P-promoter binding in the absence of antibody) and
are representative of five independent
experiments.
Using the above GATA106 -globin promoter
construct, relative contributions of the proximal -90 CACCC and
-75 CCAAT elements to transcription as induced by EPO in J2E
cells versus Me
SO in F-MEL cells were assessed.
Consistent with previous studies(25, 28) , mutation of
either element markedly decreased levels of Me
SO-induced
transcription in F-MEL cells ( Fig. 4and Table 2). In J2E
cells, however, loss of EPO-induced activity was considerably greater
for the -75 CagAT mutant (25-fold decrease) than for the
-90 aACCC mutant (6-fold decrease). Thus, these results
are consistent with differences in the roles for CCAAT and CACCC
binding factors in EPO- versus Me
SO-induced
transcription.
Figure 4:
Activities of -90 CACCC and
-75 CCAAT GATA106 promoter mutants.
Activities of -90 aACCC (pGATA106
CACCC) versus -75 CagAT (pGATA106
CCAAT) mutants within the
GATA106 promoter were assessed in
Me
SO-induced F-MEL cells (A) and EPO-induced J2E
cells (B). For each cell line, the activity of pGATA106 also
was assayed.
Figure 5:
EPO-induced transcription in J2E cells
from a [GATA]-TATA promoter. A,
activities of minimal GATA-CACCC-TATA and
[GATA]
-TATA promoters relative to the wild type
106 promoter in uninduced (solid bars) and
Me
SO-induced F-MEL cells (fill patterns).
Induction (activities in induced versus uninduced cells) also
is indexed for each construct. Levels of induction are standardized versus values for a control template, pCAT-basic. B,
activities and induction (versus pCAT-basic) of
GATA-CACCC-TATA and [GATA]
-TATA promoters in
control (solid bars) and EPO-exposed J2E cells (fill
patterns). (t testing for
p[GATA]
-TATA versus pGATA-CACCC-TATA, p = 0.03.)
Figure 6:
EPO-induced globin expression in J2E cells
is not associated with increased expression or enhanced nuclear
localization of GATA-1. A, levels of GATA-1 and
- and
-globin transcripts in J2E
cells were assayed by Northern blotting following exposure to EPO (5
units/ml; 0-64 h). Actin mRNA was assayed as a control. B, GATA-1 protein levels in J2E cells (5 units/ml EPO;
0-8 days) were assayed by Western blotting (whole cell lysates). C, GATA-1 levels in J2E cell cytosol versus nuclei
were assayed by Western blotting. Efficient separation of cytosolic versus nuclear fractions was confirmed by Western blotting
with antisera to p85/phosphatidylinositol 3-kinase and lactate
dehydrogenase (cytosolic markers) and topoisomerase II (nuclear
marker). M
standards are indexed in the left
margin (B and C).
Figure 7:
Rates of nuclear translocation of GATA-1
in control versus EPO-induced J2E cells. Following exposure to
EPO (-/+ EPO), J2E cells were labeled metabolically with
TranS-label and were chased with methionine and cystine
for 4 h in the presence (or absence) of EPO.
S-GATA-1 then
was immunoprecipitated from cytosol and nuclei using mAb N-6 and was
assayed by SDS-PAGE and autoradiography. EPO exposure (+ lanes)
resulted in a reproducible 1.2-1.5-fold increase in ratios of
nuclear:cytosolic
S-GATA-1 (range of five independent
experiments;
-scope analysis). This effect required 42-h EPO
exposure and was independent of the length of chase (0.5-4 h). M
standards are indexed in the right
margin.
Figure 8: Hemoglobinization of J2E cells in response to okadaic acid, forskolin, and 8-Br-cAMP. Hemoglobinization of J2E cells (2,7-diaminofluorine (DAF) positive cells, percent of total) was measured following 60-h exposure to the following agents: A, okadaic acid, EPO; B, isobutylmethylxanthine (IBMX), 8-Br-cAMP, forskolin, EPO. Values represent means ± S.D. for 200 cells and are representative of three independent experiments.
Mechanisms by which the glycopeptide hormone erythropoietin
(EPO) induces the coordinate expression of late erythroid genes in
pro-erythroblasts are poorly understood. Using EPO-responsive J2E cells (22, 23) and the murine -globin
promoter as a model, we presently have examined mechanisms of induction
by this hematopoietin. These studies comprise the first direct analyses
of cis-elements which mediate induction by EPO and involved
the development of a novel nontoxic conjugate for
transferrinfection(24) . In this system, the proximal
-106 to +26
promoter is shown to
comprise a direct target for activation by EPO, with a requirement for
the -346 to -107 distal domain for maximal activity.
Elements within the distal -346 to -107-bp
domain; the proximal -106 to +26-bp domain; and simple
synthetic constructs that mediate EPO-induced transcription in J2E
cells are defined and are shown to differ significantly from those
which mediate Me
SO-induced transcription in F-MEL cells.
Also, an important role for GATA-1 in EPO-induced
transcription at a -60 (G)GATAG element is indicated, and
efficient activation by EPO of a [TGATAA]-
TATA
promoter in J2E cells is demonstrated. These latter findings suggest
that EPO-induced transcription at the
promoter, and
potentially other late erythroid genes, may involve the activation of
GATA-1 (or possibly a direct co-activator). EPO-induced differentiation
in J2E cells is not associated with significant effects on GATA-1
levels, nuclear versus cytosolic compartmentalization, or
rates of nuclear translocation. However, the observed induction of
hemoglobinization in J2E cells by forskolin, 8-Br-cAMP, and okadaic
acid is consistent with the possible involvement of serine/threonine
phosphorylation pathways, potentially including the modulated
phosphorylation of GATA-1.
Given the complex sets of cis-
and trans-factors known to coordinately regulate globin gene
expression (52) together with the diverse sets of pathways
known to be activated by EPO(5, 6) , it is perhaps
remarkable that the minimal -106 to +26 promoter per se is activated efficiently by EPO in J2E
cells. The further positive effect exerted by the upstream -346
to -107 bp region of this promoter in J2E cells also is of
interest in that cis-elements within this promoter domain,
including a BB1 element at -165, previously have been shown to
repress transcription in F-MEL cells(33) . In J2E cells,
specific positively acting elements within the -346 to
-107-bp region have not yet been identified. Comparisons with
activating sequences in the human
-globin promoter in F-MEL cells (53) suggest that this effect may be exerted by a consensus
-250-bp NF1 site or a conserved -165-bp element (with
activation by CP1 versus BB1 repression), whereas a possible
role for a consensus AGATAA element at -215 (which footprints
using nuclear extract from J2E cells) was excluded via mutagenesis
studies (Fig. 2). In the human epsilon globin promoter a
-165-bp consensus GATA element which lacks proximal promoter
function has been demonstrated to mediate enhancer
activity(54) , and the -215-bp AGATAA element of the
murine
-globin promoter might be speculated to serve
a similar function.
In contrast, GATA-1 is indicated to play an
essential role in EPO-induced transcription at the proximal
-globin promoter via a nonconsensus (G)GATAG
element at -60 bp. Mutation of this element to GGgTAG
dramatically inhibited activity in EPO-induced J2E cells (but not in
non-erythroid cells), whereas mutation to TGATAG (GATA-1
consensus) increased activity 1.9-fold. Also, an upstream synthetic
TGATAA element (pGATA106-CAT) complemented this -60 GGgTAG
mutation; and specific binding of GATA-1 to the wild type GGATAG
element was demonstrated (Fig. 3). Notably, a promoter comprised
of (G)GATAG elements and a TATA box recently has been
demonstrated to be efficiently transactivated in fibroblasts by GATA-1
(but not GATA-3)(47) . The present studies demonstrate
activation from this nonconsensus (G)GATAG element within a wild
type promoter in response to EPO. Transcriptional assays in F-MEL cells
of presently constructed GGgTAG and TGATAG mutants and
previously of a GGATAGAG
GctgcagG mutant within the
-346 to +26 promoter (33) further demonstrate a
requirement for the -60-bp (G)GATAG element. Cowie and Myers (25) and Charnay et al.(28) , however, failed
to detect effects of either a GaATAG point mutation (25) or a linker scanning mutation within this -60
(G)GATAG element (CACAGGATAG
CcagatcTgG)(28) . In these studies a 300-bp
Friend virus long terminal repeat or the 3` human
-globin gene
enhancer was included to increase transcriptional activity. Thus,
effects of these mutations may have been complemented by functional
GATA elements within these enhancers (as illustrated presently by the
ability of an upstream TGATAA element to complement a -60
GGgTAG mutation).
In the course of this study we also have
defined several apparent differences in cis-elements which
mediate murine promoter activation in J2E/EPO versus F-MEL/Me
SO differentiation pathways. First,
as mentioned above, the distal -340 to -107 domain was
found to increase promoter activity in J2E cells, yet repressed
transcription in F-MEL cells (Fig. 2). This distinction may be
due to differential levels and/or activities of CP1, NF1, or the BB1
repressor protein. Second, within the proximal -106 to
+26-bp
promoter, CACCC(-90) and
CCAAT(-75) elements each contributed to the activity of a GATA106
promoter in J2E and F-MEL cells. However, in J2E cells CCAAT mutation
(CagAT) inhibited EPO-induced transcription more markedly than
CACCC mutation (aACCC) ( Fig. 4and Table 1). Thus,
the -90 CACCC element appears less important for EPO-induced
transcription in J2E cells than for
Me
SO-induced transcription in F-MEL cells. Third, this
finding is substantiated by the efficient activation of a minimal
synthetic GATA-CACCC-TATA promoter in F-MEL cells versus nominal activation of this promoter by EPO in J2E cells (Fig. 5). Although these effects are consistent with direct
action at the GATA106 and TGATAA-CACCC-TATA promoters of a dominant
CACCC binding factor in F-MEL cells (i.e. Sp1 or
EKLF(29, 30) ), a CACCC-TATA promoter was found to be
insufficient for Me
SO activation (48) . (
)By comparison with the porphobilinogen promoter, the
spacing of TGATAA and CACCC elements in the GATA106 and GATA-CACCC-TATA
promoters (i.e. 35 bp) is optimal for
co-activation(55) . Thus, a possible explanation for
differences in activities of these promoter constructs in J2E versus F-MEL cells is that GATA-1 may function predominately
as a co-factor (e.g. EKLF/Sp1)-dependent activator in F-MEL
cells and as a direct activator in the J2E/EPO pathway. The finding
that a [GATA]
-TATA promoter is efficiently
induced (>75-fold) in J2E cells following EPO exposure, yet is less
highly induced (approximately 10-fold) by Me
SO in F-MEL
cells (Fig. 5) is consistent with this model and, importantly,
indicates that transcriptional activation by GATA-1 is induced directly
and efficiently by EPO.
EPO-induced transcription of
-globin promoter constructs, however, was not
explained by induced increases in levels of GATA-1 in J2E cells (Fig. 6), as has been suggested for responsive SKT6
cells(49) . Enriched early progenitor cells have been shown to
accumulate GATA-1 transcripts upon exposure to interleukin-3 as well as
EPO, yet require exposure to EPO for subsequent terminal
differentiation(56) . This observation in normal
pro-erythroblasts likewise suggests that possible effects of EPO on
GATA-1 levels are not sufficient to promote late erythroid gene
expression.
For certain alternate trans-factors which
mediate the action of extracellular growth and differentiation signals,
including ISGF-3, NFB/rel, and Swi-5, regulated nuclear
translocation (especially via regulated phosphorylation) recently has
emerged as a common alternate activating mechanism(57) .
Because GATA-1 is present in cytosol at significant levels, possible
effects of EPO on GATA-1 translocation in J2E cells were assessed.
However, nuclear and cytosolic distributions of GATA-1 were not
affected by EPO, and rates of nuclear translocation were enhanced at
most 1.5-fold following 48 h of exposure (Fig. 6C and Fig. 7).
In J2E cells, F-MEL cells, and human erythroleukemia
K562 cells, GATA-1 occurs as a
phosphoprotein(50, 51) . ()Thus, modulated
phosphorylation (in the absence of induced increases in GATA-1 levels
or nuclear translocation) represents an alternate possible mechanism
for direct induction of the [TGATAA]
-TATA
promoter by EPO in J2E cells. Using the monoclonal antibody N-6,
attempts were made to map
P-phosphotryptic GATA-1 peptides
from control versus EPO-exposed J2E cells. However, this was
complicated by the poor solubility and limited recovery of purified
GATA-1. Consequently, activators of serine/threonine phosphorylation
pathways were tested for the ability to activate globin expression in
J2E cells. Forskolin, 8-Br-cAMP, okadaic acid, and to a lesser extent
isobutylmethylxanthine each were shown to induce hemoglobinization (Fig. 8). Induced increases in cAMP previously have been shown
to promote hemoglobinization in EPO-responsive SKT6 cells (58) and to enhance EPO-dependent activation in committed TSA8
cells(59) . While these agents possibly may act via
EPO-independent pathways, these results are at least consistent with a
role for modulated serine/threonine phosphorylation, potentially
including the regulated phosphorylation of GATA-1 by EPO. Possible
significance of GATA-1 phosphorylation at conserved sites presently is
being addressed via directed mutagenesis. Also, a recently developed
cell-free transactivation system for the assay of direct and
co-factor-dependent GATA-1 transactivation should facilitate these
studies(51) . Notably, EPO-induced hemoglobinization of J2E
cells, as well as normal murine CFU-e, occurs over an extended time
course (4-48 h). Therefore, mechanisms beyond the possible
modulated phosphorylation of primary effectors also likely contribute
to the regulated expression of late erythroid genes.