(Received for publication, April 3, 1995; and in revised form, May 18, 1995)
From the
The Id proteins function as negative regulators of
basic-helix-loop-helix transcription factors, which play important
roles in determination of cell lineage and in tissue-specific
differentiation. Down-regulation of Id1 mRNA is associated with
dimethyl sulfoxide-induced terminal differentiation of mouse
erythroleukemia cells. To examine the significance of Id1
down-regulation in erythroid differentiation, we generated stable mouse
erythroleukemia cell lines that constitutively express a
``marked'' form of the murine Id1 gene. Terminal
erythroid differentiation was inhibited in these lines, as indicated by
a block in activation of the erythroid-specific genes The mouse erythroleukemia (MEL) Members of the bHLH family share a
common amino acid sequence motif comprising a basic region just
amino-terminal to an amphipathic In this paper, we explore
the functional significance of Id1 down-regulation in differentiating
erythroid (MEL) cells. MEL cell lines that constitutively express
functional Id1 protein fail to undergo terminal differentiation; both
uninduced and induced levels of The
effect of unregulated expression of Id1 was not a promiscuous
inhibition of erythroid gene expression, as transcription of several
erythroid-specific genes (the erythropoietin receptor and the
transcription factors GATA-1, NF-E2, and EKLF) was not affected. In
contrast, a novel erythroid-enriched gene, JP19, was
activated, raising the possibility that Id1 forms heterodimers not only
with positive regulatory bHLH proteins but with repressors as well.
Continued expression of Id1 had no effect on formation or stability of
DNase I hypersensitive (HS) site HS2 within the
Figure 1:
Structure of the
Id1-er expression constructs. A, structure of EMSV-Id1-er(S).
The Id1 coding sequence was ligated to the hormone-binding domain of
the ER, yielding a predicted Id1 protein containing an additional 21
amino acids (a termination codon is located at position 22; see
``Experimental Procedures''). The chimeric gene was cloned
into the expression vector EMSVscribe, in the sense (shown here) and
antisense orientations. The vector contains the MSV long terminal
repeat and SV40 splice and polyadenylation signals (Davis et
al., 1987). The position of the sequence used to generate an
ER-specific probe for RNase protection is indicated by a smallhatchedboxbelow the map. B, junction sequence formed by ligation of the Id1 with the
HE14 ER fragment. The carboxyl-terminal arginine (R) of Id1 is circled, and the additional base that generated the frameshift
is indicated by a lowercaseg.
Id1-er and
Id1 The chimeric Id1 genes were
introduced into MEL cells and G418
Figure 2:
Id1-er RNA and protein expression in
stably transfected MEL cell lines. A, expression of endogenous
Id1 and exogenous Id1-er RNA in control (parental) and transfected MEL
cells. Northern blots were prepared using total cellular RNA (15
µg) from uninduced MEL cells (lane1, control; lanes6, 11, and 16 for
Id1-er
Figure 3:
Expression of erythroid-specific genes in
Id1-erexpressing and -non-expressing MEL cell lines. MEL cell lines
were cultured for 4 days in the absence (odd-numberedlanes) or in the presence (even-numberedlanes) of Me
We next examined the
consequences of deregulated Id1 transcription on the expression of a
variety of genes using an RNase protection assay (Fig. 3). The
basal (uninduced) level of expression of Because Id1 is
normally expressed in uninduced, cycling MEL cells, we considered the
possibility that artificially raising Id1 levels might have caused a
de-differentiation, which would preclude erythroid-specific gene
expression. To address this question, we examined the expression of
other genes, including transcription factors known to regulate the
activity of erythroid-specific enhancers and/or promoters.
Transcription of JP19, a novel erythroid-enriched gene, An alternative explanation for the absence of
Interestingly, the Id1-er-expressing lines were able to proliferate
continuously when grown in medium containing Me
Figure 4:
A, down-regulation of c-myc and
transient down-regulation of the lineage-specific transcription factors
GATA-1 and NF-E2 in parental and Id1-er-expressing MEL cell lines. RNA
samples from the experiment shown in Fig. 2A were
analyzed for expression of c-myc, GATA-1, and NF-E2 using an
RNase protection assay. In all cell lines, c-myc was
down-regulated within 2 h after addition of Me
Figure 5:
DNase I hypersensitivity of HS2 of the
mouse
In contrast with the LCR, DNase I hypersensitivity within the
Figure 6:
Dose
dependence of
In this paper, we establish a functional basis for the
correlation between the decline in Id1 levels and terminal erythroid
differentiation; constitutive expression of Id1 in
Me Interactions between different HLH
proteins are an important determinant of their regulatory functions
during cellular differentiation (for reviews, see Kadesch(1993) and
Weintraub(1993)). We have shown here that the dimerization function of
Id1 is required for its ability to antagonize erythroid
differentiation. Although Id1 has been reported to dimerize
preferentially with certain subgroups of bHLH proteins and not at all
with others (Benezra et al., 1990b; Sun et al., 1991;
Wilson et al., 1991; Hsu et al., 1994a), little is
known about the biochemistry of Id1 in the context of the erythroid
cell. Overexpression of Id1 in MEL cells antagonizes the activation of
at least one E box binding factor whose identity is unknown (Shoji et al., 1994). Id1 is capable of binding the ubiquitous bHLH
proteins E12 and E47 in vitro (Benezra et al., 1990b)
and in myogenic cells (Jen et al., 1992). However, gene
targeting experiments have shown that E12 and E47 are not required for
erythropoiesis (Zhuang et al., 1992) and suggest that Id1 may
have other dimerization partners in developing red blood cells. Indeed,
both uninduced and induced MEL cells contain multiple E box binding
activities (Hu et al., 1992; Bresnick and Felsenfeld,
1993). A number of recent studies have demonstrated
the involvement of Id proteins in control of cell growth (Barone et
al., 1994; Hara et al., 1994; Iavarone et al.,
1994; Peverali et al., 1994). Cellular proliferation may in
some cases be maintained in part through direct interactions between Id
proteins and pRb, as recently reported for Id2 in osteosarcoma cells
(Iavarone et al., 1994). However, at least in vitro,
Id1 and pRb proteins fail to interact. During the first few hours after treatment of
MEL cells with Me In addition to the early down-regulation of Id1 and
c-myc expression, we have also observed an early and transient
down-regulation of three erythroid-specific transcription factors
(GATA-1, NF-E2, and EKLF), occurring slightly later than that of Id1 and c-myc. While the pattern of expression of
these genes is not altered by ectopic expression of Id1 and is
therefore not controlled by Id1, the down-regulation of these genes,
and of Id1 and c-myc, may be related by a common
mechanism during the precommitment period. Whether the down-regulation
of GATA-1, NF-E2, or EKLF is dependent on the levels of c-Myc protein
is not known. The block in globin induction was observed in lines
which express low levels of Id1-er RNA and protein (no greater than
2-3-fold higher than endogenous Id1 in uninduced cells) as well
as those that express the chimeric gene at higher levels, suggesting
that even low level, constitutive expression of Id1 is sufficient to
inhibit differentiation and that the relative levels of HLH proteins in
the cell at a given time is crucial. This idea is supported by the
heterokaryon experiments, which show that fusion with human erythroid
cells can reverse the inhibition of mouse globin gene expression in the
Id1-er-expressing MEL cell lines in a dose-dependent fashion,
presumably through titration of Id1 by bHLH proteins provided in trans.
Similarly, Benezra and colleagues (Jen et al., 1992) concluded
that very small increases in Id1 expression may have a significant
effect on differentiation of muscle. Relative levels of bHLH proteins
have been shown to play an important role in determining the
differentiated state of muscle cells (Blau, 1992; Peterson et
al., 1990; Schfer et al., 1990;
Thayer and Weintraub, 1990). Finally, on the basis of biophysical
studies in vitro, it has been concluded that inhibition by Id
proteins need not require concentrations of Id that are significantly
in excess of the partner bHLH protein (Fairman et al., 1993).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-globin,
-globin, and band 3 and continued proliferation in the presence of
dimethyl sulfoxide. Interestingly, this block occurred even in the
presence of normal levels of the lineage-specific transcription factors
GATA-1, NF-E2, and EKLF. Constitutive expression of Id1 did
not interfere with DNase I hypersensitivity at site HS2 of the locus
control region, expression of the erythropoietin receptor gene, or
down-regulation of the endogenous Id1 or c-myc genes.
The differentiation block is reversible in these lines and can be
rescued by fusion with human erythroleukemia cells. These findings
suggest that in vivo, Id1 functions as an antagonist of
terminal erythroid differentiation.
(
)cell
(Friend et al., 1971) has provided a useful model for the
study of red blood cell differentiation and development. Treatment of
MEL cells with chemical inducers such as dimethyl sulfoxide
(Me
SO) causes them to undergo a program of terminal
differentiation similar to that of normal erythroid cells (Friend et al., 1971). A growing body of work highlights the role of
transcriptional regulators in the differentiation process and in
expression of the specialized phenotype of erythroid cells. At least
three members of the basic-helix-loop-helix (bHLH) (Murre et
al., 1989a) family of transcription factors, TAL1 (Visvader et
al., 1991), Id1 (Benezra et al., 1990b), and Id2 (Sun et al., 1991), are regulated in erythroid cells in a pattern
that implicates them in the control of red blood cell differentiation.
TAL1 (formerly SCL, tal, TCL5; reviewed by Baer (1993)) is expressed in
erythroid and related lineages and is up-regulated in MEL cells in
response to Me
SO (Visvader et al., 1991).
Experiments in cultured erythroid cells (Aplan et al., 1992;
Green et al., 1991) and targeted tal-1 gene
inactivation in embryonic stem cells (Shivdasani et al., 1995)
have demonstrated that the TAL1 protein is a positive regulator of
erythroid differentiation and clearly establish an important role for
bHLH proteins in erythropoiesis.
helix-intervening
loop-amphipathic
helix (bHLH) (Murre et al., 1989a).
This motif has been shown to function in protein dimerization and DNA
binding (reviewed by Kadesch(1993) and Weintraub (1993)). The Id-like
proteins lack the basic region but may still dimerize and thereby act
as dominant negative regulators by inhibiting DNA binding (Benezra et al., 1990b). In this way, the Id proteins serve to modulate
the activity of ubiquitous and cell type-specific bHLH proteins
(discussed by Benezra et al. (1990a) and Duncan et
al.(1992)). The prototype Id gene, Id1, was originally
cloned from a MEL cell cDNA library (Benezra et al., 1990b),
although it is not erythroid-specific. Id1 has been functionally
implicated in the differentiation of several cell types, including
muscle (Jen et al., 1992), myeloid (Kreider et al.,
1992), and B cells (Pongubala and Atchison, 1991; Wilson et
al., 1991; Sun, 1994). During Me
SO-induced terminal
MEL cell differentiation, the expression of Id1 (Benezra et
al., 1990b) as well as Id2 (Sun et al., 1991) is
down-regulated. These observations suggest that in a proliferating
proerythroblast such as an uninduced MEL cell (Friend et al.,
1971), Id inhibits the activation of the terminal differentiation
program until the cell is exposed to an appropriate inducing signal
(Benezra et al., 1990a, 1990b).
-globin,
-globin, and band 3
transcription are markedly reduced, hemoglobin synthesis is greatly
diminished, and the cells continue to proliferate in the presence of
Me
SO. These results suggest that down-regulation of Id1 is
required for terminal erythroid differentiation to proceed.
-globin locus
control region (LCR) but inhibited formation of a
Me
SO-inducible HS site within the promoter of the
maj-globin gene. The Id1-mediated block to terminal
differentiation occurred even in the presence of normal levels of three
lineage-specific transcription factors (GATA-1, NF-E2, and EKLF) and
was independent of c-myc. Furthermore, we observed that
patterns of c-myc expression during terminal differentiation
were unaffected by Id1 in these cells. Fusion of the constitutive
Id1-expressing MEL cells with human erythroleukemia cells rescued mouse
globin gene expression, probably by titrating the exogenous Id1 with
positive regulatory factors; thus, the differentiation block is
reversible. These findings support a model in which modulation of Id1
expression forms one component of the erythroid differentiation switch.
Plasmid Constructions
The wild type Id1
coding sequence (Benezra et al., 1990b) was cloned in sense
(S) and antisense (A) orientations into a derivative of EMSVscribe
(Davis et al., 1987) containing a NotI linker
inserted into the unique RI site to yield EMSV-Id(S)NotI and
EMSV-Id(A)NotI. To generate a ``marked'' Id1 gene (Id1-er), we ligated a 530-base pair polymerase
chain reaction fragment containing the entire Id1 coding sequence to a
0.95-kb estrogen receptor (ER) fragment, HE14 (Eilers et al.,
1989). The Id1 and ER coding sequences in this construct are out of
frame by +1, and the first translational stop codon is found at
the 22nd position downstream from the carboxyl-terminal residue of Id1,
yielding a predicted Id1 protein product ``marked'' by the
addition of a 21-amino acid peptide (Fig. 1). Because this
peptide does not correspond to the native amino acid sequence of ER,
the HE14 portion of the construct (er) is denoted using lower case
letters.
Generation of Constitutive Id1-expressing MEL Cell
Lines
MEL cells (subline 585S) (Baron and Maniatis, 1986)
were coelectroporated (960 microfarads, 280 mV; Bio-Rad Gene Pulser)
with a 20:1 ratio of KpnI-linearized EMSV-Id1-er (S/A) DNA and EcoRI-linearized pSV2neo. Drug selection was initiated after
24 h by addition of geneticin (G418, 0.8 mg/ml) to serially diluted
cells in 48- or 96-well plates. Medium was changed every 2-3 days
for 2-3 weeks, after which time the concentration of G418 was
reduced to 0.4 mg/ml, and individual colonies were expanded.Cell Culture
All mouse and human
erythroleukemia cell lines were maintained and induced as previously
described (Baron and Maniatis, 1986). F4N and DS19/sc9 MEL cells were
kindly provided by R. Benezra and P. Marks, respectively. In these
sublines and in the 585S subline (see above) the level of Id1 RNA
dropped sharply within 2 h after addition of MeSO to the
culture medium.
In Vitro Transcription and
Translation
Plasmids EMSV-Id1(S) and EMSV-Id1-er(S) were
linearized with KpnI and used as templates for transcription
with T3 RNA polymerase (Promega). In vitro translations of
this RNA were performed in 25-µl reactions containing
[S]methionine (DuPont NEN) and 17.5 µl of
rabbit reticulocyte lysate (Promega).
Metabolic Labeling and
Immunoprecipitation
Cells (2 10
) were
incubated for 7 h in Dulbecco's modified Eagle's medium
minus methionine and cysteine, supplemented with 10% dialyzed fetal
bovine serum and 1 mCi [
S]-Express Protein
Labeling Mix (DuPont NEN, NEG-072). The cells were then harvested, and
lysates were prepared as described (Jen et al., 1992).
Incorporation of
S was determined by precipitation with
trichloroacetic acid.
S-Labeled lysates were precleared by
overnight incubation with rabbit serum (1 µl/100 µl) and
protein A-agarose (7 µl/100 µl; Santa Cruz Biotechnology) at 4
°C. Aliquots of cleared lysate were brought up to 125 µl with
homogenization buffer (Jen et al., 1992). An equal volume of
immunoprecipitation buffer (Jen et al., 1992) containing 2
µl of
-Id1 antiserum or preimmune serum was added, and the
samples were rocked 6 h at 4 °C. For competition experiments, the
-Id1 antiserum was preincubated with glutathione S-transferase-Id1 (Jen et al., 1992) or glutathione S-transferase leader protein (Smith and Johnson, 1988) at 1
µg/µl. Protein A-agarose beads (20 µl) were then added, and
rocking was continued for 2 h. After centrifugation, immune complexes
were washed three times with 0.8 ml of RIPA buffer (Harlow and Lane,
1988), and 20 µl of dye buffer was added. Samples were
electrophoresed on a 20% discontinuous SDS-polyacrylamide gel. Gels
were stained with Coomassie Blue to visualize markers and then treated
with Entensify enhancing agents (DuPont NEN) before drying for
fluorography. Antisera and glutathione S-transferase fusion
proteins were generously provided by R. Benezra.
Preparation and Northern Blot Analysis of
RNA
Total cellular RNA was prepared as described (Chirgwin et al., 1979) and subjected to blotting analysis using
standard procedures (Sambrook et al., 1989). The probe used to
detect Id1 and Id1-er was the 0.9-kb BamHI-EcoRI
insert from pMH18R (Benezra et al., 1990b). A 2.1-kb EcoRI insert from pUCM
(Baron and Maniatis, 1986) was
used as a probe for mouse
-globin. A 0.74-kb KpnI-HindIII rat glyceraldehyde-3-phosphate
dehydrogenase insert from pBKSII
GAPDH (a gift from T.
Brown) was used as a loading control.
RNase Protection Analysis
Total cellular
RNA prepared from induced and uninduced MEL cells was analyzed by
quantitative RNase mapping as described (Baron and Maniatis, 1986,
1991). The plasmid BSK-hER, containing a 253-base pair hER HindIII-BglII fragment (Eilers et al.,
1989), was linearized with HindIII and transcribed using T3
polymerase. RNase mapping yielded a protected fragment of 253
nucleotides. A riboprobe for hybridization with antisense RNA was
prepared by T7 polymerase transcription from an XbaI-linearized BSK-hER template and yielded a protected
fragment of 118 nucleotides. The templates for maj-,
-, and
-globin and for
-actin (Baron and Maniatis, 1986)
and mouse GATA-1 (Baron and Farrington, 1994) have been described. A
riboprobe for the erythroid cell-enriched gene JP19 was
prepared by T3 polymerase transcription of HindIII-linearized
template (JP19-215) and yielded a protected fragment of 215
nucleotides. The NF-E2 and EKLF templates were prepared by subcloning
the products of polymerase chain reaction-amplified MEL cell cDNA into
pBSK
. Both templates were linearized with XbaI and transcribed with T7 polymerase to yield protected
hybridization products of 257 and 129 nucleotides, respectively. A
probe against the mouse EpoR was prepared by BglII digestion
of SP72EpoR (generously provided by B.
Mathey-Prvot) and transcription with SP6
polymerase and protected an RNA fragment of 87 nucleotides. The mouse
band 3 (Kopito et al., 1987) probe was prepared by T7
transcription from XbaI-linearized pRK145 (a kind gift from R.
Kopito), and the c-myc probe was prepared by T7 transcription
of HindIII-linearized pBSCPstI/XhoI/c-myc/exonI (kindly
provided by K. Marcu). The c-myc probe hybridizes with
transcripts initiating at each of two promoters, P1 and P2 (Remmers et al., 1986), to yield a doublet band on polyacrylamide gel
electrophoresis.
DNase I Hypersensitivity Analysis
Nuclei
isolated from uninduced and induced cells were treated with increasing
amounts of DNase I, and genomic DNA was then purified (Forrester et
al., 1990). The DNA was digested with BamHI, fractionated
on a 1% agarose gel, and blotted onto filters. Filters were probed with
a 700-base pair BamHI-SspI fragment to examine HS2 of
the LCR or with a 0.9-kb Pst fragment from the region immediately
upstream of the maj-globin gene.
Preparation of Heterokaryons
Fusion of
cells by treatment with polyethylene glycol was performed exactly as
described (Baron and Maniatis, 1986).
Generation and Characterization of
Id1-er-expressing MEL Cell Lines
To determine whether
down-regulation of Id1 is required for terminal erythroid
differentiation, we ectopically expressed the Id1 gene in MEL
cells. A chimeric gene (termed Id1-er, Fig. 1A, see also ``Experimental
Procedures'') was constructed in which murine Id1 and hER coding
sequences were shifted by +1 with respect to one another, yielding
a predicted Id1 protein containing an additional 21 amino acids (Fig. 1B). As a control, a chimeric construct
containing a mutated Id1 gene (PAH2-Id1; Pesce and Benezra,
1993) was prepared and denoted Id1-er. PAH2-Id1 contains a valine to proline substitution within the second
amphipathic helix of the HLH domain. It cannot form heterodimers with
the bHLH proteins E12 or E47 in vitro or inhibit
MyoD-dependent transactivation in vivo (Pesce and Benezra,
1993). Id1, Id1-er, and Id1
-er proteins were translated in vitro and analyzed for their activity in DNA binding
(electrophoretic mobility shift) assays. Both Id-1 and Id1-er inhibited
DNA binding by a truncated version of the bHLH protein E47 (Murre et al., 1989b) in a dosependent manner, while
Id1
-er had no effect (not shown). Therefore, inhibition
of DNA binding by Id1 or Id1-er requires a functional dimerization
surface (mutated in Id1
-er), and Id1-er, like Id1,
inhibits DNA binding directly through formation of inactive
heterodimers with other bHLH proteins.
-er were subcloned into the expression vector
EMSVscribe (here abbreviated EMSV) (14) in the sense (S) and
antisense (A) orientations. To confirm that addition of the 21 amino
acid peptide did not adversely affect Id1 activity, the chimeric Id1-er
constructs were tested for their ability to inhibit MyoD-dependent
transactivation when transfected into cultured cells (Benezra et
al., 1990b; Pesce and Benezra, 1993). As anticipated, expression
of Id1 or Id1-er completely inhibited activation of an MCK-CAT reporter
(Benezra et al., 1990b) by MyoD, in the sense (S) but not the
antisense (A) orientation (not shown). Id1
-er had no
effect in either orientation, consistent with earlier findings (Pesce
and Benezra, 1993) and indicating that 1) the activity of Id1-er is
dependent upon its ability to dimerize with other bHLH proteins and 2)
the inhibitory effect of Id1-er(S) was not related to the addition of
the 21-amino acid peptide.
clones were isolated.
Four independent Id1-er(S)expressing lines (B5, A7, A5, Fig. 2A; and A1, not shown) and one
Id1-er(A)-expressing line (B7, Fig. 3) were obtained. To
estimate the relative levels of Id1-er and Id1 RNA in the cell lines,
total RNA was prepared and examined by Northern blot analysis (Fig. 2A). Id1-er RNA (
1.5 kb) was detected in
lines B5 (Fig. 2A, lanes6-10),
A7 (lanes11-15), A5 (lanes16-20), and A1 (not shown) but not in the parental
MEL line (lanes1-5). The ratio of exogenous
Id1-er to endogenous Id1 RNA (
0.9 kb) was
10:1 in two of the
lines (B5 and A7; Fig. 2A, lanes6 and 11) and
2:1 in the other two lines (A5, lane16; and A1, not shown). Id1-er RNA levels decreased
transiently during treatment with Me
SO, though they were
always higher than endogenous Id1 levels (Fig. 2A).
Significantly, the endogenous Id1 gene was down-regulated in
parental MEL cells and in the Id1-er-expressing lines with similar
kinetics (Fig. 2A and other data not shown), suggesting
that, at least in erythroid cells, endogenous Id1 gene expression is
not positively or negatively autoregulated. The rapid decrease in
endogenous Id1 expression (within 2 h after addition of
Me
SO to the culture medium) has also been seen in other
systems (Kreider et al., 1992) and was also observed with two
other MEL cell sublines (data not shown). In some experiments, the
decrease was transient and was followed by a later and variable rise in
Id1 RNA levels (within 10-24 h).
lines B5, A7, and A5) or from cells induced to
differentiate by addition of Me
SO for 2, 4, 8, or 30 h (lanes2-5, control MEL; lanes7-10, Id1-er
lines B5, A7, and A5)
and hybridized with an Id1 probe. Positions of RNA markers are
indicated. Endogenous Id1 RNA was detected in parental and transfected
MEL cell lines (lanes1, 6, 11, 16). Id1-er RNA was detected in the four cell lines B5, A7,
A5, and A1 (cell line A1 is not shown in this figure) but not in the
parental MEL cell line. Id1-er RNA levels remained higher than those of
endogenous Id at all times. A photograph of the ethidium
bromide-stained formaldehyde gel, taken prior to blotting, is shown in
the lowerpanels. B, induction of globin
gene expression in parental MEL cells. Northern blots, as described in A, were probed with a mixture of mouse
-globin and rat
glyceraldehyde-3-phosphate dehydrogenase probes. Globin RNA was
strongly induced in the parental MEL cells (lane5)
but not in the Id1-er-expressing lines (only B5 is shown in this
figure). C, expression of Id1-er protein in Id1-er
MEL cells.
S-Labeled cell lysates were
immunoprecipitated with anti-Id1 antiserum, without competitor protein (lanes1 and 4) or with antiserum first
incubated with unlabeled glutathione S-transferase-Id1 fusion
protein (lanes2 and 5) or glutathione S-transferase leader protein alone (lanes3 and 6). For comparison,
S-labeled in
vitro translated and immunoprecipitated Id1 and Id1-er proteins
are shown in lanes8 and 9. Id1-er protein
was detected in the Id1-er-expressing line B5 (lanes4 and 6) but not in the nonexpressing control line C6 (lanes1 and 3). For both control MEL cells
and all four Id1-er-expressing lines, endogenous Id1 protein was only
very weakly detectable and did not show up well in the photographs. The asterisks represent proteins that are nonspecifically
precipitated by the immune and pre-immune sera; the faster migrating
protein resolves poorly from endogenous Id1. Levels of Id1-er protein
in lines A5 and A1 were also very low and were comparable to or
slightly higher than the levels of endogenous Id1 in undifferentiated
MEL cells.
SO. Total cellular RNA was
prepared and analyzed by RNase protection using probes for mouse
-globin, band 3,
-actin, JP19, and EpoR. MEL lines B3, C6,
D2, and B7 were negative for Id1-er and expressed levels of
maj-globin and band 3 RNA comparable to those found in the
parental MEL cells. A reciprocal relationship was observed for
-globin and JP19 RNA levels in Id1-er
and
Id1-er
lines (compare JP19, lanes5-12, with
maj in A, lanes7-14). The lineage-specific transcription factors
GATA-1 and NF-E2 and the erythropoietin receptor were expressed at
similar levels in all lines.
Constitutive Expression of Id1-er Blocks Terminal
Erythroid Differentiation
Northern blot analysis of RNA
samples from the experiment of Fig. 2A indicated that
the -globin gene was strongly induced by Me
SO
treatment of the parental MEL cell line (Fig. 2B, lanes1-5) but that this activation was blocked
in Me
SO-treated Id1-er-expressing lines (shown for line B5
in Fig. 2B, lanes6-10; also,
see below). As a first step in correlating this phenotype with Id1-er
translation, we assayed for the presence of Id1-er protein in
metabolically labeled MEL cell extracts.
S-Labeled cell
lysates were immunoprecipitated with an anti-Id1 antiserum, without
competitor protein (Fig. 2C, lanes1 and 4) or with antiserum first incubated with unlabeled,
purified glutathione S-transferase-Id1 fusion protein (lanes2 and 5) or glutathione S-transferase leader protein alone (lanes3 and 6). Id1-er protein was specifically detected in the
Id1-er-expressing line B5 (lanes4 and 6)
but not in the nonexpressing control line C6 (lanes1 and 3). Similar results were obtained for line A7, in
which Id1-er RNA levels were comparable to those of B5. For both
control MEL cells and all four Id1-er-expressing lines, endogenous Id1
protein was only very weakly detectable and does not show up well in
the photograph. Levels of Id1-er protein in lines A5 and A1 were also
very low and were comparable to or slightly higher than the levels of
endogenous Id1 in undifferentiated MEL cells (not shown). Thus, the
levels of protein expressed from the transfected genes mirrored the RNA
levels found in the four cell lines.
maj-globin in
each of the Id1-er-expressing lines (toppanel, lanes7, 9, 11, 13) was
significantly lower than that in the control (non-expressing) lines (lanes1, 3, 5) or in an antisense
line (lane15). While the Id1-er(S)-non-expressing
lines showed a robust activation of
maj-globin
transcription in response to Me
SO (lanes2, 4, 6, 16), the induced
levels in the Id1-er(S)-expressing lines (lanes8, 10, 12, 14) remained at or below the levels
seen in the uninduced controls (compare lanes8, 10, 12, 14 with lanes 1, 3, 5, 15). Essentially identical results
were obtained for
-globin (Fig. 2B and other data
not shown). This block to globin gene expression was observed in the
lines that expressed Id1-er at low levels (A1 and A5) as well as in the
lines that expressed Id1-er at higher levels (B5 and A7), suggesting
that relatively small perturbations in Id1 expression may suffice to
inhibit erythroid differentiation (see ``Discussion''). In a
similar fashion, the levels of the erythroid gene band 3 were markedly
reduced in both the uninduced and Me
SO-induced state in all
four Id1-er(S)-expressing lines (Fig. 3).
(
)was strongly activated in Id1-er(S)-expressing lines (Fig. 3, lanes7-14) compared with
control lines C6 and D2 (lanes3-6). In
contrast, mRNA levels for the erythropoietin receptor (Youssoufian et al., 1990) and the lineage-restricted transcription factors
GATA-1, NF-E2, and EKLF (see below) were identical in Id1-er-expressing
and control lines and showed little or no change upon Me
SO
induction. These observations are consistent with the notion that Id1
prevents uninduced MEL cells from initiating their pathway of
differentiation.
- and
-globin gene expression is that spontaneous
nondifferentiating variants were isolated during the derivation of
these lines. To ensure that we were not misled by clonal variations, we
analyzed a total of 23 independently isolated control lines and found
that all of these control lines differentiated in the presence of
Me
SO; in no instance was a clone encountered where globin
expression was less than
75% of the level of the C6 control line,
and in some cases it was greater (not shown). In contrast, the maximum
levels of globin expression observed for the four Id1-er-expressing
cell lines (Fig. 3A, lanes8, 10, 12, and 14) were 0.5-2% of the C6
control (determined by RNase protection of serially diluted samples).
As expected, expression of the mutated Id1-er gene
(Id1
-er) had no effect on erythroid differentiation (not
shown). We therefore conclude that the observed inhibition of terminal
differentiation is likely to be due to the continued production of
Id1-er in lines B5, A7, A5, and A1 and is not the result of spontaneous
events unrelated to Id1 expression. Moreover, the lack of inhibition by
antisense or mutated Id-er indicates that inhibition by Id1-er is
mediated by protein and requires a functional dimerization surface.
SO, while
the growth rate of control cultures slowed down considerably, and
significant cell death was observed within 6-7 days (not shown).
Proliferation was in fact enhanced by constitutive Id1 expression; the
growth rate of uninduced Id1-er-expressing lines was from 1.6 to 2.7
times faster than that of the parental MEL cells (not shown).
Constitutive expression of high levels of Id1-er appears to have little
or no effect on viability, as the Id1-er(S)-expressing lines have been
maintained stably in culture for more than 2 years. The block in
Me
SO-induced differentiation has also remained stable
throughout this time.
Down-regulation of c-myc in Differentiating MEL
Cells
Like Id1, the bHLH oncoprotein c-myc is
down-regulated early in terminally differentiating MEL cells (Lachman et al., 1985; Kirsch et al., 1986; Kume et
al., 1988) and its constitutive expression blocks MEL cell
differentiation (Coppola and Cole, 1986; Dmitrovsky et al.,
1986; Prochownik and Kukowska, 1986). We therefore asked whether
c-myc transcription is affected by deregulated expression of
Id1-er. Early down-regulation (within 2 h; Fig. 4A, toppanel) of c-myc was observed in the
parental MEL cells, with a later rise by 30 h after addition of
MeSO, as observed by others (Lachman et al., 1985;
Kirsch et al., 1986; Kume et al., 1988). This pattern
was identical in the Id1-er-expressing lines (Fig. 4A, toppanel), suggesting that Id1 is not involved in
regulation of c-myc expression. Id1 must either function
downstream of c-myc or participate in a myc-independent pathway.
SO and began
to rise again within 30 h. GATA-1 and NF-E2 were transiently
down-regulated (between 2-8 h after addition of Me
SO)
and then rose again to pre-induction levels. B,
down-regulation of EKLF. RNA samples from C6 control (lanes1-7) and an Id1-er-expressing line A7 (lanes8-14) were analyzed by an RNase protection assay
for expression of EKLF and Id1-er. Id1-er expression was detected using
an hER probe. The down-regulation of EKLF occurs in both lines and must
be independent of Id1.
Transient Down-regulation of Lineage-specific
Transcription Factors
We next examined the temporal
expression of the erythroid transcription factors GATA-1, NF-E2, and
EKLF during MEL cell differentiation. Surprisingly, these genes are
also transiently down-regulated in MeSO-treated MEL cells
and in the Id1-er-expressing lines (Fig. 4, A and B) but with somewhat slower kinetics (within 2-4 h) than
observed for Id1 or c-myc. By 30-48 h after
addition of Me
SO, transcription of all three
lineage-specific genes returned to pre-induction levels (Fig. 4A, lanes5, 10, 15, 20; Fig. 4B, lanes5, 12). Whether these changes are necessary for
globin gene activation during differentiation is not known, but they
must occur independently of Id1 during the
``precommitment period'' (see below).
DNase Hypersensitivity and Chromatin
Structure
The -globin LCR is a cis-acting regulatory
region required for expression of genes within the linked
-globin
gene cluster; it is thought to function early in erythroid
differentiation to establish an ``accessible'' chromatin
domain (reviewed by Dillon and Grosveld (1993)). Previous studies have
shown that DNase I hypersensitivity associated with the
-LCR of an
exogenous human
-globin locus is present prior to induction and is
not significantly changed during Me
SO-induced
transcriptional stimulation of MEL cells (Forrester et al.,
1989, 1990). A strong erythroid-specific enhancer within the LCR
colocalizes to a DNase I hypersensitive site termed 5`-HS2, located
approximately 9.6 kb upstream of the mouse
-globin gene (Hug et al., 1992; Moon and Ley, 1990). To determine whether
deregulated expression of Id1 affects chromatin structure of the
-globin locus in the vicinity of the HS2 enhancer, we performed a
DNase I hypersensitive site analysis in Id1-er-expressing (A7) and
-non-expressing (C6) MEL cell lines. Several DNase I hypersensitive
sites were found to map within the 6.6-kb BamHI fragment for
both lines A7 and C6 (Fig. 5A). Following induction
with Me
SO, there was no significant change at the prominent
-9.6 hypersensitive site, but a slight reduction in nuclease
sensitivity was detected for both cell lines at sites -10.4 and
-8.4 kb upstream of the (most 5`-proximal)
-like globin
gene,
. Thus, Me
SO-induced changes in chromatin at HS2
were independent of deregulated Id1 expression. These results are
consistent with previous work showing that human
-LCR is similarly
DNase I hypersensitive before and after Me
SO induction.
-LCR and
maj-globin promoter in control and
Id1-er
MEL cells. A, HS2 of the mouse
-LCR. Nuclei isolated from uninduced and induced cells were
treated with increasing amounts of DNase I. Genomic DNA was purified,
digested with BamHI, and subjected to Southern blot analysis
using a 700-base pair BamHI-SspI probe (location
within the 6.6-kb BamHI fragment indicated in schematic
diagram in lower part of figure). In both lines A7 (Id1-er-expressing)
and C6 (control), several novel bands were visible upon addition of
DNase I, including the major hypersensitive site at -9.6 kb and
minor sub-bands (indicated by asterisks) at -10.4 and
-8.4 kb upstream of the embryonic
-like globin gene (
).
The patterns of nuclease hypersensitive sites were identical in both
cell lines. B, mouse
maj-globin promoter. The
filters from A were reprobed with a 0.9-kb PstI
fragment from the region immediately upstream of the adult
maj gene. While the promoter region was weakly hypersensitive in both
A7 and C6 prior to treatment with Me
SO, an increase in
hypersensitivity following Me
SO induction was seen only in
line C6 and not A7. Coordinates are relative to the
maj-globin transcription initiation site in this
figure.
maj-globin gene promoter region has been shown to be
induced concurrent with the stimulation of globin gene transcription
(Hofer et al., 1982; Sheffery et al., 1982). To
examine the chromatin in the vicinity of the
maj-globin
gene, the same filters were hybridized with a probe from the region
immediately upstream of the
maj promoter. This region was
weakly hypersensitive in both cell lines prior to treatment with
Me
SO. After induction, an increase in hypersensitivity was
observed for the control line C6 and paralleled the transcriptional
activation of the adjacent
maj-globin gene, as previously
reported (Hofer et al., 1982; Sheffery et al., 1982).
No increase was observed for the Id1-er-expressing line A7 (Fig. 5B), indicating that ectopic expression of Id1
inhibits the Me
SOdependent formation of a fully functional
globin gene promoter.
Reversibility of the Differentiation
Block
The results presented thus far are consistent with
the idea that Id1 acts as a dominant negative regulator of terminal
erythroid differentiation by sequestering differentiation-promoting
bHLH proteins in inactive complexes and that Id1-er behaves in an
analogous fashion in our stable transfectants. In such a model, the
relative levels of positively and negatively acting HLH proteins should
determine the phenotype of the cell (Blau, 1992; Kadesch, 1993;
Weintraub, 1993). To test this prediction, we fused Id1-er-expressing
or non-expressing MEL cells with human erythroid (K562) cells to form
transient heterokaryons (Baron and Maniatis, 1986, 1991; Baron, 1993) (Fig. 6). K562 cells constitutively express globin RNAs at high
levels (see Baron and Maniatis(1986) and references therein). When a
K562-to-MEL ratio of 4:1 was used for cell fusion, activation of
maj-globin transcription was observed in the
Id1-er-expressing lines (Fig. 6A, lanes2 and 4) but not in the control line (lane6). K562 cells do not express adult
-globin genes
and do not activate adult
-globin expression in nonerythroid cell
nuclei following cell fusion (Baron and Maniatis, 1986). The activation
of mouse
maj-globin in the A7 and B5 heterokaryons most
likely results from complementation by ubiquitous and/or
erythroid-specific factors in the K562 cells. As expected, the mouse
embryonic
-like globin gene (
) was activated in these
heterokaryons, as previously reported (Baron and Maniatis, 1986) (Fig. 6A). Rescue of
maj-globin
expression was not seen if the cell ratio was decreased to 1:1 or 1:15 (Fig. 6B). De novo activation of the mouse
embryonic
-like globin (
) was still observed (Fig. 6B), consistent with the idea that different
factors (and different thresholds for activation by these factors) are
involved in regulating the adult and embryonic
-like globin genes
(Baron and Maniatis, 1986, 1991; Baron, 1993).
maj-globin gene rescue in K562
MEL
cell heterokaryons. A, K562/MEL ratio of 4:1. Human erythroid
K562 cells were induced with hemin (40 µM) for 48 h and
then fused with uninduced MEL lines A7, B5, or (as a control) C6 at a
ratio of 4:1. Cultures were harvested after 48 h, and total RNA was
analyzed for expression of the mouse
maj-globin or
embryonic
-like (
)-globin gene by quantitative RNase mapping.
The Id1-er-mediated
maj-globin gene suppression was
rescued at this ratio of donor and recipient cells. Expression of human
and mouse
-actin was detected by hybridization with a single
(human) actin probe that cross-hybridizes with RNAs from both species.
Id1-er expression is indicated by + or - above the
panels; U, unfused cells; F, fused. B,
K562/MEL ratio of 1:15. Human erythroid K562 cells were induced with
hemin for 48 h and then fused with uninduced MEL lines A7, B5, or (as a
control) C6 at a ratio of 1:15. Under these conditions, factors
provided by the K562 cells are limiting and rescue is not observed.
However, the embryonic
-like globin gene (
) is still
activated. Note that actin RNA levels reflect cell ratios in A and B.
SO-treated MEL cells blocks induction of globin RNA and
protein, band 3 gene transcription, and withdrawal from the cell cycle
in the presence of Me
SO. These effects were neither the
result of general inhibition of erythroid-specific gene expression nor
of cellular de-differentiation. Continued expression of Id1 had no
effect on transcription of four erythroid-specific genes that were
already expressed at high levels in uninduced MEL cells: GATA-1
(reviewed by Orkin(1992)), NF-E2 (reviewed by Engel(1994)), EKLF
(Miller and Bieker, 1993), and the erythropoietin receptor (Youssoufian et al., 1990). The DNase I hypersensitivity of the strong HS2
enhancer of the
-LCR, known to be a stable feature of an exogenous
human
-LCR in MEL cells both before and after Me
SO
induction (Forrester et al., 1989, 1990), was also unaffected.
In contrast, the Me
SO-dependent increase in nuclease
hypersensitivity of the
maj-globin promoter observed for
control cells was blocked by ectopic expression of Id1. Interestingly,
a potential bHLH recognition site (CAAATG, -263 to -258)
(Shehee et al., 1989) is found in the vicinity of the
maj-globin promoter HS site, and we are currently
examining the protein binding properties of this sequence in the
Id-overexpressing MEL cell lines.
Although Id1 does not heterodimerize with the
hematopoietic regulator TAL1 (Shivdasani et al., 1995) in
vitro (Sun et al., 1991) or in vivo (Hsu et
al., 1994a), it may compete with TAL1 for dimerization with
another bHLH protein such as E2-2 (ITF-2) (Henthorn et
al., 1990) or HEB (Hu et al., 1992; Nielsen et
al., 1992). Thus, Id1 may block erythroid differentiation by
disrupting TAL1/E protein complexes that are critical to growth arrest
and differentiation.
(
)Because
the enhancement of cellular proliferation by Id1 requires the presence
of an intact HLH domain, it seems most likely that Id1 functions
indirectly by titrating bHLH proteins away from a complex responsible
for cell cycle arrest (Peverali et al., 1994). The temporal
relationship between the decline in Id1 levels and the activation of
globin and other erythroid-specific genes (e.g. band 3)
during erythroid maturation is consistent with the notion that in
vivo, Id1 may help to maintain progenitor cells in an immature,
proliferative state and thereby play a role in regulating erythroid
cell differentiation.
SO (sometimes referred to as the
precommitment period) (Marks et al., 1994), early
down-regulation of the bHLH proto-oncogene c-myc is observed
and appears to be required for terminal differentiation (see Results).
At later times, c-Myc RNA levels increase again. We have observed the
same pattern of c-myc expression in control and constitutive
Id1-expressing MEL cell lines and conclude that Id1 is not involved in
c-myc regulation. Id proteins fail to interact directly with
c-Myc in vitro (Sun et al., 1991) or in vivo (Hsu et al., 1994a, 1994b), and c-myc can
inhibit myogenic differentiation independently of Id1 (Miner
and Wold, 1991). It therefore seems most likely that Id1 functions
independently of c-myc in erythroid cells. Unregulated
expression of Id1 protein had no detectable effect (positive or
negative) on expression of the endogenous Id1 gene in
uninduced cells or on its down-regulation in the presence of
Me
SO. Therefore, at least in erythroid cells, the Id1 gene does not appear to be controlled by an autoregulatory loop.
-er, mutated version of
Id1-er; kb, kilobase(s); MSV, murine sarcoma virus; ER, estrogen
receptor.
We thank Robert Benezra, Andrew Lassar, Elliott Epner,
Robert Davis, Martin Eilers, Ron Kopito, Bernard
Mathey-Prvot, Tom Brown, and Kenneth Marcu for
generously providing a number of plasmids and other reagents used in
these experiments. We are grateful to Robert Benezra and Richard Baer
for sharing results prior to publication and to Robert Benezra and
members of the Baron lab for critical reading of the manuscript. We
thank Patrick Hayes for oligonucleotide synthesis, Robert Nahf for
assistance with DNA sequencing. W. C. F. thanks Rudolf Grosschedl for
support.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.