From the CRC Department of Gene Regulation, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 9BX, United Kingdom
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ABSTRACT |
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Accumulating evidence implicates functions of the Id family of helix-loop-helix proteins in the regulation of cell growth and differentiation in metazoa. Within the mammalian hematopoietic organ, expression of the Id3 gene is restricted to the lymphoid cell compartment. We show here that in non-lymphoid hematopoietic cells, repression of transcription is correlated with hypermethylation of sequences in the vicinity of the upstream regulatory region of the Id3 gene, suggestive of a strict developmental control of expression of this gene in lymphoid versus non-lymphoid hematopoietic cells. Enforced ectopic expression of Id3 in K562 erythroid progenitor cells promotes erythroid differentiation and is correlated with a quantitative/qualitative shift in the profile of interacting TAL1 and E protein heterodimers that bind to a consensus E box sequence in in vitro band shift assays, consistent with selective targeting of E2A E protein(s) by Id3 and suggesting a possible mechanism involving TAL1-mediated differentiation. By using a Gal 4-VP16 two-hybrid competition assay and an E box-dependent reporter assay, we demonstrate directly that the E2A protein E47 preferentially associates with Id3 in vivo. These observations provide a paradigm for understanding how overlapping but distinct specificities of individual Id proteins may constitute a developmentally regulated program underlying cell determination in diverse lineages.
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INTRODUCTION |
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Among the diverse family of eukaryotic transcriptional regulatory proteins characterized by the presence of the helix-loop-helix domain, an expanding subgroup of basic helix-loop-helix (bHLH)1 proteins has become recognized which play a pivotal role in the regulation of cell growth, commitment, and differentiation (1, 2). The bHLH subgroup itself is comprised of the ubiquitously expressed (class A) E proteins encoded by the E2A, E2-2, and HEB genes, together with a larger number of more diverse (class B) cell-type specific bHLH proteins (1, 2). Examples of the latter type include MyoD (3) and its relatives (4) and the TAL1 oncoprotein, implicated in T cell leukemia (Refs. 4 and 5 and references therein 5-7) each of which performs an essential function in co-ordinately regulating gene expression during cell lineage commitment and differentiation during myogenesis (4) and hematopoiesis/erythropoiesis, (6, 7), respectively. Enforced expression of these genes promotes differentiation (3, 8, 9) while functional ablation arrests differentiation (3, 6, 7) in their respective cell lineages. In common with most other transcription factor families, bHLH proteins bind their target DNA recognition sequence (referred to as the E-box consensus sequence, CANNTG) in a dimer configuration, either as an E protein homo/heterodimer or, more commonly, as a heterodimer comprising one of several cell-type specific bHLH proteins together with an E protein family member (1, 2). DNA binding which is essential for the transcriptional activation (or repression) functions of bHLH proteins is facilitated by a short region of highly basic amino acids located immediately N-terminal to the H-1 amphipathic alpha helix (reviewed in Refs. 2 and 10).
A widely held view, yet to be rigorously verified, is that different combinatorial associations between bHLH dimer partners imparts diversity in functional modulation of distinct E-box transcriptional regulatory activities (1). This in turn is dependent on the relative abundance/activities of the cell's compliment of such bHLH proteins, and provides an elegant mechanism (at least in principle) for fine tune control of gene expression during cell lineage commitment and determination.
An important mechanism through which the activities of bHLH proteins
themselves are regulated is through heterodimerization with members of
the Id class of HLH protein (1, 2, 11). This distinct family of
regulatory HLH proteins of which there are four known members in
mammals (Id1-4) are encoded by a set of evolutionary conserved early
response genes (9, 12-17). As a consequence of better packing of the
hydrophobic core and the absence of destabilizing polar loop residues
and of repulsive charges at the monomer interface at the base of the
four -helix bundle (10, 18), Id proteins are able to form highly
stable heterodimers with selected bHLH protein targets, principally
(although not necessarily exclusively), of the E protein type (11, reviewed in Refs. 1 and 2). Because Ids lack a basic DNA-binding domain, such Id-bHLH heterodimers are thought to be functionally inert
since they are unable to transactivate E box regulatory sequences (11,
13, 19-23). In addition, Id proteins inhibit bHLH functions at a
further level since Id-bHLH heterodimerization results in
destabilization of the target bHLH protein partner (24). As functional
antagonists of bHLH transcription factors, Id proteins act at a general
level as positive regulators of cell growth and negative regulators of
cell differentiation (11, 16, 19, 21, 23, 25-30).
In several studies where the functional properties of different Id proteins have been evaluated either biochemically or biologically, they display a high level of similarity, implying at least some degree of functional redundancy as would be expected for a multigene family of this type (16, 18, 19, 26, 27, 31). Consistent with this, a number of cell types express multiple Id genes both in vitro and in vivo (Refs. 16, 19, and 32-34 and references therein). However, in other instances, expression of individual Id genes appears to be highly cell-type/lineage restricted. One example of this is provided by the hematopoietic system. The Id1 gene is widely expressed in both lymphoid and myeloid cell lineages (20, 35, 36) and its enforced ectopic expression has been shown to arrest differentiation in monocyte-macrophage (28), erythroid (29, 30), and B lymphocyte precursors (20, 27). In the latter case, inhibition of B lymphopoiesis can be accounted for solely on the basis of Id1 interaction with E2A-encoded E protein (s) since functional ablation of this bHLH E protein gene by gene targeting in vivo results in an identical phenotype to that observed in Id1 transgenic mice (27, 37). The Id2 gene is similarly highly promiscuous in its hematopoietic expression pattern (20, 35, 36), but Id3 appears to be expressed exclusively within the lymphoid compartment of hematopoietic cells (21, 35, 36) despite the fact that, like Id1/Id2 it can also function to promote cell growth/block differentiation (19) and to inhibit E box reporter gene activity at least in studies in other model cell types (14, 19, 21, 22, 24).
In evaluating the functional significance of the restricted expression pattern of Id3 in hematopoiesis we report here that repression of transcription of the Id3 gene in myeloid cells is correlated with hypermethylation of the 5' region of the gene, implying a strict developmental control of Id3 expression in lymphoid versus non-lymphoid hematopoietic cells. Enforced ectopic expression of Id3 in K562 erythroid progenitor cells promotes erythroid differentiation and is correlated with a quantitative/qualitative shift in the profile of interacting TAL1 and E protein heterodimers that bind to a consensus E box sequence in in vitro band shift assays, consistent with selective targeting of the E2A E protein(s) by Id3 and suggesting a possible mechanism involving TAL1-mediated differentiation. By using a Gal4-VP16 competitive two-hybrid assay to compare Id3 and Id1, we demonstrate directly that the E2A protein, E47 preferentially associates with Id3 in vivo. These observations provide a paradigm for understanding how overlapping but distinct specificities of individual Id proteins may constitute a developmentally regulated program underlying cell determination in diverse lineages.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
All cell lines were maintained in either
Dulbecco's modified Eagle's medium or RPMI 1640 medium supplemented
with 10% fetal calf serum and 2 mM
L-glutamine. Retroviral transduction of K562 cells was
carried out essentially as described previously (38). The vector
pBabepuro Id3, (19, 39) which was transfected into PA317 cells to
generate high titer amphotropic virus was introduced into target cells
by co-cultivation. Following selection with puromycin (2.5 µg/ml) for
10 days, individual clones were isolated by limiting dilution and
subjected to further characterization. Differentiation of K562 cells
was induced by treatment of cells for 4-5 days with cytosine
arabinoside (2 × 108 M) and monitored
by staining for hemoglobin-positive cells essentially as described
previously (40).
DNA Extraction and Southern Hybridization--
Genomic DNA was
isolated from cell lines by SDS lysis, proteinase K digestion, and
phenol extraction as described previously (14). 5-10-µg samples of
total DNA were digested to completion by using a 5-fold excess of
MspI or HpaII using conditions recommended by the
suppliers (Boehringer Mannheim). Digested DNA was electrophoresed on
1% agarose gels and transferred to nitrocellulose membranes by
capillary blotting. A 375-base pair Id3 fragment corresponding to
nucleotides 1-375 (12) was generated by PCR and radiolabeled with
[-32P]dCTP by a random primed reaction as described
above. Hybridization, post-hybridization washing, and autoradiography
was carried out as described by Murphy and Norton (15).
RNA Extraction and Analysis--
Total RNA was prepared from
cell lines using the RNAZol extraction technique (CinnaBiotech). RNA
concentration was estimated by absorption at 260 nm. Samples of 10 µg
of total cellular RNA were analyzed on 1.2% agarose, 2.2 M
formaldehyde gels and stained with ethidium bromide to monitor the
equivalence of loading by fluorescence intensity of rRNA bands. After
blotting onto "GeneScreen Plus" nylon membranes (NEN Life Science
Products Inc.), prehybridization and hybridization was carried out as
described by Murphy and Norton (15). Probes were labeled to a specific
activity of 5 × 108 cpm using a random primed
labeling kit (Boehringer Mannheim), with [-32P]dATP
(specific activity 3000 Ci/mmol; Amersham, United Kingdom). Blots were
washed at 65 °C for successive 30-min periods first in 2 × SSC, 0.1% SDS, and then in 0.2 × SSC, 0.1% SDS and exposed to
x-ray film at
70 °C with an intensifying screen.
Western Blotting-- Analysis of proteins by Western blotting was performed essentially as described by Harlow and Lane (41). Briefly, cells were detached by gentle scraping and were washed once in phosphate-buffered saline. The resultant cell pellet was re-suspended in SDS sample buffer (0.1 M Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.004% bromphenol blue) and boiled for 5 min. Cellular proteins were separated on 12% polyacrylamide-SDS gels and transferred to nitrocellulose membranes. After blocking for 1 h at room temperature in 15 mM NaCl, 10 mM Tris-HCl, pH 8, 0.05% Tween 20 (containing 5% dried milk) and then incubating with either control sera or with anti-Id3 antibody RD1 (diluted 1-100 in the above buffer) for a further hour at room temperature, the membrane was washed extensively in the same buffer and bound antibody detected using horseradish peroxidase-linked goat, anti-rabbit (Dako, diluted 1-4,000) in conjunction with the enhanced chemiluminescence detection system (Amersham). A rabbit polyclonal Id3 antiserum (RD1) was raised by a standard immunization regime using a synthetic C-terminal 15-amino acid Id3 peptide as immunogen. Immunopurified antibody cross-reacted with mouse and human Id3 proteins but did not detect Id2 or Id1 proteins in control experiments.
In Vitro Protein Binding Studies--
A PCR product was
generated using the primers, 5'-ATGGATCCATGAATGGCGCTGAGCCCGGTG-3' and
5'-AGGTGTCAGGACACGGCCGAGTCA-3' corresponding the nucleotides 368-388
and 727-748 in the human Id3 cDNA template (14), which resulted in
the addition of a BamHI site at the 5' end of the Id3 coding
sequence. PCR amplification was performed on plasmid DNA template (1 ng). Reaction conditions were 94 °C (1 min), 54 °C (1 min), and
72 °C (1.5 min) for 30 cycles. PCR reactions comprised 100 µM dATP, dCTP, dGTP, and TTP together with primers (1 µM), Taq polymerase (1 unit, Promega, Madison, WI) in reaction buffer (100 µl). PCR products were subcloned into the
PCR II vector (Invitrogen) and subsequently as a
BamHI-EcoRI fragment into the PGEX2T vector
(Pharmacia), sequenced and expressed in the Escherichia coli
strain INV F'. Bacterial protein purification was carried out
essentially as described by Kaelin et al. (42) with protein
harvest at 6 h
post-isopropyl-1-thio-
-D-galactopyranoside induction.
Purified GST fusion protein was analyzed for relative protein content
and purity by SDS-polyacrylamide gel electrophoresis.
DNA Mobility Shift Assays--
A 20-base pair double-stranded
oligonucleotide containing the wild type E box recognition sequence:
ACCTGAACAGATGGTCGGCT or mutant, ACCTGAA
CCGATTGTCGGCT (45) (the E box
element is shown in bold and the differences are underlined), was used in all reactions to assay for bHLH binding. Competition reactions with
either wild type or mutant probe (above) established the specificity of
the reaction. DNA mobility shift assays were carried out essentially as
described by Murre et al. (46). Oligonucleotides were
self-annealed, 5' end-labeled using [-32P]ATP
(specific activity 3000 Ci/mmol, Amersham) with T4 polynucleotide kinase and were subsequently purified on an acrylamide gel. Equimolar equivalents of Id3, Id1, and E47s proteins were estimated by
[35S]methionine incorporation and normalized with respect
to methionine content of the translated protein. Approximately 4 × 104 cpm of labeled oligonucleotide was incubated in a
final volume of 10 µl of programed or unprogramed rabbit reticulocyte
lysate. Each binding reaction was carried out by first incubating the lysate at 37 °C for 20 min in binding buffer comprising 10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA and subsequently
with 1 µg of poly(dI·dC). Finally the probe was added and the
incubation continued for a further 20 min at 37 °C prior to analysis
on a 5% nondenaturing acrylamide gel. Where required, GST protein was
added to the binding reactions prior to addition of labeled
oligonucleotide probe. Antibodies specific to the E2A and TAL1 proteins
(45) were generously supplied by Dr. R Baer and used in supershift
experiments, essentially as described elsewhere (43). Nuclear extracts
from cells were prepared essentially according to the method of Dignam
et al. (47).
DNA Transfection and in Vivo Reporter Assays-- Plasmids encoding Id1 (44), Id3 (14), and the E47 and E12(43) proteins were constructed by subcloning the respective cDNAs into the vector pcDNA3 (Invitrogen).
Mouse NIH-3T3 fibroblasts were transiently transfected by the calcium phosphate procedure with an E-box driven chloramphenicol acetyltransferase (CAT) reporter cassette together with the CMV driven E47 expression vector as described previously (19). The reporter was challenged with increasing inputs of either Id1 or Id3 expression constructs and all transfections were normalized for efficiency by inclusion of a luciferase reporter. Quantitation of CAT and luciferase activity in cells 24 h post-transfection was performed as described previously (19). The two-hybrid luciferase assay was carried out essentially as described previously (24), except that a dual luciferase reporter system (Stop and Glo, Promega) was used to monitor transfection efficiency. The plasmids encoding the VP16E12 and Gal4E47 hybrid polypeptides were generated by insertion of the E12 and E47 cDNAs modified by addition of a BamHI recognition sequence at the ATG start site, into BamHI-EcoRI digested PSG424, and BamHI-XbaI digested VP16 NcoI vectors, respectively (48, 49). 35S labeling and immunoprecipitation were carried out as described previously (24) using the Id1 (SC-488) and Id3 (SC-490) rabbit polyclonal antibodies (Santa Cruz Biotechnology). ![]() |
RESULTS |
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Lymphoid-specific Expression of Id3 in Hematopoietic Cell Lines-- To investigate the lymphoid restricted expression of Id3 in the hematopoietic system, we analyzed the methylation status of the human Id3 gene in the immediate vicinity of its upstream regulatory sequences (12) by using a combination of methylation-sensitive/insensitive enzyme isoschizomers, HpaII/MspI. In general, transcriptional activity, particularly of developmentally regulated genes involved in control of cell fate in metazoa, is correlated with a more open chromatin conformation which in turn is inversely correlated with the extent of methylation of cryptic cytosine residues, typically in the context of CpG dinucleotides (50). As shown in Fig. 1A, the human Id3 gene possesses several MspI/HpaII sites within 2 kb of the 5' cap site. Digestion with MspI (methylation insensitive) generated fragments of 0.87 and 0.18 kb which could be detected with a probe overlapping the 5' cap site in Southern analysis (Fig. 1, A and B). This fragment pattern was invariant among different lymphoid and non-lymphoid cell line DNAs examined (Fig. 1B). In contrast, HpaII digestion (methylation sensitive) of myeloid HL60 and K562 DNA generated, in addition, a fragment of approximate size 2.0 kb, with a corresponding reduction in intensity of the 0.87-kb fragment (Fig. 1B). None of the lymphoid cell lines tested generated this additional 2.0-kb fragment (shown for MA42L-mature B; Nalm 6 and Ramos-immature B cells in Fig. 1B). Thus, we conclude that the MspI/HpaII site bounding the 0.87- and 1.4-kb fragments (marked with an asterisk in Fig. 1A) is differentially methylated in myeloid versus lymphoid cell types, consistent with cis-repression of the Id3 gene in myeloid lineage cells.
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Effect of Enforced, Unscheduled Expression of Id3 in K562 Erythroleukemia Cells-- To assess the possible functional significance of the lymphoid restricted expression of Id3 in the hematopoietic system, we investigated the functional consequences of enforced unscheduled expression of this gene on the erythroid differentiation program of K562 cells. Following retroviral transduction of the Id3 cDNA into K562 cells using the vector, pBabepuroId3 (19, 39), a number of clones expressing a range of Id3 mRNA levels were obtained (Fig. 2A), in parallel with control clones expressing pBabe vector without insert. In contrast to the rather variable Id3 expression levels seen at the RNA level, Western analysis revealed relatively invariant Id3 protein levels among Id3 transductants (shown for clones K2 and K4 in Fig. 2B). The expression of Id3 protein in these K562 clones was also very low, being near the lower limit of detection with available antisera.
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Id3 Does Not Abrogate Id1 Function-- In addition to forming stable heterodimers with bHLH proteins such as E47 (but not Tal1), Id3 also heterodimerizes with Id1.2 Since Id1 is known to negatively regulate erythroid (and myeloid) differentiation (28-30), we reasoned that enforced expression of Id3 in K562 cells might promote erythroid differentiation by interacting with, and effectively neutralizing the functions of Id1. To directly test this hypothesis, we designed a simple experiment which exploited the shared properties of Id1 and Id3 in their abilities to heterodimerize with bHLH target E proteins (11, 13, 14, 19-22, 24), thereby preventing the association of E protein with cognate E box recognition sequence in in vitro band shift analyses. As shown in Fig. 3, increasing inputs of in vitro synthesized Id1 and Id3 proteins both efficiently inhibited the formation of E47 homodimer band shift complexes when added separately. However, when equimolar equivalents of the two proteins were added in combination, the effect on inhibition of E box binding by E47 was not significantly different from that seen with each Id separately (Fig. 3). While the two Id proteins are evidently capable of physical association at least in vitro, this experiment shows that there is no detectable functional antagonism between these Ids which might explain the differentiation-promoting effect seen with Id3 in K562 cells.
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Id3 Selectively Ablates E Box Binding of E2A Proteins in Vitro and in Vivo-- We next examined the ability of Id3 to prevent heterodimers comprising TAL1 and E protein-containing heterodimers from binding to E box DNA in in vitro band shift assays. As shown by the experiment in Fig. 4A, exogenous Id3 but not control GST protein readily abrogated E47 homodimer binding to E box sequence as seen with the in vitro synthesized Id protein in Fig. 3. In contrast, binding of TAL1-E47 heterodimer to its preferred consensus sequence was only partially titratable upon addition of exogenous Id3 (Fig. 4A). These results extend previous data for Id1 and Id2 (49, 51-53) which demonstrate that TAL1-E2A heterodimers are relatively resistant to the actions of Id proteins.
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The E47 E2A Protein Is Selectively Targeted by Id3 in Vitro and in Vivo-- The preceding experiments demonstrate that ectopic expression of Id3 in K562 cells is associated with selective loss of E2A homodimer-containing complexes as detected by binding to DNA and would be consistent with this E protein being selectively targeted by Id3 in vivo. However, the levels of endogenous Id1 (and exogenous Id3) proteins in K562 cells were found to be prohibitively low for direct analysis of their in vivo association with E protein (see Fig. 2B and data not shown). Therefore, to determine whether the selective loss of E2A homodimer binding reflects differences between Id1 and Id3 in their in vivo association with E proteins, exemplified by E2A, we compared the magnitude of different E2A (E47)-Id interactions in vitro by a GST pull-down assay, and in vivo by a Gal4-Vp16 two-hybrid assay. As shown in Fig. 5, in vitro translated E2A protein, E47, preferentially bound to Id3 in an in vitro pull-down assay. For comparison, the class B bHLH protein, MyoD, showed preferential binding to Id1 in this assay (Fig. 5), consistent with previous data (54). To evaluate Id-E2A interaction in vivo, we used a quantitative VP16-Gal4 two-hybrid assay in transiently transfected cells. In preliminary experiments, however, we found no significant differences between the ability of Id1 and Id3 to associate with E47 using VP16-Id and Gal4-E47 gene fusions in direct interaction assays,2 consistent with previous findings (54). Therefore, as an alternative more discriminating assay, we employed a competitive two-hybrid approach in which the E2A homodimer interaction was challenged with increasing inputs of each of the wild type Id proteins, as shown in Fig. 6. At the highest input of Id competitor, both Id proteins almost totally abrogated E2A homodimer formation (Fig. 6). However, at progressively decreasing inputs, the Id3 protein appeared to be significantly more potent in competing for E2A homodimer formation than Id1. To determine whether this apparent difference in the ability of Id1 and Id3 to interact with E2A protein is reflected by differences in their abilities to antagonize E2A-dependent gene expression, we examined the effect of increasing inputs of each Id on E2A-dependent trans-activation of an E-box reporter as shown in Fig. 7. As with the two-hybrid assay, Id3 inhibited E2A function more efficiently than did Id1. Thus, in cells expressing Id1 and Id3 (such as would be the case for K562 clones manipulated to express exogenous Id3) Id3 would selectively target E2A protein.
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DISCUSSION |
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Although the Id3 gene is widely expressed in cell types of multiple (non-hematopoietic) lineages both in vitro and in vivo (13, 14, 34, 55, 56), cells of the myelopoietic compartment are notable for the absence of detectable expression of this gene (35, 36). Moreover, this transcriptional inactivity is correlated with hypermethylation of sequences in the immediate vicinity of the upstream regulatory region of the Id3 gene, suggestive of a strict developmental control of Id3 gene expression in lymphoid versus non-lymphoid hematopoietic cells. To investigate the possible biological significance of this regulated expression pattern, we evaluated the functional consequences of enforced expression of the Id3 gene in progenitor erythroid cells in which Id3 is not normally expressed. Ectopic expression of the related Id1 protein in erythroid precursors is known to lead to an arrest in the differentiation program (29, 30), as occurs in several other cell types, including those of myeloid cell lineage (28). Since Id1 is normally expressed in such non-lymphoid hematopoietic cells, at least in their undifferentiated counterparts, this has led to the suggestion that the Id1 family member normally serves as a negative regulator of myeloid cell differentiation, in addition to its proposed similar role in regulation of early B lymphopoiesis (27-30). However, in our studies, all Id3-expressing K562 clones that were examined displayed an enhanced propensity to differentiate either spontaneously or in response to chemical induction when compared with controls. This observation demonstrates that the unscheduled expression of Id3 in the "non-permissive" erythroid environment interferes with the normal differentiation program and implies that at least in this cellular context, the functions of different Ids (Id1 versus Id3) are biologically distinguishable. We note in passing that despite relatively high (although variable) levels of the exogenous Id3 retroviral mRNA observed among different transduced clones, expression of the Id3 protein was only just detectable using available antisera and, moreover, this expression level displayed little variation between different Id3-transduced K562 clones. This phenomena has been previously reported in studies on ectopic Id gene expression (23), including Id3 (19), in cell line models and may well reflect a limited tolerance of particular cell types to unscheduled Id protein expression.
The observed enhancement in differentiation of K562 cells in response to transduction of Id3 is very reminiscent of the effect reported to occur in response to enforced overexpression of the TAL1 bHLH protein in these and other primitive erythroid precursor cell lines (8, 9). The TAL1 protein, whose functions are essential for normal erythropoiesis (6) interacts avidly with bHLH proteins, such as E47 (45, 51-53). TAL1 also associates in vivo with members of the LIM domain protein family, specifically LM02(RBTN2/TTG2) which is also indispensable for erythropoiesis (57-59). However, beyond this, little is known about the mechanisms/gene targets through which TAL1-E47/LM02-mediated differentiation occurs during erythropoiesis.
Although the Id3 gene is not normally expressed in erythroid progenitors and is not therefore a normal physiological regulator of erythropoiesis, an understanding of the mechanisms underlying this differentiation-promoting effect of Id3 might provide some insight into how candidate interacting regulatory partners function during normal erythropoiesis. To date some non-bHLH proteins have been described as potential Id targets, such as the retinoblastoma susceptibility gene product, Rb (and related pocket proteins) for Id2 (25, 31) and the MIDA1 protein for Id1 (60), thus far only bHLH proteins (essentially of the E protein type) have been reported to associate with Id3 (13, 14, 19, 21, 22, 24, 61). However, in addition to the E47 and TAL-1 bHLH proteins, we also examined the possibility of Id1-Id3 interactions since such Id homo-heterodimers are predicted to occur from three-dimensional modeling studies (18). Although Id3 did appear to associate with Id1 in vitro, albeit to a lesser extent than with E47,2 we could find no evidence for functional antagonism between these proteins in a functional assay of abrogation of E protein binding to an E box oligonucleotide. A simplistic explanation for Id3 promotion of erythroid differentiation based on antagonism of Id1 function therefore seems unlikely.
As with other Ids, Id3 was found to associate avidly with the E47 E protein in vitro (confirming previous data, see Refs. 13, 14, 19, 21, 22, 24, and 61) and although binding to the TAL1 bHLH protein was detectable, the ability of Id3 to abrogate E47-TAL1 heterodimer binding to an E box sequence was considerably less than that seen for E47 homodimers. Such resistance of TAL1-E protein heterodimers to Id antagonism has previously been reported for the Id1 and Id2 proteins (49, 52, 53), and what abrogation of binding occurs can be largely accounted for by sequestration of the E protein dimer partner by the Id competitor. These observations serve to further highlight the functional similarities between different Ids, as observed in in vitro assays.
In evaluating the status of E2A and TAL1-related proteins in K562 cells by band shift assay using an E box probe representing a preferred consensus for TAL1-E protein heterodimer binding (45), we found that expression of exogenous Id3 correlated with selective loss of E2A E protein homodimer binding and a quantitative shift in relative abundance of various TAL1-E protein binding complexes. Based on these observations, a reasonable hypothesis would be that Id3 promotes erythroid differentiation by selectively sequestering individual E protein partners, thereby leading to a configuration of TAL1-E proteins which functionally mimics the effect produced by over-expressing TAL1 in these cells. It may be significant in this regard that the TAL1 protein which, like Id proteins is also capable of blocking differentiation when ectopically expressed in certain cell types (62, 63) has been suggested to perturb T cell maturation by selective sequestration of E2A-related bHLH proteins (64).
We previously reported that in co-immune precipitation experiments, the Id3 protein preferentially targets the E2A, E47 bHLH protein when compared with Id1 (61). Such an observation would be compatible with data on Id3-expressing K562 cells which already express Id1. Accordingly, endogenous Id1 would normally function to block differentiation through interaction with its preferred target bHLH E proteins (and perhaps other targets) while the introduction of exogenous Id3 leads to an "inappropriate" loss of E2A function in these cells resulting in an enhanced differentiation response.
Finally by using competitive two-hybrid and E box-dependent reporter assays we were able to directly confirm this preferred functional interaction between Id3 and the E47 E2A protein in vivo. Such differences in target specificity among Id proteins for different E proteins may well serve to underlie the fine-tuned regulation of target gene expression involved in developmental control, coupled to bHLH protein function.
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ACKNOWLEDGEMENTS |
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We thank Neil Wood, Amir Hannan, and Suzie Armitage for technical assistance and the following of kind gifts of recombinant clones/reagents, Drs. X. H. Sun, Roger Watson, and Tony Green for E47 and TAL1 plasmids, and Dr. Richard Baer for supplying us with the Gal4/VP16 expression-reporter cassettes for use in the two-hybrid assay system and for generously supplying TAL1 antiserum. We also thank Drs. Tony Green and Roger Watson for helpful discussions during the initial stages of this work.
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FOOTNOTES |
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* This work was supported by the United Kingdom Medical Research Council and the Cancer Research Campaign.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: CRC Department of Gene
Regulation, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 9BX, United Kingdom. Tel.: 0161-446-3129; Fax: 0161-446-3109; E-mail:
JNorton{at}picr.man.ac.uk.
1 The abbreviations used are: bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; GST, glutathione S-transferase; PCR, polymerase chain reaction; kb, kilobase pair(s).
2 M. Jasiok, R. Deed, and J. Norton, unpublished observations.
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REFERENCES |
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