©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of an Erythroid Differentiation Switch by the Helix-Loop-Helix Protein Id1 (*)

(Received for publication, April 3, 1995; and in revised form, May 18, 1995)

James Lister (1)(§) William C. Forrester (2)(¶) Margaret H. Baron (1)(**)

From the  (1)Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138-2020 and the (2)Department of Microbiology and Immunology, University of California, San Francisco, California 94143-0414

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

The mouse erythroleukemia (MEL)()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 (MeSO) 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 MeSO (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.

Members of the bHLH family share a common amino acid sequence motif comprising a basic region just amino-terminal to an amphipathic 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 MeSO-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).

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 -globin, -globin, and band 3 transcription are markedly reduced, hemoglobin synthesis is greatly diminished, and the cells continue to proliferate in the presence of MeSO. These results suggest that down-regulation of Id1 is required for terminal erythroid differentiation to proceed.

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 -globin locus control region (LCR) but inhibited formation of a MeSO-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.


EXPERIMENTAL PROCEDURES

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.


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.



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).


RESULTS

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.

Id1-er and Id1-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.

The chimeric Id1 genes were introduced into MEL cells and G418 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 MeSO, 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 MeSO 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).


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 lines B5, A7, and A5) or from cells induced to differentiate by addition of MeSO 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.




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 MeSO. 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 MeSO treatment of the parental MEL cell line (Fig. 2B, lanes1-5) but that this activation was blocked in MeSO-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.

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 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 MeSO (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 MeSO-induced state in all four Id1-er(S)-expressing lines (Fig. 3).

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,()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 MeSO induction. These observations are consistent with the notion that Id1 prevents uninduced MEL cells from initiating their pathway of differentiation.

An alternative explanation for the absence of - 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 MeSO; 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.

Interestingly, the Id1-er-expressing lines were able to proliferate continuously when grown in medium containing MeSO, 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 MeSO-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.


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 MeSO and began to rise again within 30 h. GATA-1 and NF-E2 were transiently down-regulated (between 2-8 h after addition of MeSO) 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 MeSO, 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 MeSO-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 MeSO, 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, MeSO-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 MeSO induction.


Figure 5: DNase I hypersensitivity of HS2 of the mouse -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 MeSO, an increase in hypersensitivity following MeSO induction was seen only in line C6 and not A7. Coordinates are relative to the maj-globin transcription initiation site in this figure.



In contrast with the LCR, DNase I hypersensitivity within the 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 MeSO. 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 MeSOdependent 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).


Figure 6: Dose dependence of 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.




DISCUSSION

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 MeSO-treated MEL cells blocks induction of globin RNA and protein, band 3 gene transcription, and withdrawal from the cell cycle in the presence of MeSO. 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 MeSO induction (Forrester et al., 1989, 1990), was also unaffected. In contrast, the MeSO-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.

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). 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.

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.()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.

During the first few hours after treatment of MEL cells with MeSO (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 MeSO. Therefore, at least in erythroid cells, the Id1 gene does not appear to be controlled by an autoregulatory loop.

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).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1 GM42413 (to M. H. B.), Lucille P. Markey Charitable Trust Grant 87-24 (to M. H. B.), and March of Dimes Birth Defects Foundation Basil O'Connor Award 5-715 (to M. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Predoctoral Training Grant GM 07620.

Special Fellow of the Leukemia Society of America.

**
Lucille P. Markey Scholar in Biomedical Science. To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, 16 Divinity Ave., Cambridge, MA 02138-2020. Tel. and Fax: 617-495-1135; baron{at}biosun.harvard.edu

The abbreviations used are: MEL, murine erythroleukemia; bHLH, basic helix-loop-helix; LCR, locus control region; HS, DNase I hypersensitive site; EKLF, erythroid Kruppel-like factor; NF-E2, nuclear factor-erythroid 2; Id1-er, marked form of murine Id1 gene; Id1-er, mutated version of Id1-er; kb, kilobase(s); MSV, murine sarcoma virus; ER, estrogen receptor.

J. Lister and M. H. Baron, unpublished observations.

R. Benezra, personal communication.


ACKNOWLEDGEMENTS

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.


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