The helix-loop-helix protein Id inhibits differentiation of murine erythroleukemia cells.

Id is considered to be a negative regulator of basic helix-loop-helix proteins, which play important roles in cell type-specific transcription and cell lineage commitment. The Id gene was first cloned in murine erythroleukemia (MEL) cells, which can be induced to differentiate toward erythrocytes with Me2SO, and its mRNA decreases after differentiation in various types of cells. In this report, we demonstrate that overexpression of Id interferes with MEL cell differentiation and that inhibition of differentiation is accompanied by reduction in expression of three erythroid-specific genes. While down-regulation of Id is an early event in the differentiation process of MEL cells, E-box binding activity of these cells increases only at a later stage of differentiation, and this late increase is reduced by the overexpression of Id in the early stage. Sequential changes in the activity of several basic helix-loop-helix proteins thus appeared to be involved in erythroid differentiation.

The basic helix-loop-helix (bHLH)' proteins are a family of putative transcription factors, some of which play an important role in the differentiation to specific cell lineages (1). These proteins contain a helix-loop-helix (HLH) domain composed of two conserved amphipathic a helices separated by a variable loop region and an adjacent basic amino acid region. Acting as transcription factors, these proteins form homo-or heterodimers through HLH domains and bind to specific DNA sequences with basic regions (2). Sequences for binding and transcriptional activation for these proteins are represented by E-box motif (CANNTG), which presents promoter/enhancer elements of genes associated with a wide variety of specific cell lineages such as muscle cells (1,3), B lymphocytes (4), pancreatic /3-cells (5), and nerve cells (6). Interestingly, Benezra et al. (7) isolated a novel HLH protein Id that lacks the basic region. Id mRNA decreases in various cell lines when they are induced to differentiate (7); therefore, Id is expected to be a negative regulator of differentiation. In fact, heterodimers of Id with bHLH proteins such as MyoD and E2A (E12/E47) are unable to bind DNA in uitro, repress-* This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 address. ing transcriptional activation in transfected cells (8), and overexpression of Id suppresses muscle and myeloid differentiation (9, 10). A Drosophilin homolog of Id, extramacrochaetae, which genetically antagonizes the mutant phenotype of Drosophilia bHLH, daughterless, and achaete-scute proteins (ll), also can inhibit DNA binding activity of these bHLH proteins in uitro (12). These reports have provided a new aspect of transcriptional regulation of lineage-specific genes. For example, in the process of muscle differentiation, proliferating myoblasts express high levels of Id, which disrupts "transcriptionally active" heterodimers of MyoD and E2A. When myoblasts differentiate to myotubes, the level of Id decreases, and the newly formed heterodimers of MyoD and E2A can activate transcription of muscle-specific genes such as muscle creatine kinase and myosin light chain. Recently, two other members of the Id family have been identified in the mouse, Id2 (13) and HLH462 (14), also referred to as Id3. Every member has been reported to have similar dimerization activity and specificity in uitro; thus functional and regulative differences between the members remain to be clarified.
Murine erythroleukemia (MEL) cells, in which the Id gene was first cloned, can be induced to differentiate with Me2S0, and the decrease of Id mRNA after differentiation has been reported by Benezra et al. (7). It is believed that Id may play a regulatory role in the differentiation program of MEL cells by blocking the activity of some of the bHLH proteins, but the effect of Id on MEL cell differentiation has not yet been examined. In this work, we looked at the functions of the gene on the differentiation through its transfection and found that overexpression of the transfected Id gene interferes with this event.

MATERIALS AND METHODS
Plasmid Constructions-Murine Id cDNA was obtained from H. Weintraub (Fred Hutchinson Cancer Research Center) and inserted into the pSVneoHMT-IIA vector that had been constructed in a previous work (15). Briefly, human metallothionein IIA gene promoter and the poly(A) signal sequence from SV40 large T gene were inserted into pSV2neo vector. Then, a 0.8-kilobase SmaI/DraI fragment containing all coding regions of the Id gene was inserted between the HMT-IIa promoter and the poly(A) signal sequence. The resulting plasmid (pHMT-Id) contains the murine Id gene downstream of the HMT-11, promoter and the neomycin gene as a selective marker.
Cell Lines and Transfections-MEL cells (DS19/3) were maintained in Eagle's minimum essential medium supplemented with 12% fetal calf serum and were induced to differentiate by the addition of 1.8% (v/v) Me2S0. The plasmid was introduced into MEL cells by a protoplast fusion method (16). Escherichia coli (HB101) cells containing pHMT-Id were grown in the presence of chloramphenicol (100 mg/liter) for 12 h to amplify the plasmid, treated with lysozyme (2 mg/ml) for 10 min at 4 "C, and then added to MEL cells fixed on Petri dishes coated with poly-L-lysine. After treatment with polyethylene glycol, the cells were washed with the medium and cultured in a CO, incubator. After 2 days, G418 (100 pg/ml) was added to the 5078 Day after induction with DMSO Detection of Induced Expression of Id Gene and Protein-HMT-IIA gene promoter is known to be inducible by heavy metals and glucocorticoid hormone (17). The effect of the transfected gene in stable transfectants was examined by adding ZnClz (160 p~) to the medium. Expression of the transfected gene in these clones was first examined by Northern blotting; total RNA from each clone was prepared by the guanidinium isothiocyanate procedure (18), and 10 pg of total RNA was electrophoresed in denatured gel, transferred to nylon membrane, and hybridized to radiolabeled probes as described (19). To analyze the production of Id protein, anti-Id serum was generated by immunizing rabbits with glutathione S-transferase-fusion protein (20). Before use, this serum was affinity purified with maltose-binding protein-fusion protein (New England Biolabs, Inc.) coupled to CNBractivated Sepharose (Pharmacia LKB Biotechnology Inc.). Id mRNA-positive clones were further examined by immunoprecipitation with anti-Id antibody. 1 X IO7 cells were metabolically labeled with 150 pCi of Tran3%-label (ICN Biomedicals Inc.), and the crude extracts were made by sonication in 600 pl of lysis buffer (20 mM Tris-HC1, pH 7.4, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, 1 mM PMSF, 5 pg/ml each of antipain, bestain, leupeptin, pepstain A, and chymostatin). The extracts were mixed with 3 p1 of anti-Id antibody and incubated for 1 h at 4 "C, and then 6 pl of protein A-Sepharose beads (Pharmacia LKB Biotechnology Inc.) was added. After incubation for an additional 1 h, the beads were washed 4 times with washing buffer (50 mM Tris-HC1, pH 7.4, 0.5 mM NaC1, 1 mM EDTA, 0.25% gelatin, 0.1% SDS) and resuspended in SDS sample buffer. The proteins were recovered by boiling for 2 min and electrophoresed in 10% SDS-polyacrylamide gel.
Electrophoretic mobility shift assay for E-box motif was performed as described (7). For this assay, a 25-base pair double-stranded oligonucleotide containing the canonical E-box motif from muscle creatine kinase enhancer (22) was used. The sequence of this nucleotide is as follows: & Schuell). The filter was denatured with 6 M guanidine HCL/HBB buffer (20 mM HEPES, 5 mM MgCl,, 1 mM KCl, 5 mM DTT), renatured gradually, and blocked with 5% skim milk/HBB buffer for 1 h a t 4 "C. The filter was set on Screener Blotter (Sanplatec Corp.) and incubated with bacterially produced fusion proteins (glutathione S-transferase (4.7 mg/ml), glutathione S-transferase-Myc(314-439) (4.7 mg/ml), and glutathione S-transferase-AId (0.9 mg/ml)) in 1% skim milk/HBB buffer for 12 h at room temperature. Then the filter was washed with phosphate buffered saline containing 0.2% Triton X-100, and the glutathione S-transferase-fusion proteins bound to the filter were immunologically detected by anti-glutathione S-trans-

Decreased Expression of Id Gene Is a n Early Event in MEL
Cell Differentiation-When MEL cells were treated with Me2S0, they were committed to terminal erythroid differentiation after a latent period of 48 h (15,23). Benezra et al. (7) reported that the level of Id mRNA was low after differentiation. In examining whether a change in Id mRNA level occurs as an early event of induction, we found that the level had dropped rapidly by 48 h after Me2S0 treatment to one-tenth of the initial level and remained very low thereafter (Fig. 1). These findings support the hypothesis that Id is an important factor, especially in the early event of MEL cell differentiation.
Overexpression of Id Interferes with the Differentiation of MEL Cells-If lower Id mRNA is essential for subsequent differentiation of MEL cells, forced expression of Id should alter the cell differentiation phenotype. To test this, we introduced Id gene into MEL cells under the control of HMT-IIA promoter in the pSVneo vector. This promoter offers great advantages for analyzing the effect of the transferred gene in MEL cells because of its accurate inducibility in response to heavy metals (ZnCl2) without any damage or undesirable effects. Furthermore, considering the previously suggested growth regulation by Id (9,14), this promoter is useful in that it avoids cloning artifacts during selection of the transfected clones. MEL cells were transfected by the protoplast fusion method, and the transfectants were selected in geneticin (G418) for 2 weeks; finally, 12 independent clones were obtained.
We first surveyed the effects of Id gene on differentiation.
Each transfectant was exposed to Me2S0 in the presence or absence of Zn2+, and the extent of differentiation was monitored by the benzidine staining of hemoglobinized cells. The ratio of differentiated cells in the presence of Zn2+ to that in its absence was scored (Fig. 2), and the scores in seven nontransfected MEL cell clones (shown as parent in Fig. 2) fell between 0.7 and 1.1 due to clonal variation. The scores of transfected clones, on the other hand, were between 0.4 and 0.9, which indicated the inhibitory effect. We selected several .5-kDa protein standards, which is consistent with the previous report (9). clones with scores of less than 0.5 (inhibitory phenotype) and others with scores of more than 0.8 (noninhibitory phenotype) and examined expression of the transferred gene by Northern blotting. As expected, we were able to recognize 5-10-fold inducible Id mRNA in all these showing the inhibitory phenotype (DSId221, -261, -342, and -432), while clones from the noninhibitory phenotype (DSId352, -361, and -422) failed to induce Id mRNA (Fig. 3A). Immunoprecipitation was done using anti-Id antibody to confirm that elevated levels of Id protein were induced in the cells with inhibitory phenotype. Fig. 4 shows that Id proteins were induced with Zn2+ in all such clones but not in the parent cells (DS19/3). Classification of the seven clones into either an inducible or noninducible type based on Id gene expression clearly showed that overexpression of the gene interfered with MEL cell differentiation (Fig. 2).
Inhibition of Differentiation Depends on Timing of Id Expression-The importance of the timing of a decrease in Id mRNA in the differentiation was examined by adding Zn2+ to cultures a t different periods after Me2S0 treatment. Fig. 5 shows the results on clone DSId342, representing those in which the expression of Id gene was inducible. When Zn2+ was added either at time 0 or at 6 h after Me2S0 treatment, the inhibition was maximum. After 12 h, the extent of inhibition gradually decreased depending on the time of addition. Thus, the initial decrease in Id mRNA within 6 h after Me2S0 treatment may be critical for the induction of differentiation.
Overexpression of Id Reduces Expression of Erythroid-specific Genes-In addition the benzidine staining, which reveals hemoglobin production, we asked whether overexpression of the Id gene interferes with expression of other erythroidspecific genes in the clone DSId342. Three erythroid-specific genes, &globin, glycophorin, and erythroid-specific &aminolevulinate synthase (in each of which expression is induced after 2 days of Me2S0 treatment (19)), were employed as probes. Northern blotting analysis of the total RNAs from the cells induced with Me2S0 in the presence or absence of Zn2+ (Fig. 6) showed that in all three genes expression was reduced in the presence of Zn2+, which maintained the inhibitory effect of Id in differentiation of MEL cells. We then examined c-myc gene expression because its reduction is a prerequisite for the MEL cell differentiation and forced expression of the transferred c-myc gene blocks differentiation (15, 24-27). Fig. 6 shows that the expression of c-myc gene does not change in the presence or absence of Zn2+. Thus, inhibition of differentiation by overexpression of Id gene was not dependent on the change in c-myc gene expression. In spite of the continuous presence of Zn2+, Id mRNA was rapidly induced, but it dropped to a normal level by 2 days after Me2S0 treatment. This observation supports the idea that Id is important especially in the early event of MEL cell differentiation.
Overexpression of Id Changes E-Box Binding Activity of MEL Cells-In the muscle differentiation system, forced expression of Id inhibits the differentiation process through interaction with bHLH proteins such as MyoD and E2A (9).  The labeled E-box oligonucleotides were incubated with the nuclear extracts prepared from clone DSId342 in the presence (+) or absence (-) of Zn2+ at indicated times after Me2S0 treatment. The arrowhead indicates the bands of E-box binding activity (lanes 14), which compete with excess (500-fold) unlabeled oligonucleotide ( l a n e 7). In the right panel, bacterially produced glutathione Stransferase or glutathione S-transferase-AId fusion proteins were added to the nuclear extract. The E-box binding activity in the &day extract was reduced by the addition of increasing amounts of glutathione S-transferase-AId protein (lanes 3-7) but not glutathione S-transferase proteins (lanes 8-12).  contained an E-box from muscle creatine kinase enhancer (22). Nuclear extracts were prepared from DSId342 cells in the presence or absence of Zn2+ at three points of the differentiation process, 0.5, 2, and 4 days after Me,SO treatment (Fig. 7, leftpanel). At 0.5-days, Id expression dropped. At days 2, the extent of differentiated cells began to increase, and levels of E-box binding activities were low and the same in both extracts. At 4 days, when the extent of differentiated cells reached maximum, levels of E-box binding activities increased significantly in the absence of Zn2+ (Fig. 7, left   panel, upper band), but the increase was blocked in the presence of Zn2+.
To confirm that the block in the increase of E-box binding activity was due to Id protein, we added bacterially produced glutathione S-transferase or glutathione S-transferase-AId fusion protein to the nuclear extract in vitro. AId deletes 79 amino acids from the N terminus of Id to promote solubility in bacterial lysate and contains a complete HLH domain.
Glutathione S-transferase-AId was shown to form specific heterodimers with MyoD (data not shown). Addition of glu-

2.
3. tathione S-transferase-AId, but not of glutathione S-transferase, dramatically reduced E-box binding activity of the 4-day extract in proportion to the amount of glutathione S-transferase-AId protein added (Fig. 7, right panel). These results support the hypothesis that Id interferes with MEL cell differentiation through interaction with the protein(s), which possesses E-box binding activity. Possible Partners for Id in MEL Cells-To identify possible partner(s) for Id in MEL cells, we searched the proteins that bind specifically to Id by West-western blotting. As shown in Fig. 8, we found an approximately 30-kDa protein that binds specifically to glutathione S-transferase-AId protein under the condition where glutathione S-transferase-Myc(314-319) protein bound specifically to a protein whose molecular mass (17 kDa) was identical to that of Myn, a murine homolog of Max. The same bands with higher molecular weight observed with glutathione S-transferase-Myc(314-319) and glutathione S-transferase-AId may be due to nonspecific binding.

DISCUSSION
When MEL cells are treated with inducing agents, they are committed to terminal erythroid maturation. The first changes occur in the transport of ions (28)(29)(30)(31) and in phosphoinositol turnover (32), and changes in the expression of a group of genes including c-myc and c-myb oncogenes follow (33,34). These changes are called early events and are required for the commitment of MEL cell differentiation. In fact, c-Myc has also been shown to inhibit differentiation in various cell lineages including MEL cells (15, 24-27, 35, 36), and it apparently acts as a negative regulator for differentiation. The immediate decrease in Id mRNA after Me2S0 treatment observed in this work suggests the involvement of Id in these early events. Our results with the transfection of the Id gene into MEL cells clearly demonstrated that Id is an important factor in MEL cell differentiation, especially in the early events, because differentiation was blocked strongly when the transferred Id gene was overexpressed earlier in the differentiation process (Figs. 2-5). Since expression of the transferred gene is transient (Fig. 6) and the commitment of MEL cell differentiation is not completely synchronous, a certain population of the cell remains to differentiate. Electrophoretic mobility shift assay analysis indicated that E-box binding activities were blocked by either Id overexpressed in MEL cells or by the excess amount of bacterially produced Id protein in the nuclear extract (Fig. 7). These suggest that Id acts as a negative regulator of bHLH proteins in MEL cells in a similar manner as reported in muscle differentiation (7,9); in undifferentiated MEL cells, Id may form heterodimers with bHLH proteins and in this way inhibit DNA binding activity. When the MEL cells are treated with Me2S0, Id proteins rapidly decrease and the preexisting or newly synthesized bHLH proteins form functional homodimers or heterodimers, which are required for further differentiation. Our results, however, suggest a more complex mechanism of Id regulation in MEL cell differentiation. Instead of the rapid decrease in Id mRNA after induction, increase in the E-box binding activities was only detectable on day 4 after induction. This is inconsistent with myeloid differentiation, where an increase in the E-box binding activities follows a rapid decrease in Id (10). In addition in spite of the continuous presence of Zn2+ after Me2S0 treatment, overexpression of Id was only observed during the first day after induction (Fig.  6). The overexpressed Id in the early event did not affect the E-box binding activity but strongly reduced it on day 4 when Id levels must drop. This time lag in MEL cells suggests the following possibilities. 1) More than two bHLH proteins are involved in MEL cell differentiation; Id proteins release the first bHLH proteins in the early event, and then these proteins either stimulate transcription of a second group of bHLH proteins or release these proteins by homo-or heterodimer formation. The first bHLH proteins are undetectable because of their limited quantity or because of different DNA binding specificity. Alternatively, Id may interact with non-bHLH protein(s) as reported in MyoD (37, 38), and these non-bHLH proteins may be involved in the commitment and differentiation.
2) The rapid drop in Id releases a small amount of bHLH proteins in the early event and leads to the autonomous transcriptional activation, which results in the accumulation of the E-box binding proteins after a time lag. Although these two possibilities must be tested by identifying the bHLH proteins interacting with Id in MEL cells, the early change in Id may lead to the commitment of differentiation, and the later accumulated E-box binding proteins may facilitate the terminal differentiation.
It has been reported that several bHLH proteins, such as SCL, LYL, and TAL2 (39-41) are expressed in hematopoietic cells. SCL restricts its expression to erythroid cells (42) and promotes spontaneous differentiation when transfected into MEL cells (43); it can, therefore, be a candidate for Id's partner in MEL cells. On the other hand, neither E-box binding activity of SCL protein nor direct interactions of SCL protein with E2A have been reported (40). We investigated the possibility of direct interaction between Id and SCL in vitro, but even if present, the binding activity of Id and SCL was far weaker than that of Id and MyoD in standard immunoprecipitation analysk2 These data suggest that a distinct subset of E2A-like bHLH proteins is present and mediates regulation of SCL by Id or that unidentified bHLH proteins, which promote MEL cell differentiation, are regulated by Id. We searched for Id binding proteins in MEL cells by West-western blotting and found an approximately 30-kDa protein that binds specifically to Id (Fig. 8). Analysis of its function may clarify the regulatory mechanism of MEL cell differentiation by Id.