©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
MIDA1, a Protein Associated with Id, Regulates Cell Growth (*)

(Received for publication, June 14, 1995)

Wataru Shoji Toshiaki Inoue Tohru Yamamoto (§) Masuo Obinata (¶)

From the Department of Cell Biology, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-77, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have isolated cDNA clone encoding a protein that can associate with Id, a helix-loop-helix (HLH) protein. This protein is named MIDA1 (mouse Id associate 1), and its predicted amino acid sequence consists of Zuotin (a putative Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats similar to c-Myb oncoprotein. MIDA1 associates with the HLH region of Id with the conserved region adjacent to eukaryotic DnaJ conserved motif within the Zuotin homology region, although it does not have any canonical HLH motif. The addition of antisense oligonucleotide of MIDA1 inhibited growth of murine erythroleukemia cells without interfering with erythroid differentiation, indicating that it regulates cell growth.


INTRODUCTION

Id is a member of helix-loop-helix (HLH) (^1)proteins that play an important role in cell type-specific transcription and cell lineage commitment (1) by forming DNA-binding heterodimers such as MyoD and E2A (E12/E47). It lacks a basic region and negatively regulates other basic helix-loop-helix (bHLH) proteins by forming heterodimers that cannot bind DNA. Id gene was isolated first in murine erythroleukemia (MEL) cells (2) that could be induced to differentiate toward erythrocyte. We previously reported that Id mRNA decreased soon after induction of differentiation of MEL cells with Me(2)SO, and it was inhibited by the overexpression of Id gene(3) . Overexpression of Id was shown to inhibit induction of differentiation in other cell systems (4, 5, 6) , indicating that the same inhibitory function is involved in differentiation of various cell lineages.

In addition to the regulation of differentiation, Id was shown to act as a growth regulator. HLH462 refereed as Id3 shows immediate early response to serum stimulation(7) , and loss of Id gene expression inhibits cell cycling induced by serum stimulation(8) . Furthermore, Id2 is reported to antagonize RB protein that maintains the cells in G(0)/G(1) phase of the cell cycle(9) . Thus, Id may function as a regulator of both growth and differentiation of the cells.

In the present study, we searched the proteins that associate with Id by West-Western screening (3) to know how Id functions in the commitment of growth and differentiation of MEL cells. From an expression cDNA library constructed from mRNAs of MEL cells, we obtained a new protein that can bind Id without having HLH motif.


MATERIALS AND METHODS

West-Western Screening

The gt11 library was plated in the Y1090 bacterial strain. After incubation at 42 °C for 3 h, 25 mM isopropyl-1-thio-beta-D-galactopyranoside-impregnated nitrocellulose filters (Schleicher & Schuell) were overlaid on the plates to induce beta-galactosidase fusion protein. To get good yield of protein production and transfer, the plates were incubated for 8 h at 37 °C. Filters were marked, rinsed with TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) to remove bacterial debris, and treated successively with 6, 3, and 0 M guanidine HCl/HBB buffer (20 mM Hepes, pH 7.5, 5 mM MgCl(2), 1 mM KCl, 5 mM dithiothreitol) at 4 °C for denaturation-renaturation of transferred protein. Filters were blocked with 5% skim milk/HBB buffer and incubated with 0.2 µg/ml of bacterially produced glutathione S-transferase (GST)-DeltaId protein (3) in 1% skim milk/HBB buffer for 8 h at room temperature. DeltaId lacks the 49 N-terminal amino acids of Id to promote solubility in bacterial lysate and contains a complete HLH domain. Then they were washed 4 times with PBST (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na(2)HPO(4)bullet7H(2)O, 1.4 mM KH(2)PO(4), pH 7.3, 0.2% Triton X-100), and positive plaques were immunologically detected by successive treatment with anti-GST antibody(3) , horseradish peroxidase-anti rabbit IgG (Zymet Laboratory Inc.), and ECL Western blotting detection reagents (Amersham Corp.).

In Vitro Association Studies

In vitro transcription and translation were performed under conditions recommended by the Promega Protocols and Application Guide. S-Labeled proteins were produced in the lysate from pCITE2 vectors (Novagen) that have each cDNA inserts. To check the translation products, 1 µl from each lysate was subjected directly to SDS-polyacrylamide gel electrophoresis, and 5 µl was diluted into 500 µl of HBB buffer and incubated with either GST or GST-DeltaId affinity matrices in which approximately 5 µg of fusion protein adsorbed to 10 µl of glutathione-Sepharose beads (Pharmacia Biotech Inc.) for 1 h at 4 °C. The beads were washed 4 times with PBST at room temperature, and the bounded proteins were eluted with SDS-containing sample buffer, followed by SDS-PAGE and autoradiography.

In Vivo Association Studies

GST-Id (full length) cDNA was inserted under beta-actin promoter and was transfected into MEL cells. One of the stable transfectants, which constitutively express GST-Id protein and parental MEL cells, were used for in vivo association studies. 1 times 10^8 cells were washed with phosphate-buffered saline (PBS) and incubated at room temperature for 30 min with a cross-linking agent dithiobis(succinimidyl propionate) (Pierce) at a final concentration of 2.5 mM in PBS. The cross-linking reaction was terminated by the addition of one-tenth volume of 1 M Tris-HCl, pH 7.5, and cells were lysed by gently suspending with 5 ml of TBS-MgCl(2) buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 15 mM MgCl(2)) containing 1% Triton X-100 and proteinase inhibitors (5 µg/ml antipine, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The cleared lysate was incubated with 100 µl of glutathione-Sepharose beads (Pharmacia) for 1 h at 4 °C, and GST-Id and its associated proteins were precipitated with beads. After 4 times washing with TBS-MgCl(2) buffer, the bound proteins were eluted with SDS-containing sample buffer. In the presence of 5% beta-mercaptoethanol, S-S bonds of cross-linked protein complex were cleaved, and liberated proteins were separated in SDS-PAGE. MIDA1 was detected with Western blotting using anti-MIDA1 rabbit serum, which was obtained by immunizing bacterially produced GST-MIDA1 (residues 446-621) fusion protein. With this antiserum, MIDA1 was detected specifically as a single band of 74 kDa (data not shown).

Northern Blotting

Total RNAs from tissues and cultured cells were prepared by guanidinium-thiocyanate-phenol chloroform procedure(10) . 10 µg of RNA from each sample was electrophoresed in a denatured 1% agarose gel, transferred to a nylon membrane, and hybridized with cDNA probe radiolabeled with a random primer extension kit (E. I. du Pont de Nemours & Co., Inc.) in the hybridization buffer (50% formamide, 5 times SSC, 1 times FBP (1% Ficol, 1% polyvinylpyrrolidone, 1% bovine serum albumin), 20 mM NaHPO(3) pH 6.5, 100 µg/ml of salmon sperm DNA, 10% dextran sulfate, 0.1% SDS). After overnight incubation at 42 °C, the membrane was washed in 2 times SSC, 0.1% SDS for 10 min at room temperature and in 0.1, times SSC, 0.1% SDS for 30 min at 60 °C, and then autoradiographied.

Antisense Oligonucleotides Experiment

16-base phosphorothioanate oligomers (TCGGCAGGAGCAGCAT) from translation initiation region were synthesized by SAWADY Technology Co., Ltd. and purified with C(18) reversed-phase column (Waters Chromatography). The oligomers were lyophilized and suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4). Growing MEL cells (DS19/3) were inoculated in triplicate dishes at the initial concentration of 4 times 10^4 cells/ml in Eagle's minimum essential medium containing 12% fetal calf serum (heat-inactivated at 65 °C for 30 min). The cell number was monitored at daily intervals by hemocytometer, and the cell viability was determined by trypan blue exclusion. To monitor protein expression of MIDA1, 5 times 10^5 cells from each condition were lysed by RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS), and the lysates were subjected to Western blotting using anti-MIDA1 rabbit serum as a single band of 74 kDa. The amount of total protein from each lysate was ascertained to be equal by Coomassie Brilliant Blue staining of SDS-PAGE samples.

Cell Cycle Analysis

Cell cycle distribution of the oligomer-treated cells was determined with DNA content by flow cytometric analysis. Cells were fixed with 70% ethanol and resuspended in PBS containing 50 µg/ml of propidium iodide (Sigma) and 10 µg/ml of RNase (Sigma). Analysis was performed with Becton Dickinson FACScan and Cell Fit software. In the same experiment, DNA synthesis was monitored with BrdU incorporation using Cell proliferation kit (Amersham Corp.) according to its recommended protocol.


RESULTS

Screening of Id-associated Proteins

To screen the Id-associated proteins, we employed West-Western strategy, by which several biologically interactive proteins have been identified(11, 12) . We constructed the chimeric gene GST-DeltaId (Fig. 1a) and expressed it in Escherichia coli. DeltaId has a deletion of 49 amino acids from the N terminus of Id to promote solubility in the bacterial lysate, resulting its easier purification by glutathione affinity column. The fusion protein contained a complete HLH domain and could form specific heterodimers with MyoD (Fig. 1b). gt11 expression library was constructed to comprise 3 times 10^7 colony forming units of complexity using poly(A) mRNAs of MEL cells, and approximately 5 times 10^5 plaques were screened for their ability to interact with GST-DeltaId. We identified 14 positive clones, 9 clones of which were shown to encode the same protein by sequencing analysis. The longest 2-kilobase pairs insert contains one long open reading frame that encodes 621 amino acids as well as 5`- and 3`-noncoding regions (Fig. 2a). We named this clone MIDA1 (Mouse Id associated 1). The predicted amino acid sequence of MIDA1 revealed several interesting features. The N terminus region (residues 1-433) had significant homology to Zuotin that encodes 433 amino acids(13) , a putative Z-DNA binding protein in Saccharomyces cerevisiae (157/433 (36.3%) identical, 216/433 (49.9%) related)) (Fig. 2b). In the middle of the Zuotin homology region (residues 84-163), it had a eukaryotic DnaJ motif that suggested to attach to the Hsp70s and shared with proteins essential for protein translocation and correct folding (14) (Fig. 2c). The continuing C terminus region (residues 421-621) had two Trp-mediated repeats that may form helix-turn-helix structure and that may have sequence-specific DNA binding activity as c-Myb oncoprotein (Fig. 2d) (15, 16, 17) .


Figure 1: A protein probe for West-Western screening. a, construction of the protein probe, GST-DeltaId for West-Western screening. Schistosoma japonicum glutathione S-transferase (33) are fused with the residues 78-176 of mouse Id that contains the HLH domain. b, heterodimer formation of GST-DeltaId with MyoD. Left, in vitro translated MyoD (lane1) and c-Myc (lane2) proteins. Right, in vitro translated proteins were mixed with GST-DeltaId and anti-GST antibody for 1 h at 4 °C, and then the formed complexes were recovered by protein A-Sepharose (Pharmacia). Myc protein (lane4) was used as a negative control.





Figure 2: Structural features of MIDA1. a, nucleotide sequence of MIDA1 cDNA. The nucleic acid residues are numbered from the beginning of the longest cDNA insert, and amino acid residues are numbered from the beginning of the long open reading frame. Underlined residues are homologous region to Zuotin (lightunderline)(13) , eukaryotic DnaJ conserved protein motif (darkunderline)(14) , Trp-mediated repeat similar to c-Myb(15-17) (boxunderline). b, sequence comparison of MIDA1 and Zuotin. Conserved amino acids are marked by asterisks (matched) and closedcircle (related). 157/433 (36.3%) residues are identical, and 216/433 (49.9%) are closely related. c, sequence of J region from MIDA1, E. coli. DnaJ, and other eukaryotic members of DnaJ family. Conserved amino acids are reversed(14) . d, sequence alignment of Trp-mediated repeats from MIDA1 and c-Myb proteins from various species. Conserved amino acids are reversed.



Association of MIDA1 with Id

While MIDA1 had several interesting features in its protein structure as described above we couldn't find any canonical HLH motif in this protein. To search the binding domain of MIDA1 to Id, a series of deletion clones were constructed, and their RNAs were synthesized. [S]methionine-labeled proteins from the RNAs were translated in the rabbit reticulocyte lysate system (Fig. 3a) and asked whether they could be recovered by association with GST or GST-DeltaId affinity matrices(18) . As shown in Fig. 3b, wild-type, Delta2-71, and Delta2-188 of MIDA1 could associate with Id, but Delta2-256 and Delta2-342 could not. These results indicated that the residues 189-256, a part of the Zuotin homology region, is a responsible domain for association with Id. Interestingly, Id-binding activity of Delta2-188 was stronger than that of wild-type or Delta2-71, thus the residues 2-189 may regulate Id-binding activity of MIDA1. Then we examined whether two Trp-mediated repeats affect Id binding activity of MIDA1. Clone Delta2-188 was deleted from C terminus to lack one (Delta2-188, Delta526-621) or two (Delta2-188, Delta443-621) Trp-mediated repeats and employed in the same experiment (Fig. 3c). The results showed that deletion in the Trp-mediated repeats did not affect its binding activity to Id (Fig. 3d).


Figure 3: Id-binding activity of MIDA1 deletion mutant. a, MIDA1 mutants deleted sequentially from N terminus were constructed and translated in vitro. For example, Delta2-71 means a MIDA1 mutant that deletes residues 2-71. b, deletion mutants translated in vitro were mixed for 1 h at 4 °C with GST or GST-DeltaId protein captured by glutathione-Sepharose. Formed complexes were washed and recovered by quick centrifugation. Then they were eluted with SDS-sample buffer and electrophoresed with SDS-PAGE. Wild-type, Delta2-71, and Delta2-188 could be recovered by GST-DeltaId, but other deletion mutants could not. c, Delta2-188 was deleted from C terminus to determine whether Trp-mediated repeats contribute Id-binding activity. Delta2-188,Delta526-621 lacks one Trp-mediated repeat, and Delta2-188,Delta443-621 lacks two. d, C-terminal-deleted mutants were examined on Id-binding activity. The loss of Trp-mediated repeat did not affect Id-binding activity. e, summary of Id-binding activity of the deletion mutants and the structure of MIDA1. Id binding domain is located on residues 188-256, adjacent to the DnaJ motif.



Then we asked whether the HLH region of Id is essential for association with MIDA1. Binding assay with GST-DeltaId and GST-Delta(HLH)Id, which completely deletes its HLH domain, showed that GST-Delta(HLH)Id could not associate with MIDA1 (Fig. 4). Thus, it is concluded that the binding domain of Id is located in its HLH region.


Figure 4: HLH region of Id is essential for association with MIDA1. In vitro translated MIDA1 proteins were mixed with GST-DeltaId or GST-Delta(HLH)Id captured by glutathione-Sepharose, and recovered proteins were analyzed by SDS-PAGE. GST-DeltaId deletes 49 amino acids from the N terminus and contains complete HLH domain, while GST-Delta(HLH)Id deletes 99 amino acids from the N terminus to lack HLH region.



MIDA1 Associates with Id in Transfected MEL Cells

To examine whether MIDA1 associates with Id in vivo, MEL cells were transfected with the cDNA for GST-Id fusion protein, and the stable transfectants were obtained. The transfectants and the parent cells were cross-linked in vivo, using dithiobis(succinimidyl propionate)(9, 19) . In this procedure, we can avoid detecting reconstructed associations different from natural ones and also avoid missing unstable or transient ones by their nature. Then, the GST-Id fusion protein and its cross-linked proteins were recovered by affinity to glutathione-Sepharose. The proteins liberated from the cross-linked complex with GST-Id were successively separated by SDS-PAGE and subjected to Western blotting with the anti-MIDA1 serum made for the bacterially produced protein. As shown in Fig. 5, MIDA1 proteins, which can be detected as a 74-kDa single band, were recovered from GST-Id transfected cells but not from parent MEL cells. These results strongly suggest that MIDA1 associates with Id in vivo.


Figure 5: In vivo association between MIDA1 and Id. Stable GST-Id-transfected MEL cells and parent cells were treated with cross-linking agent dithiobis(succinimidyl propionate), and subjected to the analysis. Each lysates were incubated with glutathione-Sepharose, and GST-Id proteins were precipitated with their associated proteins. MIDA1 protein can be detected as a single band of 74 kDa with Western blotting using anti-MIDA1 serum.



Expression of MIDA1

The levels of MIDA1 expression was measured in MEL cells, and various mouse tissues by Northern blot analysis (Fig. 6). A single 2.2-kilobase pairs signal was detected by the MIDA1 cDNA probe. The highest level was observed in undifferentiated MEL cells, but it dropped after induction of differentiation, which was correlated with that of Id mRNA(2, 3) . In mouse tissues, relatively high expression was observed in spleens and testes, where population of continuously proliferating cells were abundant.


Figure 6: Expression of MIDA1 mRNA. 10 µg of total RNAs from MEL cells and mouse tissues were separated in denatured 1% agarose gel, and the RNAs were blotted on nylon membrane. The membrane was incubated in the hybridization buffer (50% formamide, 5 times SSC, 1 times FBP, 20 mM NaHPO(3) pH 6.5, 100 µg/ml of salmon sperm DNA, 10% dextran sulfate, 0.1% SDS) containing 5 times 10^5 cpm/ml of MIDA1 cDNA probe labeled with random primer extension. A single band was observed at 2.2 kilobase pairs.



Effect of MIDA1 Antisense Oligonucleotides in MEL Cells

To know the function of MIDA1 during MEL cell differentiation, we examined the effect of the antisense oligonucleotide of MIDA1. Six-base antisense or sense (control) oligomers from translation initiation site were added to the culture. During induced differentiation of MEL cells with Me(2)SO, addition of the antisense oligomer (20 µM) as well as the sense oligomer did not show any effect on the erythroid differentiation (Fig. 7a). However, quite interestingly, the antisense oligomer, but not the sense oligomer, strongly inhibited the growth of MEL cells when the cell number was monitored for 4 days at daily intervals in the uninduced culture (Fig. 7b). The levels of MIDA1 proteins monitored by Western blotting with the anti-MIDA1 serum were dropped in the presence of the antisense oligomer (Fig. 7c). We can hardly find any dead cells with trypan blue staining in all samples, so the addition of the antisense oligomer may affect cell cycle at a certain point. Flow cytometric analysis of the DNA content showed accumulation of S phase cells (48.3-53.2%) and decrease in G(2)+M phase cells (15.4-8.6%) (Fig. 8a). On the other hand, the BrdU incorporation analysis revealed strong decrease in DNA synthesizing cells (35.5-2.4%) (Fig. 8b). Thus, reduction of MIDA1 blocked the progressing DNA synthesis.


Figure 7: Effect of antisense or sense (control) oligomers. a, effect on the differentiation. MEL cells were inoculated at 4 times 10^4 cells/ml in Eagle's minimum essential medium containing 12% fetal calf serum (heat-inactivated at 65 °C for 30 min) and 1.8% Me(2)SO in the presence or absence of 20 µM oligomers, and the differentiation rate was monitored by benzidine staining(3) . b, effect on the cell growth. MEL cells were inoculated at 4 times 10^4 cells/ml in Eagle's minimum essential medium containing 12% fetal calf serum (heat-inactivated at 65 °C for 30 min) in the presence or absence of 20 µM oligomers, and were monitored for 4 days at daily intervals. down triangle, no oligomers; circle, sense oligomers; bullet, antisense oligomers. c, effect on the level of MIDA1 protein. Cell lysates were prepared from 1 times 10^5 cells in each condition on day 2 after the addition of 20 µM oligomers, and the levels of MIDA1 protein were determined by Western blotting. Total protein amount were ascertained to be equal by Coomassie Brilliant Blue staining of SDS-PAGE electrophoresed samples. Lane1, no oligomers; lane2, sense oligomers; lane3, antisense oligomers.




Figure 8: Effect of decreased MIDA1 protein on cell cycle in MEL cells. a, flow cytometric analysis. Cells were collected from day 2 after the addition of oligomers, fixed with 70% ethanol, and resuspended in PBS containing 50 mg/ml of propidium iodide and 10 mg/ml of RNase. Then DNA content was analyzed by FACScan (Becton Dickinson). The graph shows relative DNA content (horizontalline) and cell number (verticalline). Percentage of each cell cycle phase was calculated by Cell Fit software (Becton Dickinson). b, BrdU incorporation analysis. Cells were incubated with 3 mg/ml 5-bromo-2`-deoxyuridine and 0.3 mg/ml 5-fluoro-2`-deoxyuridine for 4 h at 37 °C, smeared on slides, and fixed with 5% acetic acid, 90% ethanol. Then, they were treated with anti-BrdU/nuclease mixed solution (Amersham Corp.) and detected by peroxidase conjugated antibody to mouse IgG, polymerizing diaminobenzidine in the presence of cobalt and nickel.




DISCUSSION

It had been reported that a HLH protein, Id, controls cell differentiation in several cell lineages through interacting with bHLH transcription factors such as MyoD and E2A(3, 4, 5, 6) . In addition, the role of HLH proteins for growth regulation through interacting with non-HLH proteins such as c-Jun and RB has recently been reported(9, 20, 21) . In this work, we have isolated a new protein, MIDA1, that associates with Id by West-Western screening of the MEL cell cDNA library. The Id-associated protein is expected to have HLH motif, but we could not find any canonical HLH motif in MIDA1, even within the Id-associated domain of MIDA1 identified by in vitro association analysis.

The predicted amino acid sequence of MIDA1, however, revealed several interesting features; almost two-thirds of the N terminus closely resembled Zuotin, isolated as a Z-DNA binding protein in yeast (13) (Fig. 2b). Z-DNA is a left-handed DNA conformation that is suggested to implicate in transcription, replication, and recombination of DNA(22, 23, 24) . It was attractive to expect that MIDA1 may regulate these cellular events by affecting DNA conformation, so we tried to examine Z-DNA binding ability of the recombinant MIDA1 protein by electrophoretic mobility shift analysis. Using P-labeled poly(dG-m^5dC) probe as stabilized Z-DNA, significant retardation was observed by MIDA1 protein, but Z-DNA-specific competition has not been obtained. (^2)Thus, the Z-DNA binding ability of MIDA1 remained to be clarified by further improvement of experimental conditions or sample preparation.

Zuotin showed overall homology with N terminus of MIDA1, but two conserved regions (residues 80-169 and 184-289 in MIDA1, residues 89-174 and 200-296 in Zuotin, respectively) were noted. The former conserved region contained ``J region,'' a 70-amino acid DnaJ conserved protein motif (Fig. 2c) that was found in E. coli DnaJ and several eukaryotic DnaJ members identified as essential proteins for cellular localization and folding of proteins (13, 14) . Since J region is considered to mediate interaction with Hsp70s(14, 25, 26) , MIDA1 may also interact with heat shock proteins and may affect conformation of proteins. Interestingly, we found that J region of MIDA1 is located adjacent to the Id binding domain. Since interaction between bHLH protein and heat shock proteins has already been reported(27) , it seems possible that ternary interaction among MIDA1, Id, and heat shock proteins could regulate conformation or localization of some important proteins in the turning point of growth and differentiation. The latter conserved region was almost similar to the region identified by requirement for the association with Id as demonstrated by the in vitro association analysis. Thus, the strikingly conserved region within the Zuotin homology region of MIDA1 may contain a new non-HLH protein motif that can interact with HLH proteins such as the leucine repeat of c-Jun (20) or the pocket domain of RB(9, 21) , although a new motif for protein-protein association motif remains to be clarified.

In the remaining C-terminal region, we found two Trp-mediated repeats that have similar sequence to the DNA binding domain of c-Myb oncoprotein (Fig. 2d). This protein motif contained conserved tryptophan or hydrophobic residues spaced at intervals of approximately 20 amino acids and is proposed to form helix-turn-helix structure(15, 16, 17) . Because last two Trp-mediated repeats from the three of c-Myb is sufficient for sequence specific DNA binding, two repeats of MIDA1 can be suggested to have similar function and participate transcriptional regulation. Preliminary attempts indicated that MIDA1 could not bind the 6-base pairs sequence, YAACKG, recognized by c-Myb, (^3)so searching for recognition sequence by a polymerase chain reaction-associated DNA-binding site selection (28) is now in progress.

We found that loss of MIDA1 by the addition of its antisense oligonucleotide strongly interfered with growth of MEL cells, but not with induction of differentiation. This growth suppression in antisense experiment is consistent with slow growing phenotype of Zuotin null mutant yeast(13) , suggesting that MIDA1 can be one of counterparts of Zuotin in mammals. Furthermore we tried to establish stable transfectants that express MIDA1 gene by sense or antisense orientation. Although over 50 clones for each orientation were established, none can overexpress nor decrease MIDA1 protein expression (data not shown). Observed strict control of protein expression may suggest its important function in cell growth. In the antisense experiment, cell cycle distribution showed accumulation of S phase cells and decrease in G(2)+M phase cells, while the index of BrdU incorporation significantly decreased. Since loss of MIDA1 seemed not to interfere with entry into S phase but DNA synthesis seemed to delay cell cycling especially at S phase, MIDA1 protein may function in DNA synthesis. Although molecular mechanisms of the effect of the MIDA1 in the cell growth is not clear at present, we can speculate the following mechanisms: 1) MIDA1 may affect DNA synthesis directly through Z-DNA binding activity, 2) MIDA1 may be essential for progression of cell cycle, as found in other chaperon proteins in yeast (29, 30) and Hsp70, which was indicated to implicate in S phase progression(31, 32) , 3) MIDA1 may bind to DNA in sequence-specific manner through Trp-mediated repeats and may regulate transcription for cell cycling.

Our demonstration on the growth regulation by MIDA1 as an Id-associated protein in MEL cells supports recent reports on the growth regulation by Id through its association with non-HLH proteins such as RB(9) . Id may be a bifunctional protein that regulates growth and differentiation through interaction with bHLH proteins and non-HLH proteins. Such mutual interaction will be required for the commitment event between growth and differentiation during development and cellular differentiation.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D63784[GenBank].

§
Present address: Inst. of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 102, Japan.

To whom correspondence should be addressed. Tel.: 81-22-274-1111 (ext. 3461); Fax: 81-22-272-5081.

(^1)
The abbreviations used are: HLH, helix-loop-helix; bHLH, basic HLH; MEL, murine erythroleukemia; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BrdU, 5-bromo-2`-deoxyuridine.

(^2)
W. Shoji and M. Obinata, unpublished data.

(^3)
S. Ishii, personal communication.


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