MIDA1, a protein associated with Id, regulates cell growth.

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.

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.
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 -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 rϪ bacterial strain. After incubation at 42°C for 3 h, 25 mM isopropyl-1-thio-␤-D-galactopyranoside-impregnated nitrocellulose filters (Schleicher & Schuell) were overlaid on the plates to induce ␤-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 g/ml of bacterially produced glutathione S-transferase (GST)-⌬Id protein (3)  In Vitro Association Studies-In vitro transcription and translation were performed under conditions recommended by the Promega Protocols and Application Guide. 35 S-Labeled proteins were produced in the lysate from pCITE2 vectors (Novagen) that have each cDNA inserts. To check the translation products, 1 l from each lysate was subjected directly to SDS-polyacrylamide gel electrophoresis, and 5 l was diluted into 500 l of HBB buffer and incubated with either GST or GST-⌬Id affinity matrices in which approximately 5 g of fusion protein adsorbed to 10 l 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 ␤-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 ϫ 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 g/ml antipine, 5 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The cleared lysate was incubated with 100 l 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% ␤-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 g of RNA from each sample was electrophoresed in a dena-* 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. tured 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 ϫ SSC, 1 ϫ FBP (1% Ficol, 1% polyvinylpyrrolidone, 1% bovine serum albumin), 20 mM NaHPO 3 pH 6.5, 100 g/ml of salmon sperm DNA, 10% dextran sulfate, 0.1% SDS). After overnight incubation at 42°C, the membrane was washed in 2 ϫ SSC, 0.1% SDS for 10 min at room temperature and in 0.1, ϫ 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 ϫ 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 ϫ 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 g/ml of propidium iodide (Sigma) and 10 g/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-⌬Id (Fig. 1a) and expressed it in Escherichia coli. ⌬Id 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 ϫ 10 7 colony forming units of complexity using poly(A) mRNAs of MEL cells, and approximately 5 ϫ 10 5 plaques were screened for their ability to interact with GST-⌬Id. 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 5Ј-and 3Ј-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 Trpmediated 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).
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.
[ 35 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-⌬Id affinity matrices (18). As shown in Fig. 3b, wildtype, ⌬2-71, and ⌬2-188 of MIDA1 could associate with Id, but ⌬2-256 and ⌬2-342 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, Idbinding activity of ⌬2-188 was stronger than that of wild-type or ⌬2-71, 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 ⌬2-188 was deleted from C terminus to lack one (⌬2-188, ⌬526 -621) or two (⌬2-188, ⌬443-621) 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).
Then we asked whether the HLH region of Id is essential for association with MIDA1. Binding assay with GST-⌬Id and GST-⌬(HLH)Id, which completely deletes its HLH domain, showed that GST-⌬(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.
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 sub-  (14). d, sequence alignment of Trp-mediated repeats from MIDA1 and c-Myb proteins from various species. Conserved amino acids are reversed. jected 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.
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.
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 M) 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 ϩM 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. 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 (3-6). 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 32 P-labeled poly(dGm 5 dC) 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 2 W. Shoji and M. Obinata, unpublished data.

FIG. 2-continued
Id-associated Protein Regulates Cell Growth 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 Trpmediated repeats from the three of c-Myb is sufficient for se-quence 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. 3 S. Ishii, personal communication.
FIG. 3. Id-binding activity of MIDA1 deletion mutant. a, MIDA1 mutants deleted sequentially from N terminus were constructed and translated in vitro. For example, ⌬2-71 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-⌬Id 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, ⌬2-71, and ⌬2-188 could be recovered by GST-⌬Id, but other deletion mutants could not. c, ⌬2-188 was deleted from C terminus to determine whether Trp-mediated repeats contribute Id-binding activity. ⌬2-188,⌬526 -621 lacks one Trp-mediated repeat, and ⌬2-188,⌬443-621 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.
FIG. 5. In vivo association between MIDA1 and Id. Stable GST-Id-transfected MEL cells and parent cells were treated with crosslinking 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.
FIG. 6. Expression of MIDA1 mRNA. 10 g 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 ϫ SSC, 1 ϫ FBP, 20 mM NaHPO 3 pH 6.5, 100 g/ml of salmon sperm DNA, 10% dextran sulfate, 0.1% SDS) containing 5 ϫ 10 5 cpm/ml of MIDA1 cDNA probe labeled with random primer extension. A single band was observed at 2.2 kilobase pairs. 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 ϩM 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.