(Received for publication, June 14, 1995)
From the
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) ()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
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/G
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.
Figure 1:
A protein probe for
West-Western screening. a, construction of the protein probe,
GST-Id 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-
Id with MyoD. Left, in vitro translated MyoD (lane1) and c-Myc (lane2) proteins. Right, in vitro translated proteins were mixed with GST-
Id 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.
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,
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.
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.
Figure 4:
HLH
region of Id is essential for association with MIDA1. In vitro translated MIDA1 proteins were mixed with GST-Id or
GST-
(HLH)Id captured by glutathione-Sepharose, and recovered
proteins were analyzed by SDS-PAGE. GST-
Id deletes 49 amino acids
from the N terminus and contains complete HLH domain, while
GST-
(HLH)Id deletes 99 amino acids from the N terminus to lack HLH
region.
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.
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
SSC, 1
FBP, 20 mM NaHPO
pH 6.5,
100 µg/ml of salmon sperm DNA, 10% dextran sulfate, 0.1% SDS)
containing 5
10
cpm/ml of MIDA1 cDNA probe labeled
with random primer extension. A single band was observed at 2.2
kilobase pairs.
Figure 7:
Effect of antisense or sense (control)
oligomers. a, effect on the differentiation. MEL cells were
inoculated at 4 10
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
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
10
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.
, no oligomers;
, sense oligomers;
, antisense oligomers. c, effect
on the level of MIDA1 protein. Cell lysates were prepared from 1
10
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.
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
dC) probe as
stabilized Z-DNA, significant retardation was observed by MIDA1
protein, but Z-DNA-specific competition has not been obtained. (
)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, ()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+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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D63784[GenBank].