MIDA1 is a sequence specific DNA binding protein with novel DNA binding properties.

BACKGROUND: Id proteins not only regulate cell differentiation negatively, but they also promote growth and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth. Its predicted amino acid sequence contains tryptophan-mediated repeats (Tryp-med repeats) similar to the DNA binding region of the c-Myb oncoprotein. We determined whether MIDA1 can bind to DNA in a sequence specific manner by PCR-assisted binding site selection. RESULTS: We identified a 7-base sequence (GTCAAGC) surrounded by a 1-3 bp palindromic sequence as the DNA sequence recognized by the Tryp-med repeats of MIDA1. This motif is located within the 5'-flanking sequence of several growth regulating genes. Gel shift assays revealed that this sequence and a certain length of flanking DNA are necessary for MIDA1 to bind DNA in a stable manner. Methylation interference and DNase I footprint analysis suggested that the DNA binding of MIDA1 is resistant to DNA methylation and that MIDA1 does not specifically localize on this particular motif. CONCLUSIONS: We concluded that MIDA1 is a novel sequence-specific DNA binding protein with some different properties from the usual transcription factors and that MIDA1 may act as a mediator of Id-mediated growth-promoting function through its DNA binding activity.

Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein.

Id proteins not only regulate cell differentiation negatively, but they also promote growth, immortalization, and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth (1). Its predicted amino acid sequence consists of a Zuotin (a Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats (Tryp-med repeats). MIDA1 exhibits a sequence-specific DNA binding activity through the Tryp-med repeats (manuscript in preparation). In this study, we revealed that, like Zuotin, MIDA1 can specifically bind to Z-DNA. This suggested that MIDA is a novel DNA binding protein that has two different DNA binding activities. Furthermore, association of Id1 with MIDA1 stimulated the sequence-specific DNA binding activity, while it inhibited the Z-DNA binding activity. Therefore, we concluded that MIDA1 may act as a mediator of the growth-promoting function of Id, by switching the two DNA binding activities of MIDA1.

Molecular cloning, expression, and activity of zebrafish semaphorin Z1a.

Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.

Direct association of YY-1 with c-Myc and the E-box binding protein in regulation of glycophorin gene expression.

We previously reported that YY-1, a versatile transcription factor, regulates expression of glycophorin gene by binding to its locus control region-like region (Gp-LCR) in combination with E-box binding protein during murine erythroleukemia (MEL) cell differentiation. In the present work, we demonstrated that YY-1 and c-Myc, a nuclear oncoprotein, were physically associated in vivo and that down regulation of c-Myc liberated free YY-1 from its complex, resulting in the functional binding of YY-1 to the Gp-LCR. We also showed that the E-box binding protein (EBP) which bound to E-box was physically associated with YY-1, facilitated binding of YY-1 to the neighboring site and their combinatorial binding may stimulate the GpLCR mediated enhancement of erythroid-specific transcription of glycophorin gene in MEL cells.

Zebrafish semaphorin Z1a collapses specific growth cones and alters their pathway in vivo.

The semaphorin/collapsin gene family encodes secreted and transmembrane proteins several of which can repulse growth cones. Although the in vitro activity of Semaphorin III/D/Collapsin 1 is clear, recent analyses of two different strains of semaphorin III/D/collapsin 1 knockout mice have generated conflicting findings. In order to clarify the in vivo action of this molecule, we analyzed sema Z1a, a zebrafish homolog of semaphorin III/D/collapsin 1. The expression pattern of sema Z1a suggested that it delimited the pathway of the growth cones of a specific set of sensory neurons, the posterior ganglion of the lateral line, in zebrafish. To examine the in vivo action of this molecule, we analyzed (1) the pathways followed by lateral line growth cones in mutants in which the expression of sema Z1a is altered in an interesting way, (2) response of lateral line growth cones to exogenous Sema Z1a in living embryos, and (3) the pathway followed by lateral line growth cones when Sema Z1a is misexpressed by cells along their normal route. The results suggest that a repulsive action of Sema Z1a helps guide the growth cones of the lateral line along their normal pathway.

Regulatory function of delta/YY-1 on the locus control region-like sequence of mouse glycophorin gene in erythroleukemia cells.

The far upstream region (-1.2-0.9 kilobase pairs) of the mouse glycophorin gene contains the locus control region (LCR)-like region, which acts as an erythroid-specific enhancer dependent on chromosomal integration in murine erythroleukemia (MEL) cells. In the present study, we demonstrated that this region binds six nuclear factors. The binding of GATA-1 to corresponding sites did not show any change before or after induction with dimethyl sulfoxide, but the binding of Spi-1/PU.l and an unidentified factor called glycophorin regulatory element binding factor (GRBF) showed a change during induction. While binding activity of Spi-l/PU.l dropped soon after induction, the GRBF activity increased after induction when expression of the glycophorin gene began. After identification of the consensus binding site of GRBF, we cloned cDNA for that factor by Southwestern method, and it was identified as a previously reported transcription factor, delta, a murine form of YY-l which is a versatile transcription factor. Mutation analysis in the delta/YY-1 binding site within the LCR-like region indicated that delta/YY-1 acts as a regulatory protein in combination with the E-box-binding protein that binds to the neighboring sequence.

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.

Phosphorylation of helix-loop-helix proteins ID1, ID2 and ID3.

Id is a helix-loop-helix protein which forms heterodimer with ubiquitous and/or tissue-specific basic helix-loop-helix proteins and inhibits their DNA binding. It has been noted that putative phosphorylation sites for various protein kinases exist in rat Id1, Id2 and Id3. We show here that Id1 and Id2 can be phosphorylated in vitro by cAMP-dependent protein kinase, Id2 and Id3 by cdc2 kinase, and all three Ids by protein kinase C. The phosphorylated Id1 was actually immunoprecipitated in nerve-growth-factor-stimulated PC12 cells. Gel mobility shift assays, however, demonstrated that neither phosphorylation of Id proteins by cAMP-dependent protein kinase nor phosphorylation of E47 by protein kinase C affected the inhibition of E47 homodimer formation and its DNA binding. Taken together with other observations, phosphorylation of Id proteins may play a role in regulation of cell differentiation but not directly in the dimerization and DNA binding.

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.

c-Myc selectively regulates the latent period and erythroid-specific genes in murine erythroleukemia cell differentiation.

During the latent period of murine erythroleukemia (MEL) cell differentiation, c-myc levels showed a significant change and the overexpression of the transferred c-myc gene inhibited the commitment and differentiation of MEL cells, suggesting that c-Myc may be a key molecule for the commitment. Since c-Myc may function as a DNA binding transcription factor, we examined whether c-Myc regulates the latent period genes (hsp and hsc70, MER5, Id and Spi-1 genes) and the erythroid-specific genes [beta-globin, glycophorin, delta-aminolevulinic acid synthase (ALAS-E), GATA-1 and erythropoietin receptor (EpoR)] in the MEL cell transformant having transferred c-myc gene. The overexpression of c-myc gene affected the latent period genes in different ways: hsc and hsp 70 genes and Id gene were positively regulated, while expression of MER5 gene was repressed. While c-myc is thought to be involved in DNA replication, its overexpression showed no effect on the expression of proliferating cell specific nuclear antigen or DNA polymerase a. The overexpression of c-myc repressed the expression of glycophorin, ALAS-E and beta-globin genes, of the five erythroid-specific genes, but had no effect on expression of GATA-1 or EpoR gene. These results suggest that c-Myc differentially regulates the expression of the latent period and erythroid-specific genes.

Role of c-Myc on erythroid differentiation.

In the early event of the induction of mouse erythroleukemia (MEL) cell differentiation, c-myc mRNA levels show a biphasic change. The elevated expression of a transfected c-myc gene inhibits the commitment and differentiation of MEL cell transformants. In the present work, we have introduced human c-myc mutants into MEL cells under the inducible promoter to define the functional domains of c-Myc involved in erythroid differentiation. The c-Myc domains necessary for commitment and differentiation are not colocalized; almost entire regions are required for inhibition of commitment, whereas domains II and IV that are essential for co-transforming activity with ras are required for inhibition of differentiation. Interestingly, mutants that delete domains for c-Myc dimerization motifs enhanced differentiation. Thus, c-Myc interferes with MEL cell differentiation by interacting with c-Myc partners and the induced protein(s) through dimerization domains. These results suggest that c-Myc may regulate commitment and differentiation by interacting with proteins through different domains.

Flow cytometric DNA analysis of lung cancer cell lines.

Tumor DNA content (ploidy) was analyzed by use of flow cytometry (FCM) in 17 lung cancer cell lines which were subcultured in our laboratory. The study included 6 adenocarcinomas, 2 squamous cell carcinomas, 1 adenosquamous cell carcinoma, 5 large cell carcinomas, and 3 small cell carcinomas. Of the 17 lung carcinoma cell lines, 15 revealed aneuploid patterns with DNA index above 1.1, whereas one had diploid. The mean DNA index (DI) in adenocarcinoma, was 1.34 +/- 0.09, DI 1.6, in squamous cell carcinoma, DI 1.0 in adenosquamous cell carcinoma, DI 1.70 +/- 0.66 in large cell carcinoma, and DI 1.29 in small cell carcinoma. Of the 17 cell lines, three lines showed multiploid patterns with clinically poor prognosis and indicated heterogeneity. Flow cytometric DNA analysis using lung cancer cell lines could provide further basic study of lung cancer cells and give a useful information on the degree of the malignancy clinically.