INTRODUCTION

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Differentiation is a fundamental attribute of the cells of multicellular organisms that is absolutely required for the formation of their bodies. Such an attribute, however, is not specific to the cells of multicellular organisms. The cells of many unicellular organisms often undergo differentiation to survive hostile environments. Yeast is among such organisms and carries out a process generally called sexual development. In response to mating pheromone together with or without nutrient starvation, the cells conjugate with those of opposite mating type and perform meiosis and sporulation (10, 11). The resulting spores are highly resistant to a variety of stresses, including nutritional starvation.

From the regulatory point of view, the process of cell differentiation can conceptually be divided into two steps: the commitment to differentiation and the subsequent expression of genes that determine the phenotype of differentiated cells. The control of the commitment to differentiation is crucial for the timing of differentiation, whereas the control of the subsequent gene expression is crucial for the expression of the particular differentiated phenotype. The step of the commitment to differentiation is regulated by a variety of signals and cellular conditions, including availability of differentiation factors, cell-cell contacts, and physical and chemical stresses for the higher eukaryotes versus nutritional starvation and mating pheromone for yeasts (16, 39). Since the control of differentiation commitment is less likely to be directly linked to the control of the expression of the desired differentiated cell phenotype, this regulation might be general and largely, if not entirely, conserved throughout eukaryotes.

The fission yeast Schizosaccharomyces pombe is similar to higher eukaryotes in its mode of cell division, control of the cell cycle, gene structure, regulation of gene expression, and a variety of other cellular processes (25). This organism commits conjugation and subsequent meiosis and sporulation upon nutrient starvation and the simultaneous availability of mating partners (12). A key factor involved in the commitment process is ste11⁺, a transcriptional regulator with an HMG box. Nutritional starvation induces and/or activates ste11⁺, which in turn activates a set of genes that are required for conjugation and meiosis (65). Among these are ste6⁺, which is required for mating pheromone signal transduction (26); fus1⁺, which is required for cell fusion (56); and mei2⁺, which is required for meiosis (75). The mei2⁺ gene product, however, is inactivated until conjugation takes place (76). This inactivation is achieved by the action of the Pat1 kinase (76). When a mating partner comes close, the mating pheromones released from each cell induce rep1⁺, which is essential for premeiotic DNA synthesis, and mat1⁺-P or mat1⁺-M in the partner cell, which are required for the induction of mei3⁺ (2, 42, 66). Conjugation allows the mat1⁺-P and mat1⁺-M gene products to form an active complex, which in turn induces the mei3⁺ gene that encodes an inhibitor of Pat1 kinase (42). Thus, conjugation leads to the inactivation of Pat1 kinase, thereby allowing the activation of Mei2 protein. Thus, the role of Pat1 is to block the onset of meiosis until conjugation takes place. Inactivation of Pat1, therefore, induces unconditional meiosis to heterothallic haploid cells and inevitable lethality (12). This lethality can be rescued by the inactivation of mei2⁺ or ste11⁺ which is essential for the expression of mei2⁺ and other gene absolutely required for meiosis (28, 51, 65, 66). Therefore, any genes that repress ste11⁺ or inhibit its function could suppress this lethality.

In the signal cascades for sexual development, ste11⁺ acts as a key target for the control of differentiation commitment. The cAMP-Pka1 pathway mediates glucose and nitrogen signals and negatively regulates the onset of differentiation by mainly repressing the ste11⁺ gene (65). Recently, our laboratory identified three new factors controlling the commitment to differentiation. The stress mitogen-activated protein (MAP) kinase encoded by phh1⁺/sty1⁺/spc1⁺ is required for the induction of ste11⁺ (32, 63, 80). Rcd1, a novel protein highly conserved among eukaryotes, is required for nitrogen starvation-invoked ste11⁺ expression (52). On the other hand, an RNA binding protein encoded by nrd1⁺ negatively regulates the onset of differentiation by repressing a subset of Ste11-regulated genes until cells reach a critical level of nutrient starvation (70).

Quite interestingly, mammals contain homologous counterparts of the components of this Ste11 regulatory system. Tcf-1/Lef-1 is a Ste11-like factor with the HMG motif essential for the terminal differentiation of T cells (71, 74). The cAMP-Pka1 cascade is well documented to negatively regulate differentiation in hematopoietic cells (17, 37), just as in fission yeast cells (41). p38 (24, 59), a homologue of Phh1 MAP kinase, influences differentiation (32). Mammals, plants, and nematodes contain well-conserved structural homologues of rcd1⁺ (>70% amino acid identity) (52). Thus, a basic mechanism controlling the commitment to differentiation might be conserved to a certain extent throughout eukaryotes.

Based on these facts, we assumed that some differentiation-controlling factor might be conserved at such a level that mammalian counterparts are functional in yeast cells, and we screened rat and human cDNA libraries for clones that suppress the lethality of the temperature-sensitive pat1-114^ts mutant. This screening resulted in the isolation of a functional homologue of nrd1⁺ from both libraries. Here, we report the cloning and functional analysis of this homologue, named ROD1, whose expression blocks differentiation of a human leukemia cell line as expected.

	MATERIALS AND METHODS

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S. pombe strains and media. The S. pombe strains used in this study have the genotype h pat1-114 leu1-32 and h⁹⁰ ura4-D18 nrd1::ura4⁺. Media were prepared as described previously (10, 23, 45, 48, 50).

Isolation of multicopy suppressors. trans-Complementation cloning of the ROD1 gene was performed as described previously (50) with h pat1-114 leu1-32 cells as a cloning host. A rat kidney fibroblast (NRK-49F) cDNA library and a human fibroblast cDNA library were constructed with the pcD2 vector (6) and transfected into the mutant yeast together with the pAL19 transducing vector (50). Cells were spread on minimum medium agar (MMA) plates, incubated at 23°C for 24 h, and then further incubated at 32.2°C for 4 to 5 days to select rescued cells. The colonies that formed on MMA plates were isolated and subjected to an instability test to distinguish authentic transformants from phenotypic revertants. Plasmid cDNA clones were recovered in Escherichia coli from the colonies that passed the instability test and confirmed for their suppressor activities by subsequent transfection into the host strain. DNA sequencing was performed by the dideoxynucleotide method (61) after being subcloned into M13-derived vectors and pBluescript II KS(+) (Stratagene). The sequence was confirmed by sequencing both strands.

RNA binding analysis. A nitrocellulose membrane was washed with RNase-free distilled water and then dried. The washed membrane was spotted with 40 µg of poly(A), poly(U), poly(C), and poly(G) and then dried. The RNA homopolymers were then immobilized to the membrane by UV cross-linking. The membrane was incubated for 30 min in 5% nonfat dried milk in 10 mM Tris-HCl (pH 7.2) containing 150 mM NaCl and 0.05% Tween 20 (M-TBST). A glutathione S-transferase (GST)-fused rat Rod1 protein was produced in E. coli from the pGEX2T vector (Pharmacia) containing the Dra1 fragment of the rat ROD1 cDNA. The fusion protein was purified with glutathione-agarose. A 0.5-ml M-TBST solution containing GST-rat Rod1 fusion protein at 20 µg/ml was laid on the membrane and incubated at room temperature for 2 h. The membrane was then washed twice with M-TBST for 10 min and incubated with anti-human Rod1 polyclonal antibody for 1 h. After two washes with M-TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (diluted 1:1,000) (Amersham), and signals were detected by enhanced chemiluminescence (Amersham). A negative control experiment was carried out with the same amount of GST protein, followed by detection with anti-GST polyclonal antibody (MBL).

Antibody production. The anti-human Rod1 rabbit polyclonal antibody was generated against the whole GST-fused human Rod1 protein produced in E. coli with the pGEX2T vector (Pharmacia) containing the full-length human ROD1. The anti-human Rod1 monoclonal antibody was also generated against the whole GST-fused human Rod1 protein. Both were obtained from MBL.

Conjugation assay. The mating frequency of the h⁹⁰ ura4-D18 nrd1::ura4⁺ strain was assayed as follows. Cells were grown to mid-log phase in pombe minimum (PM) medium (2% glucose), washed with sterile water, and inoculated in low-glucose (0.5%) PM, NH₄Cl-free PM, or NH₄Cl-free low-glucose (0.5%) PM medium at a density of 5 × 10⁶ cells/ml followed by incubation at 30°C. After incubation for the indicated times, 1 ml of cell suspension was removed and sonicated gently, and the numbers of zygotes were counted under the microscope. The percent mating frequencies were calculated by dividing the number of zygotes (one zygote was counted as two cells) by the number of total cells.

Northern blot analysis. Total RNA was prepared (13), and Northern blot analysis was performed as described earlier (47). The DNA probes used are the 1.3-kb PvuII fragment of ste11⁺ (65), the 3.2-kb ClaI fragment of mei2⁺ (75), the 1.9-kb cDNA fragment of rep1⁺ (66), and the 0.7-kb HindIII fragment of sxa2⁺ (29).

Cell culture, DNA transfection, and proliferation assay. The human leukemia cell lines K562 (JCRB 0019) (40), KG-1 (JCRB 9051) (35), U937 (JCRB 9021) (67), Jurkat (ATCC TIB 152) (78), and Daudi (JCRB 9071) (34) were cultured in 5% CO₂ at 37°C in RPMI 1640 medium containing L-glutamine (Irvine Scientific) supplemented with 10% heat-inactivated fetal calf serum (Gibco). HL60 (JCRB 0085) (8) was maintained in the same medium but with 20% fetal calf serum.

Detection of Rod1 protein. Approximately 10⁶ cells were washed twice with ice-cold PBS and incubated in 0.5 ml of ice-cold 10% trichloroacetic acid (TCA) solution for 30 min. Denatured cells were pelleted by centrifugation at 15,000 rpm for 5 min at 4°C with a Microfuge. The pelleted cells were lysed by gentle sonication in 80 µl of 9 M urea containing 2% Triton X-100 and 1% dithiothreitol (DTT). After the addition of 20 µl of 10% lithium dodecyl sulfate (LiDS) and 10 µl of 1 M Trizma base, cell lysates were sonicated again until their viscosity disappeared and then centrifuged to remove the insoluble material. The cell extracts (5 µl each) derived from 5 × 10⁴ cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot as described previously (31).

Analysis of megakaryocytic differentiation of K562 cells. TPA (Sigma) was stored at 20°C as a stock solution in dimethyl sulfoxide and diluted with RPMI medium just before use. K562 cells exponentially growing at 10⁵ cells/ml were incubated with culture medium containing 10 nM TPA for up to 3 days. Their megakaryocytic differentiation was monitored by measuring the expression of three independent differentiation markers. The expression of the platelet-specific cell surface glycoprotein IIb/IIIa (CD61) was determined by fluorescence-activated cell sorter (FACS) analysis (Becton Dickinson) and by scanning with an argon laser microscope after immunostaining. The cells were cultured for the indicated times or for 3 days in the growth medium containing the indicated concentrations of TPA. The cells were then harvested, washed twice with cold PBS, and incubated for 30 min on ice with 1:10-diluted fluorescein-labeled mouse anti-human CD61 monoclonal antibody (Dako Corp.) in PBS. Stained cells were washed with cold PBS and fixed in 1% (wt/vol) paraformaldehyde in PBS. Mock-treated cells were similarly analyzed as a negative control. Cells with signals higher than the negative control were considered CD61 positive, and the percent megakaryocytic differentiation was calculated by dividing the population of the CD61-positive cells by the total cell population. The expression of the ₂ integrin protein (CD49b) was assessed by argon laser microscopy after staining with 1:10-diluted fluorescein-labeled mouse anti-human CD49b antibody (Immunotech).

Analysis of erythroid cell differentiation. n-Butyric acid sodium salt (NaB) (Sigma) was dissolved in PBS, adjusted to pH 7.2 with NaOH, adjusted to a concentration of 1 M, and filter sterilized. Erythroid differentiation of K562 cells was induced by culturing them for 7 days in medium containing 1.25 mM NaB. The cells were harvested, washed twice with PBS, and pelleted. The cell pellets were lysed by vortexing in distilled water (100 µl per 10⁶ cells) containing 0.01% Nonidet P-40 at room temperature and then placed on ice. The protein amounts of the extracts were quantified by the Bradford method (Bio-Rad) and adjusted to 2.5 mg/ml with ice-cold water. The diaminofluorene (DAF) colorimetric reagent (0.6 ml) was added to each 0.2 ml of the adjusted cell extracts, followed by incubation at room temperature for 5 min. Within 10 min, the optical densities were measured with a Bio-Spec 1600 (Shimadzu) spectrophotometer at 610 nm. The DAF colorimetric reagent was made of 0.1 ml of DAF stock solution (1% [weight per volume] of DAF in 90% acetic acid), 0.1 ml of 30% H₂O₂, and 10 ml of 0.1 M Tris-HCl (pH 7.0) containing 6 M urea. For each photograph, 50 µl of each of the cell lysates was mixed with 150 µl of the DAF colorimetric reagent in the wells of a flat-bottom 96-well plate.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Isolation of ROD1 gene. To search for mammalian genes involved in differentiation control, rat and human fibroblast expression cDNA libraries were screened for genes that suppress the lethality of the pat1-114^ts mutant. By using this screening strategy, two mutually related cDNA clones were obtained. As described below, they were the rat and human counterparts of the same gene and were named ROD1 (regulator of differentiation 1). Both cDNAs rescued the pat1 lethality at restrictive temperatures of up to 34°C.

View larger version (129K): [in this window] [in a new window]   FIG. 1.   (A) The nucleotide sequence of ROD1 and the deduced amino acid sequence of the putative encoded protein. The conserved amino acid sequences of RNP1 and RNP2 are boxed. The MAP kinase and Cdk2 kinase phosphorylation consensus sites are underlined with solid and broken lines, respectively. (B) Amino acid homology between rat and human Rod1 sequences. Only part of the proteins is shown where the amino acid sequence differs. (C) Structure of Rod1 and Nrd1 proteins. They have four internal repeats of RNA binding domains. PYBP (rat hnRNP I) (4, 19) and hnRNP L (57) protein and snRNP U1A (46) sequence repeats are aligned. The conserved segments of RNA binding domains are denoted RNP1 and RNP2 (in boxes), and the other conserved amino acid residues are indicated in boldface letters. (D) Rod1 has RNA binding activities. GST-Rod1 fusion protein preferentially binds poly(G) and poly(U). The negative control is GST protein.

ROD1 shares a functional similarity with nrd1⁺. The fact that the ROD1 gene was isolated by the same screening strategy as that used for nrd1⁺ and that it encodes an RNA binding protein similar to Nrd1 prompted us to investigate the functional similarity between ROD1 and nrd1⁺. Cells deleted for nrd1⁺ commit conjugation without nutrient starvation (70). In nitrogen-rich low-glucose (0.5%) medium or in nitrogen-poor high-glucose (2%) medium, nrd1 disruptants efficiently conjugate, whereas nrd1⁺ cells remain uncommitted to conjugation or else conjugate poorly. Moreover, in nitrogen-poor low-glucose (0.5%) medium, the standard condition for inducing mating of the fission yeast, the nrd1 disruptants conjugate much earlier and to a higher extent than did nrd1⁺ cells. We compared the ability of ROD1 with that of nrd1⁺ to suppress the phenotype of the nrd1 disruptants under these three culture conditions. The ROD1 and nrd1⁺ coding sequences were inserted in the pcL vector and introduced into a homothallic h⁹⁰ nrd1 disruptant and exposed to the three culture conditions for the induction of mating. As shown in Fig. 2, in these assays, both human and rat ROD1 behaved like nrd1⁺ in their ability to suppress the mating-proficient phenotype of the nrd1 disruptant.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   ROD1 inhibits conjugation of h90 nrd1 cells in nitrogen-rich medium. h90 nrd1 cells were transfected with pcL-rat ROD1, pcL-human ROD1, pcL-nrd1+, or pcL. Transfectants were grown in PM (2% glucose) to mid-log phase and incubated in PM containing 0.5% glucose (A), nitrogen-free PM (2% glucose) (B), or nitrogen-free PM containing 0.5% glucose (C). Cells were harvested at the indicated times, and the percent conjugation was calculated by dividing the number of formed zygotes by the number of total cells. h90 cells transfected with empty pcL was used as a positive control.

View larger version (132K): [in this window] [in a new window]   FIG. 3.   Overexpression of nrd1+ or ROD1 similarly represses sxa2+ and rep1+ genes derepressed in the nrd1 disruptant. Exponentially growing h90 nrd1 cells transfected with empty pcL, pcL-nrd1+, or pcL-ROD1 were transferred into PM containing 0.5% glucose and incubated for the indicated times. The cells were harvested, and the total RNA was prepared. Then, 20 µg each of total RNA was applied to each lane for Northern blot analysis.

Rod1 protein is expressed in hematopoietic organs from the early stages of development. The structural and functional similarity of ROD1 to nrd1⁺ suggested that ROD1 might play a role controlling differentiation in mammals. We investigated this possibility. Because of high amino acid sequence conservation between human and rat Rod1 (Fig. 1B), we first generated polyclonal and monoclonal antibodies against human Rod1 and used them to analyze by Western blotting the tissue-specific expression of Rod1 in rats. Lysates from various tissues of embryonic and postnatal rats were examined. As shown in Fig. 4, Rod1 protein was detected as two bands, of 57 and 50 kDa, by SDS-PAGE. Other anti-Rod1 monoclonal and polyclonal antibodies reacting with different epitopes detected both bands, indicating that both bands were ROD1 gene products. Consequently, one or both of these double bands were likely to be generated by modification or slight truncation of the original Rod1 protein. In 4- to 7-week-old adult rats, Rod1 protein was detected specifically in the spleen, thymus, lungs, and bone marrow (Fig. 4A). In this assay, -actin protein was used as a control for detection in nonmuscle tissues. The relatively low signal of -actin, at least in the pancreas, may partly be due to the relatively high expression of muscle-type actins in these organs (18). At early stages of development, Rod1 was expressed in a variety of tissues, even in brain, muscle, and kidney, but it was expressed predominantly in the thymus and liver, where hematopoiesis occurs at these stages (9, 58) (Fig. 4B). We concluded that Rod1 is predominantly expressed in hematopoietic organs throughout development.

View larger version (60K): [in this window] [in a new window]   FIG. 4.   (A) Rod1 expression in various organs of rats at 5 weeks of age. (B) Rod1 expression in various organs in early stages of development. Lysates (10 µg of protein) prepared from various tissues of embryonic (E) and postnatal (P) rats were separated by SDS-8% PAGE, and the Rod1 protein was detected by Western blotting with the anti-human Rod1 monoclonal antibody 2B3. As a reference, -actin protein was detected by reimmunoblotting the same membrane filters that were used for Rod1 detection. The asterisks indicate tissues expressing muscle-type (non-) actin relatively abundantly (18, 54). Two arrowheads indicate Rod1 proteins migrating as ca. 57- and 50-kDa bands.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Expression of Rod1 in hematopoietic cell lines during in vitro differentiation. Rod1 protein was detected by Western blotting in the hematopoietic cell lines HL60, KG-1, K562, U937, Jurkat, and Daudi during differentiation. Lysates prepared from 5 × 104 cells were applied to each lane for SDS-PAGE, and immunoblot detection was carried out as described in Materials and Methods.

Overexpression of ROD1 inhibits TPA-induced megakaryocytic differentiation of K562 cells. Overexpression of nrd1⁺ in fission yeast cells inhibits their conjugation and meiosis without affecting the growth property (70). We therefore examined whether ROD1 exerts a similar activity in mammalian cells. For this experiment, we took the pluripotent hematopoietic human leukemia cell line K562 as a model because this cell line expressed ROD1, as already shown, and differentiates to megakaryocytes upon TPA treatment. Differentiation of this cell can be monitored by the induction of platelet-specific genes, morphological changes, and increased cell-cell and cell-substrate adhesions.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Growth properties of ROD1 overexpressors. (A) Level of Rod1 expressed in the stable ROD1 transfectants. The human ROD1 cDNA inserted into the CMV promoter-based expression vector (27) was transfected into K562 cells by electroporation. Stable transfectants were selected in medium containing Geneticin (G418 sulfate) at 1.5 mg/ml as described in Materials and Methods. Cell lysates were prepared from each stable transfectant, and Rod1 protein was detected by Western blotting with the anti-human Rod1 monoclonal antibody 2B3. Rod1-C2, Rod1-C3, Rod1-C7, and Rod1-C9 are transfectant clones expressing high levels of Rod1. Neo1 to Neo10 are also stable transfectants of an empty vector and are used as a negative control. (B) ROD1 overexpression does not affect the growth rate. Cells were plated at 105 cells/ml and incubated to monitor the growth of each Rod1 overexpressor. The cell number was counted at the indicated times. The values in the figure are the means ± the standard deviation for the Rod1 overexpressors and for Neo1, -2, -5, and -7.

View larger version (109K): [in this window] [in a new window]   FIG. 7.   Overexpression of ROD1 inhibits TPA-induced megakaryocytic differentiation of human K562 cells. The ROD1 transfectants and empty vector transfectants were cultured in growth medium containing 10 nM TPA. On the day indicated, cells were stained with anti-CD61-fluorescein isothiocyanate (FITC) monoclonal antibody followed by FACScan analysis. (A) Time course of the emergence of CD61-positive cells during the induction of megakaryocytic differentiation. The percent population of CD61-positive cells was calculated by dividing the number of FITC-positive cells by the number of total cells. The mean percents CD61-positive cells in ROD1 overexpressors (Rod1-C2, -C3, -C7, -C9, and -C10) or Neo clones were plotted on a graph with the standard deviation. The right figures show the FACS patterns of the Neo2 and Rod1-C2 after TPA treatment for various times. (B) The ROD1 overexpressors (Rod1-C2 and Rod1-C9) and negative controls (Neo2 and Neo4) were induced for differentiation by culture in medium containing 0.6 nM TPA for 3 days, then stained with anti-CD61-FITC monoclonal antibody and analyzed by using an argon laser microscope (Zeiss). (C) Rod1 overexpression blocks the induction of the PDGF gene, another early differentiation marker. The human Rod1 transfectants and negative controls were cultured in medium containing 10 nM TPA and harvested at the indicated times. Cell lysates (32 µg of protein each) were then separated by SDS-15% PAGE, and the PDGF-B was detected by Western blotting with an anti-PDGF-B polyclonal antibody. The arrowhead indicates the PDGF-B protein migrating as a 30-kDa band upon SDS-PAGE. (D) ROD1 overexpression blocks the induction of CD49b, a late marker of megakaryocytic differentiation. The human ROD1 overexpressors (Rod1-C2 and Rod1-C9) and the negative controls (Neo2 and Neo4) were cultured in medium containing 50 nM TPA. After a 3-day incubation, the cells were stained with anti-CD49b-FITC monoclonal antibody and analyzed by using an argon laser microscope (Zeiss).

View larger version (34K): [in this window] [in a new window]   FIG. 8.   Dose responses of K562 and its ROD1 overexpressors to TPA for megakaryocytic differentiation. The human ROD1 overexpressors (Rod1-C2, -C3, and -C9) and the negative controls (Neo1, -2, -4, and -5) were cultured in medium containing various concentrations of TPA for 3 days and stained with anti-CD61-FITC monoclonal antibody followed by FACScan analysis. (A) FACS patterns of Rod1-C3, Rod1-C9, and Neo2 treated with various TPA concentrations. (B) The percent cell population of megakaryocytic differentiation. Values of Neo clones and Rod1 clones are the means ± the standard deviation for four and three independent clones, respectively.

ROD1 overexpression also inhibits sodium butyrate-induced erythroid differentiation of K562 cells. The ability of Nrd1 to inhibit fission yeast differentiation is not specific to a particular nutrient starvation signal (70). We therefore examined whether the function of Rod1 is specific to particular differentiation signals or even to particular differentiation phenotypes. Treatment with NaB or hemin induces K562 to differentiate to erythroid cells (1, 60). The four ROD1 overexpressors (Rod1-C2, Rod1-C3, Rod1-C7, and Rod1-C9) and the four control clones were treated with NaB for 7 days, and erythroid differentiation was monitored by colorimetrically measuring the amount of synthesized hemoglobin, which is stained blue with DAF. As shown in Fig. 9, NaB-induced erythroid differentiation was also strongly blocked by Rod1 overproduction. Thus, Rod1 action was not specific to particular differentiation signals or differentiation phenotypes, which is the property expected for Rod1 on the basis of its similarity to Nrd1.

View larger version (47K): [in this window] [in a new window]   FIG. 9.   ROD1 blocks NaB-induced erythroid differentiation of K562 cells. The ROD1 transfectants and negative controls were induced to differentiate in culture medium containing 1.25 mM NaB for 7 days. Erythroid differentiation was measured by determining the level of produced hemoglobin by staining with DAF. (A) Actual blue staining of the cell lysates. (B) Colorimetric quantification of hemoglobin synthesized in the cells indicated in panel A. Values are the means ± the standard deviation for four clones each of Rod1 overexpressors and Neo control cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Like nrd1⁺, the ROD1 gene was isolated as a multicopy suppressor of the pat1-114 mutant performing lethal haploid meiosis at the nonpermissive temperature, and it encodes an RNA binding protein. Both Nrd1 and Rod1 molecules are similar in size and have four RNA binding domains, but they differ in the relative positions and sizes of the spacer regions. In addition, they are not identical in the base specificity for RNA binding. Nrd1 binds poly(U) (70), whereas Rod1 binds both poly(U) and poly(G) (Fig. 1D). Nevertheless, when expressed in fission yeast cells, ROD1 was functionally indistinguishable from nrd1⁺, in that ROD1 acted as a negative regulator of differentiation. The role of nrd1⁺ is to block differentiation by repressing Ste11-regulated genes until the cells reach a critical level of nutrient starvation (70). Ste11 is a transcriptional factor that plays a pivotal role in the control of differentiation in the fission yeast S. pombe (65). At present, the mechanism for the repression by nrd1⁺ is not fully understood but, in the absence of nrd1⁺, a subset of the Ste11-regulated genes, which include rep1⁺ and sxa2⁺ and require the presence of mating pheromone signals for induction, are particularly derepressed, suggesting that the function of Nrd1 might be to block the action of Ste11, particularly with regard to the genes that require mating pheromone signals for activation. ROD1 resembles nrd1⁺, not only in the suppression of differentiation but also in the ability to repress these genes that are derepressed in the cells lacking nrd1⁺, suggesting that both factors inhibit differentiation by the same molecular mechanism.

The resemblance between nrd1⁺ and ROD1 goes further. The overexpression of nrd1⁺ inhibits the differentiation of fission yeast without affecting its growth properties (70). Similarly, the overexpression of ROD1 blocked TPA-induced megakaryocytic differentiation as well as NaB-induced erythroid differentiation of K562 cells without influencing the growth properties of the cells. One remarkable aspect of nrd1⁺ is that its action is independent of the differentiation-inducing signals (70). The differentiation signal-independent or even differentiation phenotype-independent action of ROD1 further supports the similarity between nrd1⁺ and ROD1, although we do not know whether ROD1 could act as a universal blocker of differentiation or not. These similarities suggest that ROD1 might be a functional homologue of the fission yeast nrd1⁺ gene, with a role negatively controlling differentiation of hematopoietic cells where this factor is abundantly expressed throughout development.

On the other hand, the slight increase in the level of Rod1 protein during the differentiation of various hematopoietic cell lines seems in conflict with its putative role as a negative regulator of differentiation. However, such unexpected behavior may not be so unusual. The fission yeast cig2⁺/cyc17⁺ gene encoding a B-type cyclin plays a crucial role in switching between growth and differentiation (49, 82). This cyclin blocks differentiation and its timely inactivation is essential for the onset of differentiation. Strangely, this cyclin gene is induced during differentiation (49).

Although more experiments need to be done to demonstrate this conclusively, the possibility that ROD1 is a mammalian counterpart of nrd1⁺ may not be so remote. Mammalian homologues of some fission yeast genes regulating the onset of differentiation have recently been isolated, including two novel positive ste11⁺ regulators that were isolated in our laboratory. One, rcd1⁺, is essential for nitrogen starvation-invoked ste11⁺ expression (52). Very intriguingly, human, nematode, plant, and budding yeast cells all contain extremely conserved homologues of this gene (>70% amino acid identity), the human counterpart of which is predominantly expressed in reproductive as well as in hematopoietic organs (52). The second is the Phh1/Sty1/Spc1 stress MAP kinase, which is essential for stress responses and for the onset of differentiation in fission yeast cells (32, 63, 80). It induces ste11⁺ via the Atf1 transcriptional factor (63). As shown previously, p38 is a mammalian homologue of this kinase and is involved in the control of not only stress responses but also growth and differentiation of at least hematopoietic cells (15, 24, 38). Furthermore, as in the fission yeast, the cAMP-PKA pathways play an important role in controlling the differentiation of at least hematopoietic cells in mammals (17, 37).

Conservation does not seem to be restricted to these ste11⁺-regulatory factors. Mammals contain even putative homologues of the ste11⁺ gene itself. Ste11 possesses an HMG box domain, and mammals have many HMG box proteins. Generally, HMG box proteins found in various organisms are categorized into two groups based on the presence or absence of high sequence specificity for DNA binding. The sequence-nonspecific group includes HMG-1, UBF, and MT-TF1 (30, 53, 79), whereas the sequence-specific group includes S. pombe Ste11 (65) and MatMc (33), the mammalian sex-determining factor Sry (22, 64), and several putative regulators of lymphoid differentiation, including Sox-4 (72), Tcf-1 (71), and Lef-1 (69, 77). Sry has been proposed to act as a transcriptional regulator, which recognizes specific sequences in DNA. Sox-4 is expressed in T and pre-B lymphocytes and acts as a classical transcriptional activator (72). In Sox-4^/ mice, B-cell development is blocked at the pro-B-cell stage, in addition to a defect in the formation of heart valves (62). On the other hand, in Tcf1^/ mouse, T-cell differentiation is blocked at a late stage (74). The TCF/LEF family is conserved at least among the Caenorhabditis elegans, Drosophila, Xenopus, and mammalian cells (44, 73). Although much work needs to be done, the presence of mammalian factors similar to those controlling fission yeast differentiation suggests that the system controlling the commitment to cell differentiation might be conserved throughout eukaryotes.