The carboxyl-terminal domain of the granulocyte colony-stimulating factor receptor uncouples ribosomal biogenesis from cell cycle progression in differentiating 32D myeloid cells.

Translational regulation plays an important role in development. In terminally differentiating cells a decrease in translation rate is common, although the regulatory mechanisms are unknown. We utilized 32Dcl3 myeloblast cells to investigate translational regulation during granulocyte colony-stimulating factor (G-CSF)-induced differentiation. G-CSF causes a significant decrease in translation rate compared with interleukin-3, which is a mitogen for these cells. Although these two cytokines exhibit modest differences in their effect on translation factor phosphorylation, they exhibit dramatic differences in their effect on ribosomal abundance and ribosomal DNA transcription. However, because both cytokines stimulate cell cycling, G-CSF induces a dissociation of ribosomal biogenesis from cell cycle progression. This uncoupling of ribosomal biogenesis from cell cycle progression appears to be closely related to the transmission of a differentiation signal, because it is not observed in cells expressing a carboxyl-terminally truncated G-CSF receptor, which supports proliferation but not differentiation of these cells. Because a similar event occurs early in differentiation of murine erythroleukemic cells, this suggests that ribosomal content is a common target of differentiating agents.

The yeast hsp70 homologue Ssa is required for translation and interacts with Sis1 and Pab1 on translating ribosomes.

The 70-kDa heat shock proteins are molecular chaperones that participate in a variety of cellular functions. This chaperone function is stimulated by interaction with hsp40 proteins. The Saccharomyces cerevisiae gene encoding the essential hsp40 homologue, SIS1, appears to function in translation initiation. Mutations in ribosomal protein L39 (rpl39) complement loss-of-function mutations in SIS1 as well as PAB1 (poly(A)-binding protein), suggesting a functional interaction between these proteins. However, while a direct interaction between Sis1 and Pab1 is not detectable, both of these proteins physically interact with the essential Ssa (and not Ssb) family of hsp70 proteins. This interaction is mediated by the variable C-terminal domain of Ssa. Subcellular fractionations demonstrate that the binding of Ssa to ribosomes is dependent upon its C terminus and that its interaction with Sis1 and Pab1 occurs preferentially on translating ribosomes. Consistent with a function in translation, depletion of Ssa protein produces a general translational defect that appears similar to loss of Sis1 and Pab1 function. This translational effect of Ssa appears mediated, at least in part, by its affect on the interaction of Pab1 with the translation initiation factor, eIF4G, which is dramatically reduced in the absence of functional Ssa protein.

Analysis of eukaryotic translation in purified and semipurified systems.

Much of the current understanding of the sequential steps involved in translation initiation has been obtained using sucrose gradients to isolate ribosomes and ribosomal subunits, as described here. These purified components are combined with purified translation factors to analyze the formation of intermediates in translation initiation and the roles of the translation factors in vitro.

Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H.

A cDNA encoding human eukaryotic initiation factor (eIF) 4H was subcloned into a bacterial expression plasmid for purification of recombinant protein. Recombinant human eIF4H (heIF4H) was purified to greater than 95% homogeneity and shown to have similar physical characteristics to eIF4H purified from rabbit reticulocyte lysate as described previously. Functional studies have revealed that recombinant heIF4H functions identically to rabbit eIF4H in stimulating protein synthesis, and the ATP hydrolysis and helicase activities of eIF4A. More detailed enzymatic studies revealed that eIF4H increases the affinity of eIF4A for RNA by 2-fold, but has no effect on the binding of ATP by eIF4A. eIF4H stimulates the helicase activity of eIF4A at least 4-fold, and it is postulated that this stimulation occurs through increasing the processivity of eIF4A. Northern blot analysis shows that eIF4H is expressed ubiquitously in human tissues, and displays different levels of expression in given tissues relative to eIF4B. Secondary structure analysis of heIF4H by circular dichroism suggest that eIF4H has a mostly beta-sheet structure, which appears similar to other RNA recognition motif-containing proteins. Finally, it is suggested that eIF4H functions in translation initiation through protein-protein interactions that possibly stabilize conformational changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RNA duplex unwinding.

Purification and characterization of a new eukaryotic protein translation factor. Eukaryotic initiation factor 4H.

A new protein with translational activity has been identified on the basis of its ability to stimulate translation in an in vitro globin synthesis assay deficient in eukaryotic initiation factor (eIF) 4B and eIF4F. This protein has been purified to greater than 80% homogeneity from rabbit reticulocyte lysate and has been given the name eIF4H. eIF4H was shown to stimulate the in vitro activities of eIF4B and eIF4F in globin synthesis, as well as the in vitro RNA-dependent ATPase activities of eIF4A, eIF4B, and eIF4F. Three tryptic fragments of eIF4H yielded amino acid sequences that were 100% identical to a human sequence found in the GeneBankTM that codes for a previously uncharacterized protein (HUMORFU_1). The calculated molecular weight of the protein encoded by this sequence, its predicted cyanogen bromide fragmentation, and calculated isoelectric point are all consistent with those determined experimentally for eIF4H. Also, the presence of an RNA recognition motif within HUMORFU_1 suggests that eIF4H may interact with mRNA. We conclude that this newly characterized protein, eIF4H, functions to stimulate the initiation of protein synthesis at the level of mRNA utilization, and is encoded by the gene for HUMORFU_1.

The conserved 5'-untranslated leader of Spi-1 (PU.1) mRNA is highly structured and potently inhibits translation in vitro but not in vivo.

The transcription factor Spi-1 (PU.1) has a central role in regulating myeloid gene expression during hematopoietic development and its overexpression has been implicated in erythroleukemic transformation. Thus regulation of Spi-1 expression has broad significance for hematopoietic development. A comparison of human and murine cDNA sequences demonstrates that the 5'-untranslated region (5'-UTR) of Spi-1 mRNA is as highly conserved as the coding region (87% identical), suggesting that this sequence may be involved in regulating expression of this protein. The experiments presented in this manuscript provide evidence that the 5'-UTR of Spi-1 contains extensive secondary structure, including three stem-loops that precede the AUG codon. Analysis of the in vitro transcribed Spi-1 5'-UTR by partial nuclease digestion sensitivity is consistent with the existence of two of these stem-loops. The 5'-UTR decreased translation of Spi-1 transcripts in reticuloctye lysates 8- to 10-fold. A series of partial deletions of the 5'-UTR identified the sequence corresponding to the stem-loop most proximal to the initiating AUG codon as sufficient for inhibition of translation. However, the effect of the 5'-UTR on translation in vivo was negligible and resulted in only a slight reduction in the number of ribosomes that became associated with the mRNA. Further, this sequence had no affect on expression of luciferase. The disparity between in vivo and in vitro effects, coupled with the observation that endogenous Spi-1 mRNA is wholly associated with polysomes in MEL cells, suggests that additional cellular mechanisms contribute to regulation of Spi-1 expression in these cells or that conservation of these sequences serves a function that is independent of translation.

Inducers of erythroleukemic differentiation cause messenger RNAs that lack poly(A)-binding protein to accumulate in translationally inactive, salt-labile 80 S ribosomal complexes.

Translation has an established role in the regulation of cell growth. Posttranslational modification of translation initiation and elongation factors or regulation of mRNA polyadenylation represent common means of regulating translation in response to mitogenic or developmental signals. Induced differentiation of Friend virus-transformed erythroleukemia cells is accompanied by a rapid decrease in the translation rate of these cells. Although inducers do not alter initiation factor modifications, characterization of their effect on mRNA translation provides evidence that this is mediated by the poly(A)-binding protein (PABP). Inducer exposure results in an increase in the amount of mRNA that sediments at 80 S and a decrease in the amount in polysomes. Although these 80 S ribosomes have characteristics previously attributed to "vacant ribosomal couples," including lability in 500 mM KCl and an inability to incorporate amino acids into protein, we provide evidence that these 80 S complexes are not vacant but contain mRNA that is stably bound to the 40 S subunit, whereas the 60 S subunit is dissociated from the complex by high salt. The absence of eukaryotic initiation factor 2 from these complexes suggests that translation has proceeded through subunit joining. Immunoblotting demonstrates that the mRNAs in these 80 S ribosomal complexes do not contain bound PABP and that this protein is found to be almost exclusively associated with translating polysomes. These data suggest that the PABP plays a role in the accumulation of these 80 S ribosomal.mRNA complexes and may facilitate the formation of translationally active salt-stable ribosomes.

RNA polymerase II inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes erythroleukemic differentiation and transcriptional activation of erythroid genes.

Friend virus-transformed murine erythroleukemia (MEL) cells are a useful system for studying the regulation of erythroid growth and differentiation. As a manifestation of the leukemic process, these erythroblasts are blocked in their ability to terminally differentiate. However, this block is reversible as a variety of different agents are capable of inducing differentiation of these malignant erythroblasts. The mechanisms by which these agents cause differentiation remains unknown. We report here that 5,6-dichlorobenzimidazole (DRB), which inhibits RNA polymerase II by causing premature termination of transcription, induces differentiation of these cells, including the transcriptional activation of erythroid genes. The effects of DRB on nonerythroid gene expression and on cell growth are substantially different than that of the commonly used inducer, dimethyl sulfoxide (DMSO). The shared ability of DMSO, DRB, and other unrelated agents to induce erythroid gene expression in MEL cells while having differing effects on nonerythroid gene expression and on cell growth suggests that expression of the terminally differentiated phenotype represents a common pathway that can be triggered by different mechanisms.

Expression of the transcription factor, Spi-1 (PU.1), in differentiating murine erythroleukemia cells Is regulated post-transcriptionally. Evidence for differential stability of transcription factor mRNAs following inducer exposure.

Increased expression of the transcription factor Spi-1 (PU.1) results from retroviral insertion in nearly all Friend spleen focus-forming virus-transformed murine erythroleukemia cell lines and exposure of these cells to Me2SO, induces their differentiation and decreases Spi-1 mRNA level by 4-5-fold. While these results suggest that alterations in Spi-1 expression have significant effects on erythroblast growth and differentiation, neither the cause nor the effect of the decrease in Spi-1 expression that follows Me2SO exposure has been established. The experiments described here demonstrate that the effect of inducers on Spi-1 expression is regulated post-transcriptionally. Nuclear run-off transcriptions demonstrated that Spi-1 transcription was not decreased following Me2SO exposure. Additionally, expression of a recombinant Spi-1 mRNA under transcriptional control of a constitutively active Rous sarcoma virus promoter was regulated identically to endogenous Spi-1 mRNA. The ability of Me2SO to destabilize Spi-1 mRNA was selective, as the stability of the erythroid transcription factors GATA-1 and NF-E2 were not similarly effected. The effect of Me2SO on the stability of Spi-1 mRNA provides a novel means of altering gene expression in these cells and is likely to have significance for the differentiation of these cells.

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis.

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.

Preferential synthesis of the 78-Kd glucose-regulated protein in glucocorticoid-treated S49 mouse lymphoma cells.

To investigate the mechanism of glucocorticoid-induced lymphocytolysis, we used two-dimensional gel electrophoresis to analyze the effect of dexamethasone on the synthesis of individual proteins in S49 mouse lymphoma cells. We found that synthesis of a 78-Kd protein is preferentially maintained following dexamethasone treatment, at a time when the synthesis of most other cellular proteins is decreased. Synthesis of this protein could also be induced by tunicamycin, suggesting that it might be the 78-Kd glucose-regulated protein (GRP78). The identity of the 78-Kd protein with GRP78 was confirmed by limited chymotryptic mapping and immunoprecipitation analysis. Preferential synthesis of GRP78 in dexamethasone-treated cells was not secondary to alterations in the glycosylation of cellular proteins. Significantly, steady-state levels of GRP78 mRNA were unchanged following dexamethasone treatment. Preferential synthesis of GRP78 in glucocorticoid-treated S49 cells may reflect the unique property of GRP78 mRNA to be translated under conditions that interfere with the translation of most other cellular mRNAs. GRP78 is a highly conserved protein that is essential for cell viability. Preferential synthesis of GRP78 may be a protective response to metabolic events that interfere with normal mRNA translation in glucocorticoid-treated mouse lymphoma cells.

Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid-specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

DNA binding of heat shock factor to the heat shock element is insufficient for transcriptional activation in murine erythroleukemia cells.

The heat shock response is among the most highly conserved examples of regulated gene expression, being present in all cellular organisms. Transcriptional activation of heat shock genes by increased temperature or other cellular stresses is mediated by the binding of a heat shock factor (HSF) to a conserved nucleotide sequence (the heat shock element) present in the promoter of heat-inducible genes. Despite the high degree of conservation of this response, embryonic stages of development are characterized by the absence of a heat shock response. Murine erythroleukemia (MEL) cells also lack this response, and we report here a detailed characterization of this defect for one of the most highly conserved of these genes, hsp70. Surprisingly, heat-induced transcriptional activation of this gene does not occur, despite the induction of a protein with the binding specificity of murine HSF. However, the MEL HSF differs slightly in apparent size from the HSF in 3T3 cells, which exhibit a normal heat shock response. These data suggest that activation of mammalian HSF by heat requires at least two separate steps: an alteration of binding activity followed by further modification that activates transcription. MEL cells do not respond to heat shock because they lack the ability to perform this secondary modification. These cells provide a useful system for characterizing heat shock activation in mammals.

Decreased expression of the stress protein HSP70 is an early event in murine erythroleukemic cell differentiation.

Two-dimensional protein gels were used to systematically assess changes in gene expression in Friend erythroleukemia cells after exposure to inducers of differentiation. A rapid decrease in expression of the stress protein HSP70 was observed after exposure to inducers. The kinetics of this change suggest that it may be related to the cellular events that regulate the onset of differentiation.

A transient increase in histone H2A ubiquitination is coincident with the onset of erythroleukemic cell differentiation.

Murine erythroleukemia cells are useful for studying the regulation of erythroid differentiation since these malignant pronormoblasts differentiate to orthochromatic normoblasts when treated with a variety of inducing agents. Changes in chromatin proteins have been described following inducer exposure. The significance of these changes, which are greatest in terminally differentiated cells remains unknown. Ubiquitin is a highly conserved 8.5 kilodalton peptide that is covalently linked to up to 10% of histone H2A. We demonstrate that following exposure of MEL cells to inducers of differentiation, a transient increase in ubiquitination of H2A occurs. This change is coincident with the onset of differentiation. This result suggests that ubiquitination of H2A may have a role in the nuclear changes necessary for erythroleukemic cell differentiation.