Ernst W. Müllner

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

Establishment of normal, terminally differentiating mouse erythroid progenitors: molecular characterization by cDNA arrays

Expression profiling with cDNA arrays is an excellent tool for molecular analysis of complex processes such as terminal erythroid differentiation. The shortcomings of the currently available erythroid in vitro differentiation models, however, severely impaired the usefulness of this approach to study erythropoiesis. Here, we describe a novel, murine erythroid cell system closely corresponding to in vivo erythroid progenitors. Mortal, long‐term proliferating erythroid progenitors of fetal liver or immortal strains of p53‐deficient erythroblasts were established in culture. Both cell types proliferated in serum‐free medium and were strictly dependent on physiologically relevant cytokines and hormones, stably retaining a diploid set of chromosomes. If exposed to physiological differentiation factors (erythropoietin plus insulin), cells synchronously recapitulated the normal in vivo differentiation program to mature terminally into enucleated erythrocytes and expressed stage‐specific erythroid transcription factors in the expected temporal order. Using cDNA arrays, we found a large number of genes differentially expressed at time points during differentiation. Already 6 h after differentiation induction, 17% of the expressed genes showed significant alterations in mRNA abundance, increasing to 53% (12% up‐regulated, 41% down‐regulated genes) by 48 h. Cluster analysis of mRNA expression kinetics during differentiation identified six distinct expression patterns. All genes on the array with a known function in erythropoiesis showed the expected variations in expression. The genes identified also allowed first insights into the sequence of events within the regulatory network responsible for erythroid maturation. In mortal wild‐type as well as immortal p53‐/‐ erythroblasts, changes in mRNA abundance of several well‐regulated gene products was verified at the protein level. Taken together, this novel hematopoietic cell system faithfully executes essential steps of normal erythropoiesis and allows us to dissect and characterize molecular mechanisms involved in erythropoiesis.

Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis.

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53−/− mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.

Isolation of translationally controlled mRNAs by differential screening

Translational regulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation‐regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome‐free and polysome‐bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome‐free fraction in resting T cells to the polysome‐associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty‐two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.–Mikulits, W., Pradet‐Balade, B., Habermann, B., Beug, H., Garcia‐Sanz, J. A., Müllner, E. W. Isolation of translationally controlled mRNAs by differential screening. FASEB J. 14, 1641–1652 (2000)

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GRdim/dim), EpoR- (EpoRH) or STAT5ab function (Stat5ab–/–) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GRdim/dim, EpoRH, and Stat5ab–/– mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab–/– erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-XL. This defect could be fully rescued by exogenous Bcl-XL. These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation

Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.

Insights into erythroid differentiation obtained from studies on avian erythroblastosis virus

Analysis of the oncogenes v-erbB and v-erbA and their normal proto-oncogene counterparts has revealed several novel aspects of erythroid differentiation. A new erythroid progenitor capable of extended self-renewal has been described, tyrosine kinase receptors and steroid hormone receptors have been found to cooperate in controlling self-renewal, and dramatic alterations in the cell cycle have been found to accompany induction of terminal differentiation.

Overexpression of Thymidine Kinase mRNA Eliminates Cell Cycle Regulation of Thymidine Kinase Enzyme Activity

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G-S transition and stays high throughout G until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cells but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G and presumably also G.

Cell size control: new evidence for a general mechanism.

Continuously proliferating cells have to precisely double their size during each cycle to maintain constant volumes. Time and again, this fact raised questions on the existence of an active cell size control mechanism in eukaryotic cells, which would prevent delayed or premature cell division at inadequate mass. We addressed this open issue by recapitulating in animal cells several long-standing experiments which had identified such a mechanism in yeast. As a model, mainly chicken erythroblasts were used, whose proliferation can be driven either by a constitutively active oncogene (v-ErbB) or the physiological cytokines stem cell factor + erythropoietin. V-ErbB-driven cells proliferated faster than Epo/SCF-driven cells (doubling time 13 vs. 22 hours) and exhibited a 1.4-fold increased cell volume, due to a two-fold higher rate of global protein synthesis. Rapid and complete phenotypic reversion was achieved by exchanging the respective factors. To analyze the switch from one proliferation mode to the other in detail, we followed cell cycle progression of cells re-cultivated after synchronization by centrifugal elutriation. The results indicated that altered protein synthesis rates exclusively influenced G1 phase duration. Additional experiments with chicken erythroblasts and mammalian fibroblasts treated with low doses of aphidicolin (artificially prolonging S-phase) also pointed to the existence of a general size sensing mechanism in G1, ensuring cell size maintenance over many divisions, probably similar to the situation in yeast but certainly regulated at additional levels in higher eukaryotes.

Mouse thymidine kinase stability in vivo and after in vitro translation

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.