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Division of Molecular Biology

Department of Medical Biochemistry

Division of Molecular Biology

Research Groups

 

TERMINAL ERYTHROPOIESIS

Group Leader:         Ernst W. Müllner

Postdocs:                 Matthias Schranzhofer

PhD Students:         Cornelia Leberbauer
                                Florian Grebien

Diploma students:    Daniel Genz
                                Manfred Schifrer

Main interests   |    The cell system   |    Expression Profiling   |    Publications   |    Textbooks   |    Textbook Chapters   |    PhD theses   |    Diploma   |    Grants   |    Cooperations   |    Expression-profiling table   |    Lectures

Main interests
- Our group is interested (i) to analyse differential gene expression in differentiating erythroblasts or normal versus leu-kaemic cells of mouse, chicken and human origin, using cDNA and oligonucleotide based DNA-filters and -chips; (ii) to identify mRNAs translationally controlled upon erythroid maturation; (iii) verify the biological signifi-cance of putative targets at the level of protein production, polysome gradients and biologi-cal interference, using retroviral vectors (over-) expressing (chimeric) proteins of interest.
In addition, we use the same cell systems (i) to study the molecular basis for the "erythroid" mode of iron metabolism; (ii) to search for specific mRNA binding proteins other than IRP; (iii) to characterise the regulation of the endonuclease involved in TfR mRNA turnover; (iv) to assess differences in the mode of translational control of eALAS mRNA in avian versus mammalian species; (v) to de-lineate transformation (v-ErbA)-specific alterations in regulation of iron utilisation.

The cell system
- During embryonic development as well as in adult organisms: cell differentiation is a major topic in contemporary biology. Typical approaches focus on the delineation of signal transduction pathways underlying cell fate determination and their distortion in the development of disease but also on the regulation of gene expression of products characteristic for the differentiated state.
Our particular interest focuses on the development of the erythroid compartment. Until some time ago only erythroleukemic cells (mainly mouse and human), which show abnormal growth properties, fail to express major erythrocyte proteins and are resistant to apoptosis in case of conflicting growth factor signals, were available for these studies. The lack of suitable primary cell systems still is a general problem in the work on hematopoietic disorders, which constitute a large group of frequently life-threatening diseases. Thus, the ability to maintain and differentiate normal chicken erythroid progenitors in mass cultures in vitro was a prerequisite to define crucial molecular differences between normal and leukaemic phenotypes. Appropriate condi-tions were worked out in the group of H. Beug at the neighbouring Institute of Molecular Pathology (IMP) [e.g. Hayman et al., Cell 74: 157], an effort to which we could contribute [Dolznig et al., Cell Growth Diff 6: 1341; Mikulits et al., DNA Cell Biol 16: 849]. It could be shown that there is an intricate cooperation between receptor tyrosine kinases (like c-Kit), the erythropoietin receptor, class II nuclear hormone receptors (like the glucocorticoid receptor, GR) and respective viral oncogene counterparts (v-ErbA/thyroid hormone receptor and v-ErbB/epidermal growth factor receptor). The interplay between these factor tips the balance between a limited proliferative phase (termed self renewal), permanent proliferation with differentiation arrest (in the case of transformation), or terminal maturation [for review see e.g. Beug et al., Biochim. Biophys. Acta 1288: 35].

More recently, again in close collaboration with the Beug laboratory, we established permanently growing erythroblasts derived from fetal livers of p53 deficient mice [Dolznig et al., unpublished; and Deiner et al., unpublished]. Kept in serum free media, these cells remain fully factor dependent for proliferation and can undergo differentiation into cells indistinguishable from peripheral blood erythrocytes. Moreover, the same conditions were also suitable to expand primary erythroblasts from bone marrow as well as fetal liver of normal mice for a limited period (14 days) until crisis and finally apoptosis occurs. Currently, we participate in experiments to improve serum free culture of erythroid and other hematopoietic progenitors derived from human um-bilical cord blood, involving an ongoing cooperation with the General Hospital Vienna (AKH) and the Erasmus University in Rotterdam [see also vonLindern et al., Blood 94: 550].

With these advanced cell systems in hand, at present we concentrate on two major issues:
· First, to characterise the majority of significant alterations in gene expression during the transition from self renewal to terminal maturation of primary erythroblasts (expression profiling)

· Second, on one major functional aspect of erythropoiesis, namely the regulation of iron metabolism, which differs from that of any other cell type due to the vast demand for iron to assure efficient hemoglobin synthesis

Expression Profiling
- The use of filter/glass chip-bound cDNA- or oligonucleotide probes allows multiplex analysis of gene expression, comparing suitable cell pairs, e.g. at different stages of differentiation or normal versus disease phenotype [Brown and Botstein, Nat. Genet. 21: 33; DeRisi et al., Nat. Genet. 14: 457]. The advances in characteri-sation of ESTs ensures that a high proportion of all genes expressed in a particular cell type (about 20.000) is detected, making expression profiling much more sensitive than alternative methods for the analysis of differential gene expression like differential display [Lian and Pardee, Curr. Opin. Cell Biol. 7: 274] or suppression subtractive hybridisation [Diatchenko et al., Proc. Natl. Acad. Sci. USA 93: 6025]. Although not all technical problems of sensitivity, reproducibility and background in gene profiling are solved yet, there is a flurry of ongoing activity around this technique, reminding of the early days of PCR. We have several reasons to believe that we can do competitive research with this new methodology.

· As outlined above, we are in an almost unique position to follow the differentiation process of primary erythroid cells but also to compare "normal" with "leukemogenic" variants.

· Second, as we have shown previously, it is important to measure not only differences in total mRNA levels but to analyse alterations in polysome bound mRNA concentrations instead [Garcia-Sanz et al., FASEB J. 12: 299]: A significant proportion (about 25%) of all changes observed in gene expression upon T-lymphocyte activation was actually due to variations in translation efficiency. A cor-responding patent application was filed recently [case 14/048 DI Fa/dc, European Patent Office]. At present, there is an ongoing screen for mRNAs subject to translational control upon erythropoiesis.

· In recent experiments, we could verify that several of the target genes differentially ex-pressed in self renewing versus differentiating erythroid cells according to the cDNA filters, were appropriately regulated at the level of protein abundance. This involved cell cycle regulators, growth factor receptors, proto-oncogenes and in particular one lymphokine re-ceptor, whose relevance for proliferation of erythroid progenitors was not appreciated so far but could now be proven biologically [Dolznig, Mikulits, Boulmé et al., unpublished].

· Technically, we could improve one of the recommended protocols for probe labelling, thus greatly decreasing exposure times of the cDNA filters (Clontech) and facilitating re-utilisa-tion [13]. Moreover, due to our cooperation with the Beug group, we have recently gained access to oligonucleotide based DNA chips (Affymetrix), started efforts to produce custom-made chips and begun development of an improved software for signal evaluation.

· Within the context of an EU Training and Mobility Research Network, we participate in similar studies dealing with maturation and activation of monocytes/macrophages and T lymphocyte activation.

Iron Metabolism
- In contrast to the "broad" approach outlined in the previous section, primary erythroblasts are also an ideal subject to study a much more specific aspect of functional differentiation, namely the regulation of iron metabolism . In most proliferating cell types, iron uptake (via transferrin receptor; TfR) iron utilisation (in cytochromes or non-heme iron proteins) and storage (in ferritin for detoxification) have to be carefully balanced to avoid toxicity. A specific mRNA binding protein (IRP) affecting TfR mRNA stability and ferritin mRNA translation efficiency in an iron-dependent manner, has been shown to be critically involved in this process [Müllner et al., Cell 58: 373].
Since erythroid cells have a vast demand for iron to undergo proper hemoglobinisation, it came as no real surprise that they would regulate iron metabolism differently (the work described in the following was done mainly with chicken cells but will be extended to mouse erythroblasts in the near future):

· Ferritin mRNA translation was found to be drastically impaired in erythroid cells, presuma-bly to avoid iron storage at times of extensive consumption [Mikulits et al., BLOOD, in press]

· TfR transcripts (and protein) were hyperexpressed due to a massive increase in mRNA sta-bility [Lobmayr et al., unpublished]

· Surprisingly, both phenotypes appeared to be independent of IRP mRNA binding activity and, even more interestingly,

· both phenotypes were abolished upon leukemogenic transformation with avian erythro-blastosis virus (AEV), reverting the cells back to a "normal" IRP-dependent mode of regu-lation of iron metabolism.

Currently, we try to characterise the signal transduction pathways leading from one of the oncogenes of AEV (v-ErbA, a mutated thyroid hormone receptor) to the specific endonuclease that is required for rapid TfR mRNA turnover. Moreover, we want to find out, how the mRNA for eALAS (the erythroid isoform of delta-aminolevulinic acid synthase; the first and rate limiting enzyme in heme biosynthesis) escapes translational inhibition in erythroid cells, although it harbours the same iron-responsive element like ferritin mRNA, which is effectively blocked. In other words, primary erythroblasts may have a factor "X", which (i) interferes with translation initiation of ferritin but not eALAS mRNA and (ii) is absent in AEV-transformed cells.

Publications

  1. Deiner, E.-M., Dolznig, H., Stangl, K., Moriggl, R., Kolbus A., Kieslinger, M., Müllner, E.W., and Beug, H. (2002). Primary cells from genetically modified mice reveal molecular mechanisms in erythroid progenitor renewal and differentiation. Under revision for “The Journal of Cell Biology”

  2. Dolznig, H., Habermann, B., Stangl, K., Deiner, E.-M., Moriggl, R., Beug, H., and Müllner, E.W. (2002). Apoptosis protection by the Epo target Bcl-XL allows factor-independent differentiation of primary erythroblasts. Curr. Biol. 12, 1076-1085.

  3. Lobmayr, L., Sauer, T., Killisch, I., Schranzhofer, M., Wilson, R.B., Ponka, P., Beug, H., and Müllner, E.W. (2002). Transferrin receptor hyperexpression in primary erythroblasts is lost upon transformation by the avian erythroblastosis virus. BLOOD 100, 289-298.

  4. von Lindern, M., Deiner, E.-M., Dolznig, H., Parren-van Amelsvoort, M., Hayman, M., Müllner, E.W., and Beug., H. (2001). Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signalling pathways activated by the EpoR and c-Kit in stress erythropoiesis. Oncogene 28, 3651-3664.

  5. Dolznig, H., Boulmé, F., Stangl, K., Deiner, E.-M.,Mikulits, W., Beug, H., and Müllner, E.W. (2001). Establishment of normal, terminally differentiating mouse erythroid progenitors: Molecular characterization by cDNA arrays. The FASEB J. 15, 1542-1544. full text at “FASEB Journal Express Article 10.1096/fj.00-0705fje“

  6. Pradet-Balade, B., Boulmé, F., Müllner, E.W., and Garcia-Sanz, J.-A. (2001). Reliability of mRNA profiling: Verification for samples with different complexities. BioTechniques 30, 1352-1357.

  7. Pradet-Balade, B., Boulmé, F., Beug, H., Müllner, E.W., and Garcia-Sanz, J.A. (2001). Translation control: Bridging the gap between genomics and proteomics. Trends Biochem. Sci. 18, 325-329.

  8. Mikulits, W., Schranzhofer, M., Deiner, E.-M., Beug, H., and Müllner, E.W. (2000). Regulation of ferritin mRNA translation in primary erythroblasts: Exogenous c-Kit plus EpoR signaling mimics v-ErbA oncoprotein activity. Biochim. Biophys. Res. Comm. 275, 292-294.

  9. Mikulits, W., Pradet-Balade, B., Habermann, B., Beug, H., Garcia-Sanz, J.A., and Müllner, E.W. (2000). Isolation of translationally controlled mRNAs by differential screening. FASEB J. 14, 1641-1652.

  10. Ghysdael, J., Tran-Quang, C., Deiner, E.-M., Dolznig, H., Müllner, E.W., and Beug, H (2000). Erythroid cell development and leukemic transformation: Interplay between signal transduction, cell cycle control and by oncogenes. Pathol. Biol. 48, 211-226.

  11. Mikulits, W., Schranzhofer, M., Bauer, A., Dolznig, H., Lobmayr, L., Infante, A., Beug, H., Müllner, E.W. (1999). Impaired ferritin mRNA translation in primary erythroid progenitors: shift to iron-dependent regulation by the v-ErbA oncoprotein. Blood 94, 4321-4332.

  12. Mikulits, W., Schranzhofer, M., Beug, H., and Müllner, E.W. (1999). Post-transcriptional control via iron-responsive elements: The impact of aberrations in hereditary disease. Mut. Res. 437, 219-230.

  13. Mikulits, W., Dolznig, H., Hofbauer, R., and Müllner, E.W. (1999). Reverse strand priming: A versatile cDNA radiolabeling method for differential hybridization on nucleic acid arrays. Biotechniques 26, 846-850.

  14. Garcia-Sanz, J.A., Mikulits, W., Livingstone, A., Lefkovits, I., and Müllner, E.W. (1998). Translational control: a general mechanism of gene regulation during T cell activation. FASEB J. 12, 299-306.

  15. Mikulits, W., Sauer, T., Infante, A.A., Garcia-Sanz, J.A., and Müllner, E.W. (1997). Structure and function of the iron-responsive element from human ferritin L chain mRNA. Biochem Biophys. Res. Comm. 235, 212-216.

  16. Mikulits, W. Dolznig, H., Edelmann, H., Sauer, T., Ballou, L., Beug, H., and Müllner, E.W. (1997). Recultivation of cells synchronised by centrifugal elutriation ensures artefact-free cell cycle studies. DNA Cell Biol. 16, 849-859.

  17. Mikulits, W., Knöfler, M., Wintersberger, E., and Müllner, E.W. (1997). Mouse thymidine kinase stability in vivo and after in vitro translation. Biochim. Biophys. Acta 1338, 267-274.

  18. Beug, H., Bauer, A., Dolznig, H., van Lindern, M., Lobmayr, L., Mellitzer, G., Steinlein, P., Wessely, O., and Müllner, E.W. (1996). Avian erythropoiesis and erythroleukemia: towards understanding the role of the biomolecules involved. Biochim. Biophys. Acta (Reviews on Cancer) 1288, M35-M47.

  19. Müllner, E.W., Dolznig, H., and Beug, H. (1996). Cell cycle regulation during erythroid differentiation. Current Topics in Microbiology and Immunology 212, 175-194.

  20. Beug, H., Metz, T., Müllner, E.W., and Hayman, M.J. (1996). Self renewal and differentiation in primary avian hematopoietic cells: An alternative to mammalian in vitro models? Current Topics in Microbiology and Immunology 211, 29-39.

  21. Mikulits, W., Sauer, T., and Müllner, E.W. (1996). Overexpression of thymidine kinase mRNA eliminates cell cycle regulation of thymidine kinase enzyme activity. J. Biol. Chem. 271, 853-860.

Textbooks

"Chemische Rechenübungen für Mediziner”, (Chemical calculations for students of medicine). E. Müllner, G. Pischek and E. Wawra. 1st edn. 1999, 205 pp, WUV-Verlag, Vienna, ISBN 3-085076-493-1; 3000 copies sold so far.

“Chemie verstehen” (Understanding Chemistry); E. Wawra, H. Dolznig and E. Müllner; 1st edn. 2001, 270 pp, Universitäts-Taschenbuch Verlag Deutschland (UTB) ISBN 3-8252-8205-8 / Facultas University Press Vienna, ISBN 3-85076-542-3; 1000 copies sold so far.

“Chemie berechnen” (Calculating Chemistry); E. Müllner, G. Pischek und E. Wawra, 1st edn. 2002, 270 pp, Universitäts-Taschenbuch Verlag Deutschland (UTB) ISBN 3-8252-8205-8 / Facultas Universitäts Verlag Wien ISBN 3-85076-542-3; sales started August 2002

Textbook Chapters

Garcia-Sanz, J.A., and Müllner, E.W. (1997). mRNA expression, I.: Introduction and historical background. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 383-388. Academic Press, London, San Diego.

Müllner, E.W., and Garcia-Sanz, J.A. (1997). mRNA expression, II.: Preparation of RNA. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 389-405. Academic Press, London, San Diego.

Müllner, E.W., and Garcia-Sanz, J.A. (1997). mRNA expression, III.: Analysis of RNA expression by Northern blotting. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 407-423. Academic Press, London, San Diego.

Garcia-Sanz, J.A, and Müllner, E.W. (1997). mRNA expression, IV.: RNAse protection and primer extension. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 425-438. Academic Press, London, San Diego.

Müllner, E.W., Seiser, C., and Garcia-Sanz, J.A. (1997). mRNA expression, V.: Run-on assays. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 439-448. Academic Press, London, San Diego.

Shyu, A.B., Müllner, E.W., and Garcia-Sanz, J.A. (1997). mRNA expression, VI.: Analysis of mRNA decay. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 449-456. Academic Press, London, San Diego.

Müllner, E.W., and Garcia-Sanz, J.A. (1997). mRNA expression, VII.: Polysome gradients. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 457-462. Academic Press, London, San Diego.

Müllner, E.W., Seiser, C., and Garcia-Sanz, J.A. (1997). mRNA Expression, VIII.: Additional uses of RNA probes. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 463-476. Academic Press, London, San Diego.

Garcia-Sanz, J.A., and Müllner, E.W. (1997). mRNA expression, IX.: Outlook. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 477-480. Academic Press, London, San Diego.

Garcia-Sanz, J.A., and Müllner, E.W. (1997). mRNA expression, X.: Recommended Reading. In: Immunology Methods Manual (Ivan Lefkovits, Ed.), pp. 481-482. Academic Press, London, San Diego.

PhD theses since 1996

Mag. Wolfgang Mikulits: Translational Control in Mammalian Cells, defended 1996

Mag. Lioba Lobmayr: Regulation of Iron Metabolism during Normal Erythropoiesis", defended 1999

Mag. Helmut Dolznig: The Making of a Red Cell, defended 2000

Mag. Doris Chen: Cell Cycle Regulation during Terminal Maturation of Primary Erythroid Progenitors, defended 2002

Mag. Matthias Schranzhofer: since 1999

Mag. Katharina Stangl: since 2000 (jointly supervised with group of H. Beug)

Mag. Pavel Vaclavik: since 2000 (jointly supervised with group of J. Nimpf)

Mag. Cornelia Strobl: since 2002

Diploma since 1996

Doris Chen: Post-Transcriptional Regulation of Thymidine Kinase: Analysis of the Molecular Basis, submitted 1996

Harald Sigmund: Isolation of Lactoferrin from Chicken Heterophilic Granulocytes and Expression of the VLDL-I receptor, submitted 1997

Matthias Schranzhofer: Inhibition of Ferritin mRNA Translation in Primary Erythroblasts, submitted 1999

Cornelia Strobl: Expansion of Human Erythroid Progenitors: The Role of Steroids, submitted 2002

Sonja Haider: seit 2001 (jointly supervised with group of J. Nimpf)

Florian Grebien: since 2002

Daniel Genz: since 2002

Work in this lab (since 1996) is / was supported by grants from the

1996-1999: grant from the Herzfelder Family Foundation

1998-2002: grant from the EU within a "Research Network" of the TMR Programme (Co-coordinator), Contract # FMRX-CT98-0197

1998-2001: grant from the Austrian National Bank, OENB project number 7291-2

1998-2001: renewal for 2nd period of the "Spezialforschungsbereich" (SFB) "Molecular Mechanisms of Cell Differentiation and Cell Growth" FWF project number F00605;

2000-2002: grant from the Herzfelder Family Foundation

2001-2005: renewal for 3rd period of the "Spezialforschungsbereich" (SFB)"Molecular Mechanisms of Cell Differentiation and Cell Growth", FWF project number F00605; 436.000 €

2002-2005: direct grant from the Austrian Ministry of Science, on cord-blood derived stem cells; 435.000 € equally shared with H. Beug, IMP and J. Huber, AKH

Cooperations since 1996

Christopher G. Proud, University of Dundee, Scotland, EU-TMR project “Signal Transduction and Translation”, FMRX-CT98-0197 Enric Espel, University of Barcelona, Spain, FMRX-CT98-0197

Günther Schütz, German Cancer Research Center (DKFZ), Germany, EU-TMR project “Signal Transduction and Translation”, FMRX-CT98-0197

Jose Alberto Garcia-Sanz, Centro Nacional de Biotecnologia, CSIC, Universidad Autonoma, Madrid, EU-TMR project “Signal Transduction and Translation”, FMRX-CT98-0197

Boehringer Ingelheim Pharma, Biberach, Germany, EU-TMR project “Signal Transduction and Translation”, FMRX-CT98-0197 and FWF SFB-605

Marieke von Lindern, Erasmus University, Rotterdam, The Netherlands, EU-TMR project “Signal Transduction and Translation”, FMRX-CT98-0197 and FWF SFB-605 Jacques Ghysdael, Institute Curie, Orsay, France, FWF SFB-605

Hartmut Beug, Institute of Molecular Pathology, Vienna, FMRX-CT98-0197, FWF SFB-605 and direct grant from Minstry of Science

Prem Ponka, Lady Davis Institute, McGill University, Montreal, Canada, Herzfelder Family Foundation

Johannes Nimpf; Inst. of Medical Biochemistry, Division of Molecular Genetics, Vienna Biocenter (VBC), FWF SFB-605

Johannes Huber, Dept. Gynaecological Endocrinology, General Hospital, Vienna, direct grant from Minstry of Science

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