Supplementary Table 1 from Tissue-Wide Expression Profiling Using cDNA Subtraction and Microarrays to Identify Tumor-Specific Genes

The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP- and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis.

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, we searched for novel Pax2-regulated genes by cDNA microarray analysis of FACS-sorted GFP+ mid-hindbrain cells from wild-type and Pax2–/– embryos carrying a Pax2GFP BAC transgene. Here, we report the identification of five genes that depend on Pax2 function for their expression in the mid-hindbrain boundary region. These genes code for the transcription factors En2 and Brn1 (Pou3f3), the intracellular signaling modifiers Sef and Tapp1, and the non-coding RNA Ncrms. The Brn1 gene was further identified as a direct target of Pax2, as two functional Pax2-binding sites in the promoter and in an upstream regulatory element of Brn1were essential for lacZ transgene expression at the mid-hindbrain boundary. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicated Brn1 in Fgf8 gene regulation. Together,these data defined novel functions of Pax2 in the establishment of distinct transcriptional programs and in the control of intracellular signaling during mid-hindbrain development.

L'invention concerne un procede qui permet de classer un prelevement de cellules comme cellules tumorales, selon lequel on detecte une difference dans l'expression par ledit prelevement de cellules d'au moins un gene du tableau 4 par rapport a au moins une cellule temoin, et on classe le prelevement de cellules comme cellules tumorales si ledit gene du tableau 4 presente une expression egale a 1,5 fois celle de la cellule temoin.

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.

In Alzheimer's disease and amyotrophic lateral sclerosis deregulation of cyclin-dependent kinase 5 (CDK5) causes hyperphosphorylation of tau and neurofilament proteins, respectively, leading to neuronal cell death. We have demonstrated recently that pharmacological inhibition of CDK5 protects neurons under various stressful conditions (Weishaupt J. H., et al., Molec. Cell. Neurosci. 2003, 24, 489-502). To get an overview on the cellular mechanisms of action we analyzed global changes in protein phosphorylation in cultured cerebellar granule neurons by [(32)P]orthophosphate labeling after administration of a CDK5 inhibitor. Since CDK5 has recently been shown to phosphorylate and inactivate transcription factor MEF2, we included gene expression profiling using cDNA microarrays. By two-dimensional gel electrophoresis and matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry we identified several phosphoproteins that were modulated by compound administration. Among them syndapin I which is involved in vesicle recycling, and dynein light intermediate chain 2 which represents a regulatory subunit of the dynein protein complex. These findings are consistent with the known physiological function of CDK5 in synaptic signaling and axonal transport. Moreover, we detected phosphoproteins acting in neuronal surival and/or neurite outgrowth, such as cofilin and collapsin response mediator protein. Subsequent testing in cell cultures revealed that the CDK5 inhibitor blocked mitochondrial translocation of pro-apoptotic cofilin in cerebellar granule neurons and enhanced neurite outgrowth in dorsal root ganglia. Numerous genes exhibiting MEF2 consensus binding sequences were modulated by CDK5 inhibitor treatment. Among them some that may contribute to neurite elongation or neuronal survival, but also several genes functioning in synaptic transmission. Taken together, phosphoproteome and transcriptome analysis indicate that the compound promotes both neuronal survival and neurite outgrowth, but also may affect synaptic function in cultured neurons.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES) cells. After in vitro differentiation, some of their progeny cells are capable of mediating long term hematopoietic repopulation after transplantation into immunodeficient recipient mice, in vivo. This is especially efficient when the homeodomain transcription factor HOXB4 is ectopically expressed (1). We have recently shown that HOXB4-ES-cell derivatives behave similar to bone marrow cells also expressing this transcription factor ectopically, both in vitro and in vivo (2). Here we demonstrate that long term repopulation (>6 months) in Rag2(−/−)γ C(−/−) mice can be achieved with ES-cell derived hematopoietic cells (ES-HCs) obtained from single, molecularly characterized ES-clones, in which the insertion sites of the retroviral expression vector had been defined. Clones expressing HOXB4 above a certain level showed a high extent of chimerism in the bone marrow of transplanted mice (average 75%; range 45–95%, n=4) whereas ES-HC clones expressing lower levels only repopulated with very low efficiency (average 2.5% chimerism, range 1–4%, n=6 mice). These results suggest that the capability of long-term repopulation, in vivo, is highly dependent on the expression levels of HOXB4 in the transplanted clones. Only mice reconstituted with ES-HC clones expressing high amounts of HOXB4 and thus showing substantial chimerism, recapitulated the morphohistological phenotype observed in polyclonally reconstituted mice. This included the bias towards myelopoiesis, “benign” myeloid proliferation in spleen and the incompatibility of HOXB4 expression with T-cell poiesis (2). In summary, we demonstrate that repopulation of the hematopoietic system can be achieved with preselected clones of genetically manipulated stem cells in which a) the insertion site of the retroviral (gene therapy) vector has been characterized prior to transplantation and b) in which ectopic HOXB4 has to be expressed above a certain threshold level. Thus, ES cells carry the potential for performing safe somatic gene therapy when using integrating gene therapy vectors. Nevertheless, advanced cell therapy will certainly require the expression of HOXB4 in a regulated manner to avoid unwanted effects such as disturbed lineage differentiation.

Abstract With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues. To achieve this goal, we combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarrays. Seven subtractive libraries consisting of ∼9250 clones were established and enriched for tumor-specific transcripts. These clones, together with ∼1750 additional tumor-relevant genes, were used for cDNA microarray preparation. Hybridizations were performed using a pool of 16 critical normal tissues as a reference in all experiments. In total, we analyzed 20 samples of breast cancer, 11 of LSCC, 11 of LAC, and 8 of RCC. To select for genes with low or even no expression in normal tissues, expression profiles of 22 different normal tissues were additionally analyzed. Importantly, this tissue-wide expression profiling allowed us to eliminate genes, which exhibit also high expression in normal tissues. Similarly, expression signatures of genes, which are derived from infiltrating cells of the immune system, were eliminated as well. Cluster analysis resulted in the identification of 527 expressed sequence tags specifically up-regulated in these tumors. Gene-wise hierarchical clustering of these clones clearly separated the different tumor types with RCC exhibiting the most homogenous and LAC the most diverse expression profile. In addition to already known tumor-associated genes, the majority of identified genes have not yet been brought into context with tumorigenesis such as genes involved in bone matrix mineralization (OSN, OPN, and OSF-2) in lung, breast, and kidney cancer or genes controlling Ca2+ homeostasis (RCN1,CALCA, S100 protein family). EGLN3, which recently has been shown to be involved in regulation of hypoxia-inducible factor, was found to be highly up-regulated in all RCCs and in half of the LSCCs analyzed. Furthermore, 42 genes, the expression level of which correlated with the overall survival of breast cancer patients, were identified. The gene dendogram clearly separates two groups of genes, those up-regulated such as cyclin B1, TGF-β3, B-Myb, Erg2, VCAM-1, and CD44 and those down-regulated such as MIG-6, Esp15, and CAK in patients with short survival time.

Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E- and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A. Hence, this protein might have a much wider effect on gene expression in arrested mouse fibroblasts than hitherto suspected. This may have a profound part in the known capacity of Py to form tumors in mice. Therefore, it was interesting to gain an insight into the spectrum of transcriptional deregulation by Py tumorantigens. Accordingly, we performed microarray analysis of quiescent mouse fibroblasts in the absence and presence of small or large tumorantigen. We found that the viral proteins can induce or repress a great variety of genes beyond those involved in the S phase induction and DNA synthesis. The results of the microarray analysis were confirmed for selected genes by several methods, including real-time PCR. Interestingly, a mutation of the binding site for pocket proteins in case of LT and for PP2A in case of ST has a variable effect on the deregulation of genes by the viral proteins depending on the gene in question. In fact, some genes are transactivated by LT as well as ST completely independent of an interaction with their major cellular targets, pocket proteins and PP2A, respectively.

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. Besides their characterization in colony assays, protocols exist for the cultivation of lymphoid, myeloid, and erythroid cells. With the possible exception of mast cells, however, long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here, we describe for the first time an efficient yet easy strategy to generate mass cultures of pure, immature erythroid progenitors from mouse ES cells (ES-EPs), using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived, adult, definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions, ES-EPs differentiated into mature, enucleated erythrocytes. Importantly, ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition, the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells.

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multi-stage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify turnour-associated genes, we studied the influence of short-term TPA-treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA-treated dorsal skin samples. Of these genes, 54 were up- and 35 were down-regulated upon TPA application. Additionally, we performed suppression subtractive hybridisation (SSH) with respective RNA pools to generate and analyse a cDNA library enriched for TPA-inducible genes. Expression data of selected genes were confirmed by quantitative real-time PCR and Northern blot analysis. Comparison of microarray and SSH data revealed that 26% of up-regulated genes identified by expression profiling matched with those present in the SSH library. Besides numerous known genes, we identified a large set of unknown cDNAs that represent previously unrecognised TPA-regulated genes in murine skin with potential function in tumour promotion. Additionally, some TPA-induced genes, such as SprrIA, Saa3, junB, II4ralpha, Gp38, RalGDS and Slpi exhibit high basal level in advanced stages of skin carcinogenesis, suggesting that at least a subgroup of the identified TPA-regulated genes may contribute to tumour progression and metastasis. (C) 2003 Wiley-Liss, Inc.

Erythroid progenitors undergo renewal (proliferation without apparent differentiation) in response to erythropoietin (Epo), stem cell factor (SCF), and glucocorticoids (dexamethasone) (Dex). SCF and Dex cooperate with Epo to promote proliferation and inhibit differentiation of erythroid progenitors, while Epo alone is required to protect erythroid cells from apoptosis during terminal red cell maturation. To examine the mechanism of the synergistic interactions of Epo, SCF, and Dex, we analyzed gene expression patterns using DNA chip-based large-scale comparative gene profiling using microarrays enriched in hematopoietic transcripts or containing randomly selected genes. Differentially regulated genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). The results reveal cooperative regulation of gene expression by glucocorticoids and Epo/SCF on a number of genes, such as CIS, BTG1, VDUP1, CXCR4, GILZ, and RIKEN29300106B05. While Epo and SCF never showed opposite effects on gene expression, Dex either enhanced or attenuated the effect of Epo and/or SCF. Several glucocorticoid receptor (GR)-target genes were regulated by Dex only in the presence of Epo and/or SCF, suggesting that the GR functions in the context of a larger transactivation complex to regulate these genes. The data also suggest that modulation of cytokine-induced signals by the GR is an important mechanism in erythroid progenitor renewal.

Transactivation/transformation-domain associated protein (TRRAP) is a component of several multi-protein HAT complexes implicated in both transcriptional regulation and DNA repair. We recently identified Trrap, the murine ortholog of TRRAP, as an essential protein implicated in mitotic progression control, although its target genes are not known. In the present study, we analyzed the expression profiles of Trrap-responsive genes, using cDNA microarray in mitotic cells. From a panel of 17 664 transcript elements, we found that loss of Trrap leads to expression alteration of a large fraction of genes at mitotic stage. Functional classification of these genes indicates that Trrap influences a variety of cellular processes including cell cycle progression, cytoskeleton and cell adhesion, protein turnover, metabolism and signal transduction. The majority (71%) of differentially expressed genes was down-regulated in Trrap- deficient cells, whereas the rest were up-regulated, suggesting that Trrap may also play a role in transcriptional silencing. ChIP analysis revealed that Trrap might regulate gene expression by participating in acetylation of histone H4 and/or H3 depending on target genes and cell cycle stage. Our study indicates that Trrap regulates the expression of a wide range of genes in both quiescence and mitotic stages. Removal of the Trrap protein is associated with both increased and decreased gene expression.

Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochloro-benzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the beta-galactosidase (betagal) gene into M3-GM cells. Their administration activated betagal-specific, L-d-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-betagal injection sites revealed the presence of cells presenting the respective L-d-binding betagal peptide epitope. Based on their capacity to activate betagal-specific CTL, they were identified as being CD11c(+) dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.