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International audience; T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

The heterogeneity of cancer cells, in part maintained via the expression of multiple isoforms, introduces significant challenges in designing effective therapeutic approaches. In this regard, isoforms of the immune checkpoint HLA-G have been found in most of the tumors analyzed, such as ccRCC, the most common human renal malignancy. In particular, HLA-G∆α1, which is the only HLA-G isoform described that lacks the α1 extracellular domain, has been newly identified in ccRCC and now here in trophoblasts. Using a cellular model expressing HLA-G∆α1, we have uncovered its specific and overlapping functional roles, relative to the main HLA-G isoform, i.e., the full-length HLA-G1. We found that HLA-G∆α1 has several particular features: (i) although possessing the α3 domain, it does not associate with β2-microglobulin; (ii) it may not present peptides to T cells due to absence of the peptide-binding groove; and (iii) it exerts immune-stimulatory activity towards peripheral blood NK and T cells, while all known isoforms of HLA-G are immune-inhibitory checkpoint molecules. Such immune-stimulatory properties of HLA-G∆α1 on the cytotoxic function of peripheral blood NK cells are individual dependent and are not exerted through the interaction with the known HLA-G receptor, ILT2. Importantly, we are faced here with a potential antitumor effect of an HLA-G isoform, opposed to the pro-tumor properties described for all other HLA-G isoforms, which should be taken into account in future therapeutic designs aimed at blocking this immune checkpoint.

Understanding the emergence of lymphoid committed cells from multipotent progenitors (MPP) is a great challenge in hematopoiesis. To gain deeper insight into the dynamic expression changes associated with these transitions, we report the quantitative transcriptome of two MPP subsets and the common lymphoid progenitor (CLP). While the transcriptome is rather stable between MPP2 and MPP3, expression changes increase with differentiation. Among those, we found that pioneer lymphoid genes such as Rag1, Mpeg1, and Dntt are expressed continuously from MPP2. Others, such as CD93, are CLP specific, suggesting their potential use as new markers to improve purification of lymphoid populations. Notably, a six-transcription factor network orchestrates the lymphoid differentiation program. Additionally, we pinpointed 24 long intergenic-non-coding RNA (lincRNA) differentially expressed through commitment and further identified seven novel forms. Collectively, our approach provides a comprehensive landscape of coding and non-coding transcriptomes expressed during lymphoid commitment.

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G – cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.

Despite some success, many patients do not benefit from immunotherapy. New strategies to improve clinical efficacy include identification of novel immune-checkpoint (IC) targets or a combination of immunotherapy with antiangiogenic treatments. Here, we propose the therapeutic use of IC, HLA-G/LILRB, and explore its enhanced synergistic antitumor activity when combined with antiangiogenic therapies.

International audience; Clear cell renal cell carcinoma (ccRCC) constitutes the most common renal cell carcinoma subtype and has long been recognized as an immunogenic cancer. As such, significant attention has been directed toward optimizing immune-checkpoints (IC)-based therapies. Despite proven benefits, a substantial number of patients remain unresponsive to treatment, suggesting that yet unreported, immunosuppressive mechanisms coexist within tumors and their microenvironment. Here, we comprehensively analyzed and ranked forty-four immune-checkpoints expressed in ccRCC on the basis of in-depth analysis of RNAseq data collected from the TCGA database and advanced statistical methods designed to obtain the group of checkpoints that best discriminates tumor from healthy tissues. Immunohistochemistry and flow cytometry confirmed and enlarged the bioinformatics results. In particular, by using the recursive feature elimination method, we show that HLA-G, B7H3, PDL-1 and ILT2 are the most relevant genes that characterize ccRCC. Notably, ILT2 expression was detected for the first time on tumor cells. The levels of other ligand-receptor pairs such as CD70:CD27; 4-1BB:4-1BBL; CD40:CD40L; CD86:CTLA4; MHC-II:Lag3; CD200:CD200R; CD244:CD48 were also found highly expressed in tumors compared to adjacent non-tumor tissues. Collectively, our approach provides a comprehensible classification of forty-four IC expressed in ccRCC, some of which were never reported before to be co-expressed in ccRCC. In addition, the algorithms used allowed identifying the most relevant group that best discriminates tumor from healthy tissues. The data can potentially assist on the choice of valuable immune-therapy targets which hold potential for the development of more effective anti-tumor treatments.

Immune checkpoints are powerful inhibitory molecules that promote tumor survival. Their blockade is now recognized as providing effective therapeutic benefit against cancer. Human leukocyte antigen G (HLA‐G), a recently identified immune checkpoint, has been detected in many types of primary tumors and metastases, in malignant effusions as well as on tumor‐infiltrating cells, particularly in patients with clear cell renal cell carcinoma (ccRCC). Here, in order to define a possible anticancer therapy, we used a molecular approach based on an unbiased strategy that combines transcriptome determination and immunohistochemical labeling, to analyze in‐depth the HLA‐G isoforms expressed in these tumors. We found that the expression of HLA‐G is highly variable among tumors and distinct areas of the same tumor, testifying a marked inter‐ and intratumor heterogeneity. Moreover, our results generate an inventory of novel HLA‐G isoforms which includes spliced forms that have an extended 5′‐region and lack the transmembrane and alpha‐1 domains. So far, these isoforms could not be detected by any method available and their assessment may improve the procedure by which tumors are analyzed. Collectively, our approach provides the first extensive portrait of HLA‐G in ccRCC and reveals data that should prove suitable for the tailoring of future clinical applications.

HLA-G is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother, thus critically contributing to fetal-maternal tolerance. The first functional finding constituted the basis for HLA-G research and can be summarized as such: HLA-G, membrane-bound or soluble, strongly binds its inhibitory receptors on immune cells (NK, T, B, monocytes/dendritic cells), inhibits the functions of these effectors, and so induces immune inhibition. HLA-G function may therefore be beneficial because when expressed by a fetus or a transplant it protects them from rejection, or deleterious because when expressed by a tumor, it also protects it from antitumor immunity. This is the primary HLA-G function: that of a checkpoint molecule. Great work has been done in the past years to characterize HLA-G itself, its regulation, its functions and mechanisms of action, and its pathological relevance. We will review this here, focusing on transplantation and oncology because these pathological contexts have been studied the most and also because they best represent the two opposite sides of HLA-G: beneficial to be promoted, or deleterious to be blocked.

With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.

Hematopoietic stem cells (HSC) are essential for maintaining the integrity of complex and long-lived organisms. HSC, which are self-renewing, reconstitute the hematopoietic system through out life and facilitate long-term repopulation of myeloablated recipients. We have previously demonstrated that when mice are exposed to sublethal doses of ionizing radiation, subsets of the stem/progenitor compartment are affected. In this study we examine the role of thrombopoietin (TPO) on the regenerative capacities of HSC after irradiation and report the first demonstration of efficacy of a single injection of TPO shortly after in vivo exposure to ionizing radiation for reducing HSC injury and improving their functional outcome. Our results demonstrate that TPO treatment not only reduced the number of apoptotic cells but also induced a significant modification of their intrinsic characteristics. These findings were supported by transplantation assays with long-term HSC that were irradiated or unirradiated, TPO treated or untreated, in CD45.1/CD45.2 systems and by using luciferase-labeled HSC for direct bioluminescence imaging in living animals. Of particular importance, our data demonstrate the skull to be a highly favorable site for the TPO-induced emergence of hematopoietic cells after irradiation, suggesting a TPO-mediated relationship of primitive hematopoietic cells to an anatomical component. Together, the data presented here: provide novel findings about aspects of TPO action on stem cells, open new areas of investigation for therapeutic options in patients who are treated with radiation therapy, and show that early administration of a clinically suitable TPO-agonist counteracts the previously observed adverse effects.

HLA-G is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother, thus critically contributing to fetal-maternal tolerance. The first functional finding constituted the basis for HLA-G research and can be summarized as such: HLA-G, membrane-bound or soluble, strongly binds its inhibitory receptors on immune cells (NK, T, B, monocytes/dendritic cells), inhibits the functions of these effectors, and so induces immune inhibition. HLA-G function may therefore be beneficial because when expressed by a fetus or a transplant it protects them from rejection, or deleterious because when expressed by a tumor, it also protects it from antitumor immunity. This is the primary HLA-G function: that of a checkpoint molecule. Great work has been done in the past years to characterize HLA-G itself, its regulation, its functions and mechanisms of action, and its pathological relevance. We will review this here, focusing on transplantation and oncology because these pathological contexts have been studied the most and also because they best represent the two opposite sides of HLA-G: beneficial to be promoted, or deleterious to be blocked.

To analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the IIb murine promoter of the gene encoding the IIb-subunit of the platelet integrin IIbβ3 (Tropel et al, Blood90:2995, 1997). Administration of ganciclovir (GCV) to these mice resulted not only in an acute cessation of platelet production due to the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high proportion of Sca-1+ and CD34+ cells, with CD45R+ cells of the lymphopoietic lineage being maintained. Stromal cells were also preserved. Blood thrombopoietin levels were high. At 4 days of the recovery phase, Sca-1 and CD34 antigen expression decreased with intense proliferation of cells of the three lineages, with megakaryocyte (MK) progenitors being identified by their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the IIb gene promoter was present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin apertures. Proplatelet production was visualized in the vascular sinus. After 15 days, circulating platelet levels had increased to approximately 65% of normal. Transgenic IIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis. © 1998 by The American Society of Hematology.

The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the α subunit of the platelet integrin αIIbβ3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the αIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5′-flanking region of the murine αIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the αIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.

The genetic programs that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage remain poorly defined and require appropriate in vivo models. Using a cell-specific obliteration technique, a transgenic mouse model was produced where perturbations of megakaryocytopoiesis and platelet production may be induced on demand. This was achieved by targeting the expression of the herpes virus thymidine kinase (HSV-tk) to megakaryocytes using the regulatory regions of the gene coding for the alphaIIb gene, an early marker of megakaryocytopoiesis, which encodes the alpha subunit of the platelet integrin alphaIIb beta3. The HSV-tk gene is not toxic by itself, but sensitizes the target cell to the effect of ganciclovir (GCV), leading to the inhibition of DNA synthesis in dividing cells. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alphaIIb-tk) with GCV. After 10 days of treatment, the platelet number was reduced by greater than 96.5% and megakaryocytes were not detectable in the bone marrow (BM). After discontinuing GCV, BM was repopulated with megakaryocytes, and the platelet count was restored within seven days. The recovery was accelerated by the administration of interleukin 11. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic bipotent progenitor cells. The reversibility and facility of this system provide a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development, and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by nonarrayed DNA barcodes subsequently analyzed by high-throughput DNA sequencing. However, key limitations include statistical biases compromising quantification and the lack of applicability to deconvolute individual cell fate in vivo after pooling single cells differentially exposed to different conditions ex vivo. Here, we have derived an arrayed lentiviral library of DNA barcodes and obtained a proof-of-concept of its resolving capacity by quantifying hematopoietic regeneration after engraftment of mice with genetically modified autologous cells. This method has helped clarify and bridge the seemingly opposed clonal-succession and continuous-recruitment models of hematopoietic stem cell behavior and revealed that myeloid-lymphoid biases are common occurrences in steady-state hematopoiesis. Arrayed lentiviral barcoding should prove a versatile and powerful approach to deconvolute cell dynamics in vivo with applications in hematology, embryology, and cancer biology.

Embryonic stem cells (ESC) are mostly derived from the undifferentiated inner mass cells of a blastocyst. These cells give rise during development of the embryo to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. They do not contribute to the extra-embryonic membranes or the placenta. Ex-vivo, they can be cultured for extended periods of time and under the appropriate conditions, they can be also directed to differentiate into many specialized types of cells. These particular features are being exploited to use ESC as starting material for treatment of degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, the quite small production of ESCderived cells obtained or the active immune rejection of the ESC-derived graft.

International audience; Ionizing radiation causes rapid and acute suppression of hematopoietic cells that manifests as the hematopoietic syndrome. However, the roles of molecules and regulatory pathways induced in vivo by irradiation of different hematopoietic cells have not been completely elaborated. Using a strategy that combined different microarray bioinformatics tools, we identified gene networks that might be involved in the early response of hematopoietic cells radiation response in vivo. The grouping of similar time-ordered gene expression profiles using quality threshold clustering enabled the successful identification of common binding sites for 56 transcription factors that may be involved in the regulation of the early radiation response. We also identified novel genes that are responsive to the transformation-related protein 53; all of these genes were biologically validated in p53-transgenic null mice. Extension of the analysis to purified bone marrow cells including highly purified long-term hematopoietic stem cells, combined with functional classification, provided evidence of gene expression modifications that were largely unknown in this primitive population. Our methodology proved particularly useful for analyzing the transcriptional regulation of the complex ionizing radiation response of hematopoietic cells. Our data may help to elucidate the molecular mechanisms involved in tissue radiosensitivity and to identify potential targets for improving treatment in radiation emergencies.

International audience; Purpose: To identify transcriptional gene-networks involved in the early in vivo response of liver cells to radiation exposure and improve our understanding of the molecular processes responsible for tissue radiosensitivity.Materials and methods: Transcriptome variations of liver RNA samples were measured 3 hours post-irradiation using microarray technology. The results were confirmed and extended using real-time polymerase-chain-reaction (RT-PCR).Results: We identified quantitative changes in the expression of 126 genes, most of which were observed for the first time. We show that some modifications, such as the upregulation of the cyclin-dependent kinase inhibitor 1A (Cdkn1A) gene, persisted for at least two months after the initial exposure. Other genes regulated by the transformation-related protein 53 (Trp53/p53) such as Bcl2-associated X protein (Bax) or etoposide-induced-2.4 (Ei24/PIG8) were not upregulated. Grouping differentially expressed genes into functional categories revealed that the primary response of liver cells to radiation exposure was the enhancement of oxidoreductase activity and inhibition of cell proliferation, involving cell cycle progression and apoptosis-related genes.Conclusions: The data provides evidence of gene expression modifications associated with the hepatic response to radiation exposure. One of the main differences observed with radiation-sensitive tissues such as the spleen was cell proliferation. The comparison of our data with transcriptome modifications in different biological models enabled the identification of networks of genes that might be co-regulated. Overall, our expression data revealed genes and cellular pathways that might help to improve our understanding of the molecular basis underlying tissue radiosensitivity and to identify possible targets for novel therapeutic strategies.

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbalpha). Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbalpha-VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbalpha. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbalpha or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.

Polyploidization is a part of the normal developmental process leading to platelet production during megakaryocyte (MK) differentiation. Ploidization is mainly involved in cell enlargement, but it is not clear whether gene expression is modified during MK ploidization. In this study, human MKs were grown from CD34+ cells in the presence of thrombopoietin and sorted according to their ploidy level. A pangenomic microarray technique was applied to compare gene expression in 2N-, 4N-, 8N-, and 16N-sorted MKs. Using hierarchical clustering, we demonstrated that 2N and 4N MKs or 8N and 16N MKs are 2 different close populations with 105 discriminating genes. In the second approach, we determined the profile of genes that were continuously down- and up-regulated during polyploidization. Among the 100 down-regulated genes, 24 corresponded to genes involved in DNA replication and repair. The great majority of up-regulated genes corresponded to genes directly involved in platelet functions, such as genes encoding specific platelet glycoproteins and α-granule proteins, actin and microtubule cytoskeleton, factors involved in signaling, and transport proteins. Together, these results suggest that MK polyploidization per se does not regulate gene expression but is intrinsically included in the differentiation process.

Our knowledge of the molecular mechanisms that regulate hematopoiesis in physiologic and pathologic conditions is limited. Using a molecular approach based on cDNA microarrays, we demonstrated the emergence of an alternative pathway for mature bone marrow cell recovery after the programmed and reversible eradication of CD41+ cells in transgenic mice expressing a conditional toxigene targeted by the platelet αIIb promoter. The expression profile of the newly produced CD41+ cells showed high levels of transcripts encoding Ezh2, TdT, Rag2, and various immunoglobulin (Ig) heavy chains. In this context, we identified and characterized a novel population of Lin-Sca-1hic-Kit- cells, with a lymphoid-like expression pattern, potentially involved in the reconstitution process. Our study revealed novel transcriptional cross talk between myeloid and lymphoid lineages and identified gene expression modifications that occur in vivo under these particular stress conditions, opening important prospects for therapeutic applications.

Mouse embryonic stem (ES) cells can be propagated in vitro while retaining their properties of pluripotency and self-renewal under the continuous presence of leukemia inhibitor factor (LIF). An essential role has been attributed to subsequent activation of the Stat3 transcription factor in mediating LIF self-renewal response. To date, however, downstream target genes of Stat3 in ES cells are still unknown. To isolate these genes, we performed a microarray-based kinetic comparison of LIF-stimulated (undifferentiated) ES cells versus ES cells induced to differentiate by shutting down Stat3 activity through either LIF deprivation or, more specifically, expression of a Stat3 dominant-negative mutant. In each case, we chose the earliest time at which ES cells lose their self-renewal properties, as illustrated by a decrease in the number of embryoid bodies and blast cell colony formation as well as germ layer marker expression. Comparison of the two independent approaches revealed similarly regulated genes that are likely to be involved in the Stat3 effects on ES cell self-renewal. For instance, upregulation of growth factors such as the transforming growth factor-P relative Lefty1 or transcriptional regulators such as Id1 and Id2 and downregulation of the groucho-like protein Aes1 (grg5) were found. Promoter analysis of the aes1 gene revealed three functional Stat3 consensus sites, as shown by luciferase assays. Furthermore, chromatin immunoprecipitation experiment demonstrated that Stat3 is recruited to the promoter of aes1 in ES cells. These data demonstrated that the aes1 gene is a direct transcriptional target of Stat3 in ES cells. STEM CELLS 2005;23:1634-1642.

Summary In microarray experiments, accurate estimation of the gene variance is a key step in the identification of differentially expressed genes. Variance models go from the too stringent homoscedastic assumption to the overparameterized model assuming a specific variance for each gene. Between these two extremes there is some room for intermediate models. We propose a method that identifies clusters of genes with equal variance. We use a mixture model on the gene variance distribution. A test statistic for ranking and detecting differentially expressed genes is proposed. The method is illustrated with publicly available complementary deoxyribonucleic acid microarray experiments, an unpublished data set and further simulation studies.

International audience; p53 can induce apoptosis in various ways including transactivation, transrepression and transcription-independent mechanisms. What determines the choice between them is poorly understood. In a rat embryo fibroblast model, caspase inhibition changed the outcome of p53 activation from standard Bcl-2-regulated apoptosis to caspase-independent and Bcl-2-insensitive cell death, a phenomenon not described previously. Here, we show that caspase inhibition affects cell death commitment decisions by modulating the apoptotic functions of p53. Indeed, in the Bcl-2-sensitive pathway, transactivation-dependent signalling is activated leading to a rapid MDM2-mediated degradation of p53. In contrast, in the Bcl-2-insensitive pathway, p53 is stable and this is associated with transrepression-dependent signalling. A study with microarrays identified these genes regulated by p53 in the absence of active caspases.

Gene expression in aging kidney and pituitary was determined by subtractive hybridization, DNA micro arrays and RT-PCR. Kidneys and pituitary were removed from 10- and 30-month-old female WAG/Rij rats, which were free from chronic progressive nephrosis and had a low incidence of pituitary tumors with age. From 350 cDNA fragments isolated by subtractive hybridization, just one showed a more than twofold change in expression between 10 and 30 months. The use of a specific micro array with 4050 rodent genes also failed to detect downregulation lower than 0.5 or upregulation larger than 2.0 in aging rat kidney. Similarly,mRNA content for vasopressin V2 and V1 receptors, aquaporin 2 and 3, and adenylyl cyclase type VI was not significantly modified with age as determined by RT-PCR. In contrast,microarray analysis of pituitary mRNA expression showed upregulation of 11 genes with ratios equal to or greater than 2.0 and downregulation of 6 genes with ratios equal to or less than 0.5. Two cDNA sequences of unknown genes from the kidney subtractive library were part of the age-related up- and downregulated genes of the pituitary. Other genes were mainly related to cell differentiation, control of homeostasis, cellular signaling, endoplasmic reticulum trafficking and metabolism. These data indicated that mRNA expression is barely modified in aging kidney free from chronic progressive nephrosis, at least in the 0.5–2.0 range, in contrast to pituitary. They also suggest that the downregulation of proteins reported in aging kidneys free from gross disease is related to post-transcriptional changes.

The molecular mechanisms by which a stem cell is committed to individual lineage are largely unknown. Two different models, though not mutually exclusive, are currently debated. The first describes the temporal and hierarchical coordination of lineage-specific transcriptional programs. The second suggests that multilineage genes are expressed in a self-renewing and undifferentiated cell prior to lineage commitment. To challenge these two models in in vivo-appropriate conditions, the expression of an exogenous toxigene was used to create transgenic animals in which an inducible, reversible cell knock-out at a specific stage of differentiation could be achieved. Both additional transgenesis using the megakaryocyte specific alphaIIb promoter and targeted transgenesis were used to express the herpes virus thymidine kinase (tk) gene in the megakaryocytic lineage. When the tk gene was targeted to the locus of the megakaryocyte-specific alphaIIb gene, a typical Glanzman thrombasthenic syndrome was created. Despite this bleeding disorder, the lack of expression of the alphaIIb gene did not affect the development of the mice. In both transgenic and targeted animals, all progenitor cells were sensitive to the effect of the gancyclovir (GCV), both in vivo and ex vivo. Long-term bone marrow cell cultures on stromal layers indicated that most of the very early progenitor cells expressed the enzyme. All the results obtained with this inducible toxic phenotype indicated that genetic programs that are in control of the expression of lineage-specific genes are operative in a totipotent stem cell prior to lineage commitment and strongly support the concept that stem cells express a multilineage transcriptome.

We have previously characterized an abundant male-specific mRNA from the submaxillary gland (SMG) of rats, encoding the SMR1 (androgen-regulated) protein, which has the structure of a prohormone and is processed by maturation enzymes to release a small peptide in the blood and saliva. We have now characterized three SMR1 -related cDNAs in the SMG of Balb/c mice. These cDNAs encode three novel proteins, designated MSG1, MSG2 and MSG3. They are 639, 662 and 471 nucleotides (nt) long, respectively, and the corresponding mRNAs appear to be expressed only in the SMG. The putative polypeptides they encode carry an N-terminal secretory peptide sequence and are, therefore, presumably secreted into saliva. Although closely related, the three mRNAs show striking differences: a particularly different expression pattern and an extremely high degree of variability observed in the central part of the molecules. The MSG1 and MSG3 cDNAs are identical, except for a 173-bp insert found only in MSG1 . This insert contains three Pro-rich repeats (GPGIGRPPPPPP), reminiscent of the most abundant multigenic family of the SMG, the Pro-rich proteins (PRP). Although MSG1 shares several common features with PRP, it is structurally related to SMR1. The unusually high ratio of replacement/silent nt changes provides a basis to address complex aspects concerning the molecular events leading to the emergence of new proteins in the SMG.