Kernel-based testing for single-cell differential analysis.

Single-cell technologies offer insights into molecular feature distributions, but comparing them poses challenges. We propose a kernel-testing framework for non-linear cell-wise distribution comparison, analyzing gene expression and epigenomic modifications. Our method allows feature-wise and global transcriptome/epigenome comparisons, revealing cell population heterogeneities. Using a classifier based on embedding variability, we identify transitions in cell states, overcoming limitations of traditional single-cell analysis. Applied to single-cell ChIP-Seq data, our approach identifies untreated breast cancer cells with an epigenomic profile resembling persister cells. This demonstrates the effectiveness of kernel testing in uncovering subtle population variations that might be missed by other methods.

Investigating the role of the experimental protocol in phenylhydrazine-induced anemia on mice recovery.

Production of red blood cells involves growth-factor mediated regulation of erythroid progenitor apoptosis and self-renewal. During severe anemia, characterized by a strong fall of the hematocrit followed by a recovery phase, these controls allow a fast recovery of the hematocrit and survival of the organism. Using a mathematical model of stress erythropoiesis and an ad hoc numerical method, we investigate the respective roles of anemia-inducing phenylhydrazine injections and physiological regulation on the organism's recovery. By explicitly modeling the experimental protocol, we show that it mostly characterizes the fall of the hematocrit following the anemia and its severeness, while physiological process regulation mainly controls the recovery. We confront our model and our conclusions to similar experiments inducing anemia and show the model's ability to reproduce several protocols of phenylhydrazine-induced anemia. In particular, we establish a link between phenylhydrazine effect and the severeness of the anemia.

Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

The primary CD8 T cell immune response constitutes a major mechanism to fight an infection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developed a mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end of the response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential when looking at the predictions of the model several months after the infection.

The role of spatial organization of cells in erythropoiesis.

Erythropoiesis, the process of red blood cell production, occurs mainly in the bone marrow. The functional unit of mammalian erythropoiesis, the erythroblastic island, consists of a central macrophage surrounded by adherent erythroid progenitor cells (CFU-E/Pro-EBs) and their differentiating progeny, the erythroblasts. Central macrophages display on their surface or secrete various growth or inhibitory factors that influence the fate of the surrounding erythroid cells. CFU-E/Pro-EBs have three possible fates: (a) expansion of their numbers without differentiation, (b) differentiation into reticulocytes that are released into the blood, (c) death by apoptosis. CFU-E/Pro-EB fate is under the control of a complex molecular network, that is highly dependent upon environmental conditions in the erythroblastic island. In order to assess the functional role of space coupled with the complex network behavior in erythroblastic islands, we developed hybrid discrete-continuous models of erythropoiesis. A model was developed in which cells are considered as individual physical objects, intracellular regulatory networks are modeled with ordinary differential equations and extracellular concentrations by partial differential equations. We used the model to investigate the impact of an important difference between humans and mice in which mature late-stage erythroblasts produce the most Fas-ligand in humans, whereas early-stage erythroblasts produce the most Fas-ligand in mice. Although the global behaviors of the erythroblastic islands in both species were similar, differences were found, including a relatively slower response time to acute anemia in humans. Also, our modeling approach was very consistent with in vitro culture data, where the central macrophage in reconstituted erythroblastic islands has a strong impact on the dynamics of red blood cell production. The specific spatial organization of erythroblastic islands is key to the normal, stable functioning of mammalian erythropoiesis, both in vitro and in vivo. Our model of a simplified molecular network controlling cell decision provides a realistic functional unit of mammalian erythropoiesis that integrates multiple microenvironmental influences within the erythroblastic island with those of circulating regulators of erythropoiesis, such as EPO and glucocorticosteroids, that are produced at remote sites.

V-erbA generates ribosomes devoid of RPL11 and regulates translational activity in avian erythroid progenitors.

The v-erbA oncogene transforms chicken erythrocytic progenitors (T2EC) by blocking their differentiation and freezing them in a state of self-renewal. Transcriptomes of T2EC, expressing either v-erbA or a non-transforming form of v-erbA (S61G), were compared using serial analysis of gene expression and some, but not all, mRNA-encoding ribosomal proteins were seen to be affected by v-erbA. These results suggest that this oncogene could modulate the composition of ribosomes. In the present study, we demonstrate, using two-dimensional difference in gel electrophoresis, that v-erbA-expressing cells have a lower amount of RPL11 associated with the ribosomes. The presence of ribosomes devoid of RPL11 in v-erbA-expressing cells was further confirmed by immunoprecipitation. In order to assess the possible impact of these specialized ribosomes on the translational activity, we analyzed proteomes of either v-erbA or S61G-expressing cells using 2D/mass spectrometry, and identified nine proteins present in differing amounts within these cells. Among these proteins, we focused on HSP70 because of its involvement in erythroid differentiation. Our results indicate that, in v-erbA-expressing cells, hsp70 is not only transcribed but also translated more efficiently, as shown by polyribosome fractionation experiments. We demonstrate here, for the first time, the existence of ribosomes with different protein components, notably ribosomes devoid of RPL11, and a regulation of mRNA translation depending on v-erbA oncogene expression.

Modeling erythroblastic islands: using a hybrid model to assess the function of central macrophage.

The production and regulation of red blood cells, erythropoiesis, occurs in the bone marrow where erythroid cells proliferate and differentiate within particular structures, called erythroblastic islands. A typical structure of these islands consists of a macrophage (white cell) surrounded by immature erythroid cells (progenitors), with more mature cells on the periphery of the island, ready to leave the bone marrow and enter the bloodstream. A hybrid model, coupling a continuous model (ordinary differential equations) describing intracellular regulation through competition of two key proteins, to a discrete spatial model describing cell-cell interactions, with growth factor diffusion in the medium described by a continuous model (partial differential equations), is proposed to investigate the role of the central macrophage in normal erythropoiesis. Intracellular competition of the two proteins leads the erythroid cell to either proliferation, differentiation, or death by apoptosis. This approach allows considering spatial aspects of erythropoiesis, involved for instance in the occurrence of cellular interactions or the access to external factors, as well as dynamics of intracellular and extracellular scales of this complex cellular process, accounting for stochasticity in cell cycle durations and orientation of the mitotic spindle. The analysis of the model shows a strong effect of the central macrophage on the stability of an erythroblastic island, when assuming the macrophage releases pro-survival cytokines. Even though it is not clear whether or not erythroblastic island stability must be required, investigation of the model concludes that stability improves responsiveness of the model, hence stressing out the potential relevance of the central macrophage in normal erythropoiesis.

Cholesterol synthesis-related enzyme oxidosqualene cyclase is required to maintain self-renewal in primary erythroid progenitors.

OBJECTIVES: Molecular mechanisms controlling cell fate decision making in self-renewing cells are poorly understood. A previous transcriptomic study, carried out in primary avian erythroid progenitor cells (T2ECs), revealed that the gene encoding oxidosqualene cyclase (OSC/LSS), an enzyme involved in cholesterol biosynthesis, is significantly up-regulated in self-renewing cells. The aim of the present work is to understand whether this up-regulation is required for self-renewal maintenance and what are the mechanisms involved. MATERIALS AND METHODS: To investigate OSC function, we studied effects of its enzymatic activity inhibition using Ro48-8071, a specific OSC inhibitor. In addition, we completed this pharmacological approach by RNAi-mediated OSC/LSS knockdown. The study of OSC inhibition was carried out on both self-renewing and differentiating cells to observe any state-dependent effect. RESULTS: Our data show that OSC acts both by protecting self-renewing T2EC cells from apoptosis and by blocking their differentiation program, as OSC inhibition is sufficient to trigger spontaneous commitment of self-renewing cells towards an early differentiation state. This is self-renewal specific, as OSC inhibition has no effect on erythroid progenitors that have already differentiated. CONCLUSIONS: Taken together, our results suggest that OSC/LSS expression and activity are required to maintain cell self-renewal and may be involved in the self-renewal versus differentiation/apoptosis decision making, by keeping cells in a self-renewal state.

A multi-scale model of erythropoiesis.

In this paper, a multi-scale mathematical model of erythropoiesis is proposed in which erythroid progenitors are supposed to be able to self-renew. Three cellular processes control erythropoiesis: self-renewal, differentiation and apoptosis. We describe these processes and regulatory networks that govern them. Two proteins (ERK and Fas) are considered as the basic proteins participating in this regulation. All erythroid progenitors are divided into several sub-populations depending on their maturity level. Feedback regulations by erythropoietin, glucocorticoids and Fas ligand (FasL) are introduced in the model. The model consists of a system of ordinary differential equations describing intracellular protein concentration evolution and cell population dynamics. We study steady states and their stability. We carry out computer simulations of an anaemia situation and analyse the results.

Stem cell antigen 2: a new gene involved in the self-renewal of erythroid progenitors.

OBJECTIVES: Stem cell antigen 2 (SCA2), also known as TSA1 and LY6E, is a glycosylphosphatidylinositol-anchored molecule that belongs to the Ly-6 family and whose function remains largely unknown. We have previously shown that SCA2 is overexpressed in self-renewing avian erythroid progenitors (T2ECs) as opposed to differentiating T2ECs. The aim of this study was to define the role of SCA2 in the switch between self-renewal and differentiation of erythroid progenitors. MATERIALS AND METHODS: We have investigated the cellular processes controlled by SCA2 in T2ECs by RNA interference and overexpression approaches. Moreover, we have used a SAGE Querying and analysis tools developed in our laboratory, to investigate the expression level of SCA2 gene in different human cell types. RESULTS: We demonstrate the regulation of SCA2 expression by TGF-beta, a growth factor essential for self-renewal of T2ECs. We establish that SCA2 knockdown by RNA interference reduced the proliferation and promoted the differentiation of T2ECs. In contrast, SCA2 overexpression inhibited differentiation of T2ECs only. Furthermore, by using a bioinformatic approach, we found that SCA2 is highly expressed in a variety of human cancer cells. We confirmed this result by quantitative PCR on human colon and kidney tissues. CONCLUSIONS: Altogether, these findings imply that SCA2 may function in a dose-dependent manner to support the self-renewal state and that its deregulation might contribute to the development of some human cancers.

Involvement of the TGF-beta and mTOR/p70S6Kinase pathways in the transformation process induced by v-ErbA.

v-ErbA is the oncogenic form of TRalpha/c-ErbA which transforms chicken erythrocytic progenitors by blocking differentiation. The oncogenic property of v-ErbA has been correlated with its ability to antagonize ligand-dependent gene activation by TRalpha/c-ErbA and retinoic acid receptors. Nevertheless, its cytoplasmic retention suggests that v-ErbA could interfere with intracellular signaling pathways. We demonstrate that only the transforming form of v-ErbA confers to chicken erythroid progenitors a TGF-beta independency and induces an activation of the mTOR/p70S6K pathway. In these cells, TGF-beta and mTOR/p70S6K pathways regulate the expression of a known target gene of v-ErbA, band3. This is the first demonstration that v-ErbA is able to modulate specifically some signaling pathways leading to changes in the expression level of a gene involved in transformation.

The v-ErbA oncoprotein quenches the activity of an erythroid-specific enhancer.

v-ErbA is a mutated variant of thyroid hormone receptor (TRalpha/NR1A1) borne by the Avian Erythroblastosis virus causing erythroleukemia. TRalpha is known to activate transcription of specific genes in the presence of its cognate ligand, T3 hormone, while in its absence it represses it. v-ErbA is unable to bind ligand, and hence is thought to contribute to leukemogenesis by actively repressing erythroid-specific genes such as the carbonic anhydrase II gene (CA II). In the prevailing model, v-ErbA occludes liganded TR from binding to its cognate elements and constitutively interacts with the corepressors NCoR/SMRT. We previously identified a v-ErbA responsive element (VRE) within a DNase I hypersensitive region (HS2) located in the second intron of the CA II gene. We now show that HS2 fulfils all the requirements for a genuine enhancer that functions independent of its orientation and position with a profound erythroid-specific activity in normal erythroid progenitors (T2ECs) and in leukemic erythroid cell lines. We find that the HS2 enhancer activity is governed by two adjacent GATA-factor binding sites. v-ErbA as well as unliganded TR prevent HS2 activity by nullifying the positive function of factors bound to GATA-sites. However, v-ErbA, in contrast to TR, does not convey active repression to silence the transcriptional activity intrinsic to a heterologous tk promoter. We propose that depending on the sequence and context of the binding site, v-ErbA contributes to leukemogenesis by occluding liganded TR as well as unliganded TR thereby preventing activation or repression, respectively.

TGF-beta cooperates with TGF-alpha to induce the self-renewal of normal erythrocytic progenitors: evidence for an autocrine mechanism.

Simultaneous addition of both TGF-alpha and TGF-beta induces the sustained, long-term outgrowth of chicken erythrocytic progenitor cells, referred to as T2ECs from both chick bone marrow and 2-day-old chicken embryos. By analysis for differentiation antigens and gene expression, these cells were shown to represent very immature haematopoietic progenitors committed to the erythrocytic lineage. T2ECs differentiate into almost pure populations of fully mature erythrocytes within 6 days, when TGF-alpha and TGF-beta are withdrawn and the cells exposed to anaemic chicken serum plus insulin. Outgrowth of these cells from various sources invariably required both TGF-alpha and TGF-beta, as well as glucocorticoids. Proliferating, established T2ECs still require TGF-alpha, but are independent of exogenous TGF-beta. Using a TGF-beta-neutralizing antibody or expressing a dominant-negative TGF-beta receptor II, we demonstrate that T2ECs generate an autocrine loop involving TGF-beta during their establishment, which is required for sustained proliferation. Using specific inhibitors, we also show that signalling via Mek-1 is specifically required for induction and maintenance of cell proliferation driven by cooperation between the TGF-alpha and -beta receptors. These results establish a novel mechanism by which self-renewal of erythrocytic progenitors is induced and establish avian T2ECs as a new, quasi-optimal model system to study erythrocytic progenitors.

Role of the different RAR isoforms in controlling the erythrocytic differentiation sequence. Interference with the v-erbA and p135gag-myb-ets nuclear oncogenes.

Little is known as to how the nuclear oncogenes v-erbA and p135gag-myb-ets do transform cells. The elucidation of their molecular mechanisms of action requires the identification of relevant target genes. We analysed the possibility for the RARbeta gene to represent such a target gene. We first show that the RARbeta gene induction is a specific and direct process, requiring the continuous presence of retinoids and under the control of the RARalpha isoform exclusively. We then show that the expression of either the v-erbA or the p135gag-myb-ets oncogene is not sufficient to block the RARbeta gene induction. We confirmed the loss of RARbeta gene response in certain cell lines but we discarded the possibility that this loss might represent a necessary step for cell lines immortalization. We further show that the RARalpha isoform activation is necessary and sufficient to induce the growth inhibition and the differentiation stimulation characteristic for the commitment-inducing ability of retinoids in chicken erythrocytic progenitor cells. We therefore propose a model showing that RARalpha but not RARbeta is the key mediator for commitment to differentiation and that it should control two different set of genes whose expression is differentially affected by the v-erbA and the p135gag-myb-ets oncogenes.

Myb-Ets fusion oncoprotein inhibits thyroid hormone receptor/c-ErbA and retinoic acid receptor functions: a novel mechanism of action for leukemogenic transformation by E26 avian retrovirus.

The E26 and avian erythroblastosis virus (AEV) avian retroviruses induce acute leukemia in chickens. E26 can block both erythroid and myeloid differentiation at an early multipotent stage. Moreover, E26 can block erythroid differentiation at the erythroid burst-forming unit/erythroid CFU (BFU-E/CFU-E) stage, which also corresponds to the differentiation stage blocked by AEV. AEV carries two oncogenes, v-erbA and v-erbB, whereas E26 encodes a single 135-kDa Gag-Myb-Ets fusion oncoprotein. v-ErbA is responsible for the erythroid differentiation arrest through negative interferences with both the retinoic acid receptor (RAR) and the thyroid hormone receptor (T3R/c-ErbA). We investigated whether Myb-Ets could block erythroid differentiation in a manner similar to v-ErbA. We show here that Myb-Ets inhibits both RAR and c-ErbA activities on specific hormone response elements in transient-expression assays. Moreover, Myb-Ets abrogates the inactivation of transcription factor AP-1 by RAR and T3R, another feature shared with v-ErbA. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to retinoic acid and T3. Analysis of a series of mutants of Myb-Ets reveals that the domains of the oncoprotein involved in these inhibitory activities are the same as those involved in oncogenic transformation of hematopoietic cells. These data demonstrate that the Myb-Ets oncoprotein shares properties with the v-ErbA oncoprotein and that inhibition of ligand-dependent RAR and c-ErbA functions by Myb-Ets is responsible for blocking the differentiation of hematopoietic progenitors.

A rapid and convenient method to prepare DIG-labelled RNA probes for use in non-radioactive in situ hybridization.

We describe here the use of PCR-generated templates incorporating T3 polymerase sites in order to prepare digoxigenin (DIG)-labelled cRNA probes against any gene of known sequence. This method was applied to the preparation of probes specific for chicken glyceraldehyde-3-phosphate dehydrogenase messenger RNAs and we demonstrate that such probes can be used for in situ hybridization (ISH). This technique therefore represents a rapid and convenient means to prepare DIG-labelled cRNA probes for use in a non-radioactive ISH. It adds speed and convenience of probe preparation to the previously described advantages of non-radioactive detection techniques.

Antagonistic role of vitamin D3 and retinoic acid on the differentiation of chicken hematopoietic macrophages into osteoclast precursor cells.

An in vitro culture model of osteoclast differentiation is described which is derived from homogeneous populations of chick yolk sac and peripheral blood macrophages. In primary cultures, both types of macrophages undergo a proliferative phase, become quiescent after reaching high cell densities, then aggregate and eventually form large multinucleated giant cells (MNGCs), presumably by fusion. These MNGCs can be characterized as premature osteoclasts on the basis of several morphological and biochemical criteria, although they do not undergo the final differentiation step rendering them competent to resorb bone in vitro. Clonal analysis of single cell-derived colonies indicates that all macrophages have the potential to differentiate into these osteoclast-like cells under these culture conditions. Both retinoic acid and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] modulate macrophage growth, but in an antagonistic manner. Although retinoic acid strongly promotes macrophage proliferation and impedes MNGC formation, 1,25-(OH)2D3 inhibits proliferation and changes the kinetics of MNGC formation. Combination experiments reveal that the proliferative signals induced by retinoic acid can override the signal to differentiate induced by 1,25-(OH)2D3. Our results indicate that even though retinoic acid and vitamin D3 act through homologous receptors, they have dramatically opposing effects on macrophage differentiation toward osteoclast progenitors.

The v-erba oncogene - a superb tool for dissecting the involvement of nuclear hormone receptors in differentiation and neoplasia (review).

The v-erbA oncogene has been discovered as one of the two viral oncogenes carried by the avian leukemia retrovirus AEV. It is derived from the c-erbA protooncogene which encodes the alpha form of the nuclear receptor for the thyroid hormone triiodothyronine (T3R). This receptor belongs to a large family of nuclear hormone receptors that function as ligand-regulated transcription factors and the v-erbA oncoprotein has been shown to function as an antagonist of normal T3R and related receptors in the control of transcription. It is thus the first dominant negative transcription factor acting as an oncogene described to date. Functional and biochemical dissections of this oncogene have brought many informations on the mechanisms of action of normal receptors and on the ways through which altered receptors can contribute to oncogenic transformation. The v-erbA model is widely used as a reference to investigate the involvement of nuclear hormone receptors in the development of human cancers.

c-erbA alpha/T3R and RARs control commitment of hematopoietic self-renewing progenitor cells to apoptosis or differentiation and are antagonized by the v-erbA oncogene.

In AEV-transformed erythroleukemic cells the v-erbA gene product is likely to antagonize the function of triiodothyronine (T3) and retinoic acid (RA) receptors and thereby to block cell differentiation. We have thus investigated the effects of T3 and RA on normal early erythrocytic progenitor cells. Here we show: (1) that either RA or T3 play an essential role during the early commitment to erythrocytic differentiation, (2) that both T3 and RA induce death by apoptosis and a strong inhibition of self-renewal in progenitor cells grown in the absence of differentiation-inducing agents and (3) that the v-erbA oncogene renders erythrocytic progenitor cells insensitive to apoptosis and to self-renewal inhibition induced by RA or T3. The behaviour of a non-transforming mutant of v-erbA suggests that this v-erbA-induced protection is related to its transforming potential.

v-erbB oncogene expression accounts for most variations in protein synthesis after avian erythroblastosis virus infection of chicken embryo fibroblasts: a two-dimensional electrophoresis study.

The effect of the v-erbA and/or v-erbB oncogenes on cellular gene expression was investigated after separation by two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labelled proteins from chicken embryo fibroblasts (CEF), infected by either the avian erythroblastosis virus (AEV) carrying both oncogenes, or by viruses carrying only one of them. We observed significant changes in the synthesis of 34 proteins in AEV-transformed CEF as compared with control cells. The synthesis of 24 of them was increased while the synthesis of the other 10 proteins was decreased. The expression of v-erbB alone is necessary and sufficient to induce changes in the synthesis of 27 proteins while the 7 remaining modifications are observed only in cells expressing v-erbB together with v-erbA. Moreover, the deregulation of protein synthesis by v-erbB-expressing viruses was correlated with the morphological transformation state of cells.