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.

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.

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.

Decreased glycolytic metabolism contributes to but is not the inducer of apoptosis following IL-3-starvation.

IL-3 regulates the glycolytic pathway. In Baf-3 cells IL-3 starvation leads to a decrease in glucose uptake and in lactate production. To determine if there is a link between the decreased metabolism induced by growth factor-starvation and the induction of cell death, we have compared the cell death characteristics and the metabolic modifications induced by IL-3-deprivation or glucose-deprivation in Baf-3 cells. We show that in both conditions cells die by an apoptotic process which involves the activation of similar Caspases. Different metabolic parameters (i.e. intracellular ATP levels and lactate accumulation in the culture medium) were measured. We show that IL-3 deprivation leads to a partial decrease in lactate production in contrast to glucose deprivation that completely inhibits lactate production. Similarly following IL-3-starvation a significant drop in the intracellular ATP levels in live cells is observed only after 16 h when a large fraction, more than 50 per cent of cells, is already apoptotic. On the contrary, glucose deprivation is followed by an abrupt decrease in ATP levels in the first 2 h of treatment. However, in the presence of IL-3, cells are able to survive for an extended time in these conditions since 70% of cells survived with low ATP levels for up to 16 h. This was not due to partial inhibition of the apoptotic process by the low level of ATP as glucose-deprivation in the absence of IL-3 led to faster death kinetics of Baf-3 cells compared with IL-3 starvation only. These results indicate that the drop in ATP levels and the triggering of apoptosis can be dissociated in time and that when the glycolytic pathway is strongly inhibited, cells are able to survive with relatively low ATP levels if IL-3 is present. Finally we show that induction of bcl-x by IL-3 protects cells from glucose-deprivation induced cell death.