Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair1. In addition, antigens present within necrotic cells can sometimes provoke a specific immune response2-4 and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection5, 6. In the mouse, the CD8α+ subset of dendritic cells (DC) phagocytoses dead cell remnants and crossprimes CD8+ T cells against cell-associated antigens7. Here, we show that CD8α+ DC utilise CLEC9A (DNGR-1), a recently-characterised C-type lectin8-10, to recognise a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair uptake of necrotic cell material by CD8α+ DC but specifically reduces crosspresentation of dead cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue within its intracellular tail that allows recruitment and activation of the tyrosine kinase Syk, which is also essential for crosspresentation of dead cell-associated antigens. Thus, CLEC9A functions as a Syk-coupled C-type lectin receptor to mediate sensing of necrosis by the principal DC subset involved in regulating crosspriming to cell-associated antigens.

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

In contrast to cancer cells and embryonic stem cells, the lifespan of primary human cells is finite. After a defined number of population doublings, cells enter in an irreversible growth-arrested state termed replicative senescence. Mutations of genes involved in immortalization can contribute to cancer. In a genetic screen for cDNAs bypassing replicative senescence of normal human prostate epithelial cells (HPrEC), we identified CBX7, a gene that encodes a Polycomb protein, as shown by sequence homology, its interaction with Ring1 and its localization to nuclear Polycomb bodies. CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus. CBX7 does not inter-function or colocalize with Bmi1, and both can exert their actions independently of each other as shown by reverse genetics. CBX7 expression is downregulated during replicative senescence and its ablation by short-hairpin RNA (shRNA) treatment inhibited growth of normal cells though induction of the Ink4a/Arf locus. Taken together, these data show that CBX7 controls cellular lifespan through regulation of both the p16(Ink4a)/Rb and the Arf/p53 pathways.

Allelic exclusion of immunoglobulin genes ensures the expression of a single antibody molecule in B cells through mostly unknown mechanisms. Large-scale contraction of the immunoglobulin heavy-chain (Igh) locus facilitates rearrangements between Igh variable (V H ) and diversity gene segments in pro-B cells. Here we show that these long-range interactions are mediated by 'looping' of individual Igh subdomains. The Igk locus also underwent contraction by looping in small pre-B and immature B cells, demonstrating that immunoglobulin loci are in a contracted state in rearranging cells. Successful Igh recombination induced the rapid reversal of locus contraction in response to pre-B cell receptor signaling, which physically separated the distal V H genes from the proximal Igh domain, thus preventing further rearrangements. In the absence of locus contraction, only the four most proximal V H genes escaped allelic exclusion in immature μ-transgenic B lymphocytes. Pre-B cell receptor signaling also led to rapid repositioning of one Igh allele to repressive centromeric domains in response to downregulation of interleukin 7 signaling. These data link both locus 'decontraction' and centromeric recruitment to the establishment of allelic exclusion at the Igh locus.The diverse antigen receptor repertoire of lymphocytes is generated by V(D)J recombination, which assembles the variable regions of immunoglobulin and T cell receptor genes from discontinuous variable (V), diversity (D) and joining (J) gene segments during B and T cell development1,2. These gene segments are flanked by recombination signal sequences that function as recognition sites for the V(D)J recombinase consisting of recombination activating gene 1 (RAG1) and RAG2 proteins. After pairing of two compatible recombination signal sequences, the RAG1-RAG2 complex introduces doublestrand DNA breaks between the recombination signal sequences and flanking gene segments, followed by processing and religation of the DNA ends by repair factors of the nonhomologous end-joining machinery1,2. V(D)J recombination is tightly controlled in a lineage-and stage-specific way. Immunoglobulin and T cell receptor genes are rearranged only in B and T lymphocytes, respectively1,2. In the B lymphoid lineage, the immunoglobulin heavy-chain (Igh) locus

An increased dependency on glycolysis for ATP production is considered to be a hallmark of tumor cells. Whether this increase in glycolytic activity is due mainly to inherent metabolic alterations or to the hypoxic microenvironment remains controversial. Here we have transformed human adult mesenchymal stem cells (MSC) using genetic alterations as described for differentiated cells. Our data suggest that MSC require disruption of the same pathways as have been shown for differentiated cells to confer a fully transformed phenotype. Furthermore, we found that MSC are more glycolytic than primary human fibroblasts and, in contrast to differentiated cells, do not depend on increased aerobic glycolysis for ATP production during transformation. These data indicate that aerobic glycolysis (the Warburg effect) is not an intrinsic component of the transformation of adult stem cells, and that oncogenic adaptation to bioenergetic requirements, in some circumstances, may also rely on increases in oxidative phosphorylation. We did find, however, a reversible increase in the transcription of glycolytic enzymes in tumors generated by transformed MSC, indicating this is a secondary phenomenon resulting from adaptation of the tumor to its microenvironment. adult stem cells ͉ glycolysis ͉ Warburg effect

Chromobox 7 (CBX7) is a chromobox family protein and a component of the Polycomb repressive complex 1 (PRC1) that extends the lifespan of cultured epithelial cells and can act independently of BMI-1 to repress the INK4a/ARF tumor suppressor locus. To determine whether CBX7 might be oncogenic, we examined its expression pattern in a range of normal human tissues and tumor samples. CBX7 was expressed at high levels in germinal center lymphocytes and germinal center-derived follicular lymphomas, where elevated expression correlated with high c-Myc expression and a more advanced tumor grade. By targeting Cbx7 expression to the lymphoid compartment in mice, we showed that Cbx7 can initiate T cell lymphomagenesis and cooperate with c-Myc to produce highly aggressive B cell lymphomas. Furthermore, Cbx7 repressed transcription from the Ink4a/Arf locus and acted epistatically to the Arf-p53 pathway during tumorigenesis. These data identify CBX7 as a chromobox protein causally linked to cancer development and may help explain the low frequency of INK4a/ ARF mutations observed in human follicular lymphoma.Ink4a-ARF ͉ oncogene ͉ Polycomb ͉ follicular lymphoma ͉ p53

Control of cell proliferation by Polycomb group proteins (PcG) is an important facet of cellular homeostasis and its disruption can promote tumorigenesis. We recently described CBX7 as a novel PcG protein controlling the growth of normal cells. In an attempt to identify a putative role of CBX7 in tumorigenesis, we analysed CBX7 expression in a panel of cancer cell lines and primary tissues. CBX7 was highly expressed in three different prostate cancer cell lines and present at elevated levels in normal prostate. Ablation of CBX7 expression using short hairpin RNAs (shRNA) resulted in upregulation of p16 Ink4a and p14 Arf in both LNCaP and PC-3 prostate cell lines. CBX7 knockdown caused an impairment of cell growth that was dependent on the status of the p14 Arf /p53 and p16 Ink4a /Rb pathways in both normal and cancer prostate cells. CBX7 overexpression in LNCaP cells resulted in a slight growth advantage in both androgen-dependent andindependent conditions. Moreover, CBX7 expression cooperated with c-Myc in rendering LNCaP cells insensitive to growth arrest by androgen receptor inhibition. Together, these data suggest that CBX7 represses p16 Ink4a and p14 Arf expression in normal and tumor-derived prostate cells, affecting their growth depending on the status of the p16 Ink4a /Rb and the p14 Arf /p53 pathways.

Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.