Genetic programs promoting cell cycle progression, DNA repair, and survival are coordinately induced in developing T cells and require rapid turnover of effector molecules. As the COP9 signalosome (CSN) has been placed at the crossroads of these programs in lower organisms, we addressed its role by conditionally deleting CSN5/JAB1 , its catalytic subunit, in developing thymocytes. CSN5/JAB1 del/del thymocytes show defective S phase progression and massive apoptosis at the double-negative (DN) 4 -double-positive (DP) transition stage, which is paralleled by altered turnover of selected CSN-controlled substrates, including p53, I B-␣ , and ␤ -catenin. Combined dysregulation of the p53 and NF-B pathways affects thymocyte survival by altering the mRNA and protein levels of selected Bcl-2 family members. Genetic complementation analysis performed on p53 ؊ / ؊ , Bcl-xL/Bcl-2A1, or T cell receptor transgenic backgrounds indicates that CSN5/JAB1 acts at distinct developmental stages to coordinate proliferation, survival, and positive selection of thymocytes by controlling the induction of defi ned genetic programs acting downstream of CSN-regulated transcription factors. CSN5/JAB1-defi cient mice ( CSN5/JAB1 del/del ). The lack of CSN5/JAB1 mRNA and protein in DN and DP thymocytes was demonstrated by semiquantitative RT-PCR and Western blot analysis (Fig. S1 B). In addition, genomic DNA prepared from sorted DN and DP/single-positive (SP) cells was analyzed by PCR using primers detecting WT, fl oxed, or deleted alleles (Fig. S1 C). Most T cells in CSN5/JAB1 del/del mice showed a complete CSN5/JAB1 deletion, as indicated by the presence of the deleted allele in thymocytes and peripheral T cells. At the DN stage, CSN5/JAB1 del/del thymocytes yielded exclusively the PCR product corresponding to deleted allele. In peripheral T cells, however, we detected considerable levels of the CSN5/ JAB1 fl ox allele, suggesting that the residual mature T cells had escaped Cre-mediated deletion (Fig. S1 D). In support of this interpretation, peripheral T cells, although severely reduced in number, displayed a predominant memory-eff ector phenotype (CD44 high CD62L Ϫ ) in CSN5/JAB1-defi cient animals, suggesting homeostatic expansion of a small pool of phenotypically normal, mature T lymphocytes (Fig. S2).Thymic morphology, thymocyte counts, and subset distribution were comparatively assessed 5 -6 wk after birth in CSN5/JAB1 fl ox/fl ox mice expressing the LckCre transgene (herein defi ned as CSN5/JAB1 del/del ) and in littermates lacking LckCre . Thymi from CSN5/JAB1 del/del animals showed a severely hypoplastic medulla ( Fig. 1 A ), a marked decrease in the proportion of DP and SP thymocytes ( Fig. 1 B ), and an 80 -90% reduction in thymocyte number ( Fig. 1 C ), whereas the various subsets of DN thymocytes, as determined by the expression of the CD44 and CD25 stage-specifi c markers, were not signifi cantly altered compared with the CSN5/JAB1 fl ox/fl ox littermate controls, both percentage-wise ( Fig. 1 B ) and in absolute numbers ( F...

SummaryThe evolutionarily conserved transcriptional cofactor Jab1 plays critical roles in cell differentiation, proliferation, and apoptosis by modulating the activity of diverse factors and regulating the output of various signaling pathways. Although Jab1 can interact with the bone morphogenetic protein (BMP) downstream effector Smad5 to repress BMP signaling in vitro, the role of Jab1 in BMP-mediated skeletogenesis in vivo is still poorly understood. As a key regulator of skeletogenesis, BMP signaling regulates the critical Ihh-Pthrp feedback loop to promote chondrocyte hypertrophy. In this study, we utilized the loxP/Cre system to delineate the specific role of Jab1 in cartilage formation. Strikingly, Jab1 chondrocyte-specific knockout Jab1 flox/flox ; Col2a1-Cre (cKO) mutants exhibited neonatal lethal chondrodysplasia with severe dwarfism. In the mutant embryos, all the skeletal elements developed via endochondral ossification were extremely small with severely disorganized chondrocyte columns. Jab1 cKO chondrocytes exhibited increased apoptosis, G2 phase cell cycle arrest, and increased expression of hypertrophic chondrocyte markers Col10a1 and Runx2. Jab1 can also inhibit the transcriptional activity of Runx2, a key regulator of chondrocyte hypertrophy. Notably, our study reveals that Jab1 is likely a novel inhibitor of BMP signaling in chondrocytes in vivo. In Jab1 cKO chondrocytes, there was heightened expression of BMP signaling components including Gdf10/Bmp3b and of BMP targets during chondrocyte hypertrophy such as Ihh. Furthermore, Jab1 cKO chondrocytes exhibited an enhanced response to exogenous BMP treatment. Together, our study demonstrates that Jab1 represses chondrocyte hypertrophy in vivo, likely in part by downregulating BMP signaling and Runx2 activity.

a b s t r a c tThe COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.Structured summary: MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006) Ó

Jab1 constitutes a regulatory molecule that integrates laminin211 signals in Schwann cells to govern cell cycle, cell number, and differentiation.