The maintenance of cycling cell lineages relies on undifferentiated subpopulations consisting of stem and progenitor pools. Features that delineate these cell types are undefined for many lineages, including spermatogenesis, which is supported by an undifferentiated spermatogonial population. Here, we generated a transgenic mouse line in which spermatogonial stem cells are marked by expression of an inhibitor of differentiation 4 (Id4)-green fluorescent protein (Gfp) transgene. We found that Id4-Gfp + cells exist primarily as a subset of the type A single pool, and their frequency is greatest in neonatal development and then decreases in proportion during establishment of the spermatogenic lineage, eventually comprising~2% of the undifferentiated spermatogonial population in adulthood. RNA sequencing analysis revealed that expression of 11 and 25 genes is unique for the Id4-Gfp + /stem cell and Id4-Gfp -/progenitor fractions, respectively. Collectively, these findings provide the first definitive evidence that stem cells exist as a rare subset of the A single pool and reveal transcriptome features distinguishing stem cell and progenitor states within the mammalian male germline.

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Here, we demonstrate that Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis.

Spermatogenesis is a classic model of cycling cell lineages that depend on a balance between stem cell self-renewal for continuity and the formation of progenitors as the initial step in the production of differentiated cells. The mechanisms that guide the continuum of spermatogonial stem cell (SSC) to progenitor spermatogonial transition and precise identifiers of subtypes in the process are undefined. Here we used an Id4-eGfp reporter mouse to discover that EGFP intensity is predictive of the subsets, with the ID4-EGFP Bright population being mostly, if not purely, SSCs, whereas the ID4-EGFP Dim population is in transition to the progenitor state. These subsets are also distinguishable by transcriptome signatures. Moreover, using a conditional overexpression mouse model, we found that transition from the stem cell to the immediate progenitor state requires downregulation of Id4 coincident with a major change in the transcriptome. Collectively, our results demonstrate that the level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition between the different functional states.

ASC-2, a multifunctional coactivator, forms a steady-state complex, named ASCOM (for ASC-2 COMplex), that contains the histone H3-lysine-4 (H3K4)-methyltransferase MLL3 or its paralogue MLL4. Somewhat surprisingly, given prior indications of redundancy between MLL3 and MLL4, targeted inactivation of the MLL3 H3K4-methylation activity in mice is found to result in ureter epithelial tumors. Interestingly, this phenotype is exacerbated in a p53 ؉/؊ background and the tumorigenic cells are heavily immunostained for ␥H2AX, indicating a contribution of MLL3 to the DNA damage response pathway through p53. Consistent with the in vivo observations, and the demonstration of a direct interaction between p53 and ASCOM, cell-based assays have revealed that ASCOM, through ASC-2 and MLL3/4, acts as a p53 coactivator and is required for H3K4-trimethyation and expression of endogenous p53-target genes in response to the DNA damaging agent doxorubicin. In support of redundant functions for MLL3 and MLL4 for some events, siRNA-mediated down-regulation of both MLL3 and MLL4 is required to suppress doxorubicin-inducible expression of several p53-target genes. Importantly, this study identifies a specific H3K4 methytransferase complex, ASCOM, as a physiologically relevant coactivator for p53 and implicates ASCOM in the p53 tumor suppression pathway in vivo.

peroxisome proliferator-activated receptor ␥ ͉ transcription ͉ activating signal cointegrator-2 ͉ coactivator ͉ ASCOM

Apoptosis is genetically controlled cell death that is essential for development and homeostasis of multicellular organisms. Both excessive and insufficient cell death can lead to anomalies or diseases. The molecular machinery involved in apoptosis is highly conserved across divergent species. The apoptotic death of cells requires proteolytic activation of caspases that are synthesized as latent proenzymes. Once activated, caspases cleave a wide range of molecules that eventually result in the dismantlement of cells. Caspases are therefore tightly controlled within the cells. Active caspases can be specifically inhibited by the inhibitor of apoptosis (IAP), 1 a family of proteins containing one to three copies of characteristic baculovirus IAP repeat (BIR) (1). The BIR domains, in some cases together with the intervening linker regions, directly bind and inhibit caspases.IAP proteins are counteracted by Reaper, Hid, Grim, Sickle, and Jafrac2 in Drosophila, and by Smac/DIABLO, Omi/HtrA2, and GSPT1/eRF3 in mammals (2-13). Despite the overall sequence differences, these IAP antagonists share a conserved N-terminal IAP-binding motif (IBM) or Reaper/Hid/Grim motif. This motif is indispensable and sufficient for counteracting IAP inhibition. The small subunit of active caspase-9 also binds to XIAP through the same IBM produced by auto-processing (14). This mechanism allows these IAP antagonists to compete with caspases for IAP binding and consequently relieves caspases and promotes cell death.Some IAPs also regulate apoptosis through the ubiquitinprotein isopeptide ligase (E3) activity of their RING domain (1, 15). These IAPs are capable of targeting the poly-ubiquitination of IAP-binding proteins such as caspases and IAP antagonists (16 -21). On the other hand, the E3 activity of IAP also leads to the ubiquitination of IAPs themselves, and such autoubiquitination can be enhanced by Reaper, Hid,). An alternatively spliced form of Smac, Smac3, was recently reported to promote XIAP ubiquitination and degradation (28). These observations demonstrate that these IAP antagonists accelerate the disposal of IAPs in addition to releasing captive caspases from IAPs. Moreover, the mammalian IAP-binding protein Omi/HtrA2 can directly degrade IAP molecules through its serine protease activity (29 -31).Five of the eight human IAPs have a C-terminal RING domain (32). However, little is known about the characteristics of the E3 activity of various IAPs. Even though the ubiquitination and proteasomal degradation of XIAP have been reported to be enhanced by Smac3 rather than Smac, it is not known if c-IAP1 and c-IAP2 are also modulated by their cognate antagonists via ubiquitin/proteasomal degradation. In this paper we show that Smac stimulates the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although Smac does promote auto-ubiquitination of all four of these human IAPs. These human IAPs require the same ubiquitin-conjugating enzymes (E2) UbcH5a and UbcH6 for their E3 activity in vitro. We also demonstra...

Baculoviral inhibitor of apoptosis repeat-containing (Birc)6 gene͞ BIRC6 (Bruce͞APOLLON) encodes an inhibitor of apoptosis and a chimeric E2͞E3 ubiquitin ligase in mammals. The physiological role of Bruce in antiapoptosis is unknown. Here, we show that deletion of the C-terminal half of Bruce, including the UBC domain, causes activation of caspases and apoptosis in the placenta and yolk sac, leading to embryonic lethality. This apoptosis is associated with up-regulation and nuclear localization of the tumor suppressor p53 and activation of mitochondrial apoptosis, which includes upregulation of Bax, Bak, and Pidd, translocation of Bax and caspase-2 onto mitochondria, release of cytochrome c and apoptosis-inducing factor, and activation of caspase-9 and caspase-3. Mutant mouse embryonic fibroblasts are sensitive to multiple mitochondrial death stimuli but resistant to TNF. In addition, eliminating p53 by RNA interference rescues cell viability induced by Bruce ablation in human cell line H460. This viability preservation results from reduced expression of proapoptotic factors Bax, Bak, and Pidd and from prevention of activation of caspase-2, -9, and -3. The amount of second mitochondrial-derived activator of caspase and Omi does not change. We conclude that p53 is a downstream effector of Bruce, and, in response to loss of Bruce function, p53 activates Pidd͞caspase-2 and Bax͞Bak, leading to mitochondrial apoptosis. P rogrammed cell death or apoptosis is critical for development and homeostasis in metazoans (1). The inhibitor of apoptosis (IAP) proteins antagonize cell death by suppressing active caspases. This inhibition can be reversed by IAP antagonists Reaper, Grim, and Hid in flies and the second mitochondrial-derived activator of caspase (Smac)͞Diablo family of proteins in vertebrates (2-5). Despite their well known anti-death function in vitro, IAPs have not been shown to be required for apoptosis inhibition in vertebrate development. Gene ablation of baculoviral IAP repeat-containing (Birc)4 (XIAP), the most potent caspase inhibitory IAP in cultured cells, does not perturb mouse development (6). In addition, Birc1a (Naip)-deficient mice do not manifest developmental abnormalities or spinal muscular atrophy, even though the deletion of BIRC1 (NAIP) correlates with the severity of this disease in a significant proportion of spinal muscular atrophy patients (7,8). Although tissue-specific ablation of Birc5 (survivin) in mice has demonstrated that Birc5 is required for thymocyte development (9) and the resistance of keratinocytes to UVB-induced apoptosis (10), Birc5-null embryos die primarily because of deficient mitotic division instead of apoptosis (11,12). Taken together, the physiological function of mammalian IAP family proteins in antagonizing apoptosis remains elusive in vivo.Birc6 (Bruce) is a large mouse IAP with a molecular mass of 528 kDa (13). Similar to other IAPs, Bruce promotes cell survival. It inhibits apoptosis by binding to caspases through its BIR domain, and its caspase inhibitory activity...

SUMMARYContinuity of cycling cell lineages relies on the activities of undifferentiated stem cell-containing subpopulations. Transition to a differentiating state must occur periodically in a fraction of the population to supply mature cells, coincident with maintenance of the undifferentiated state in others to sustain a foundational stem cell pool. At present, molecular mechanisms regulating these activities are poorly defined for most cell lineages. Spermatogenesis is a model process that is supported by an undifferentiated spermatogonial population and transition to a differentiating state involves attained expression of the KIT receptor. We found that impaired function of the X chromosome-clustered microRNAs 221 and 222 (miR-221/222) in mouse undifferentiated spermatogonia induces transition from a KIT -to a KIT + state and loss of stem cell capacity to regenerate spermatogenesis. Both Kit mRNA and KIT protein abundance are influenced by miR-221/222 function in spermatogonia. Growth factors that promote maintenance of undifferentiated spermatogonia upregulate miR-221/222 expression; whereas exposure to retinoic acid, an inducer of spermatogonial differentiation, downregulates miR-221/222 abundance. Furthermore, undifferentiated spermatogonia overexpressing miR-221/222 are resistant to retinoic acid-induced transition to a KIT + state and are incapable of differentiation in vivo. These findings indicate that miR-221/222 plays a crucial role in maintaining the undifferentiated state of mammalian spermatogonia through repression of KIT expression.