Apoptosis is mediated by the caspase family of proteases that act as effectors of cell death by cleaving many cellular substrates. Caspase-2 is one of the most evolutionarily conserved caspases, yet its physiological function has remained enigmatic because caspase-2-deficient mice develop normally and are viable. We report here that the caspase-2 ؊/؊ mouse embryonic fibroblasts (MEFs) show increased proliferation. When transformed with E1A and Ras oncogenes, caspase-2 ؊/؊ MEFs grew significantly faster than caspase-2 ؉/؉ MEFs and formed more aggressive and accelerated tumors in nude mice. To assess whether the loss of caspase-2 predisposes animals to tumor development, we used the mouse E-Myc lymphoma model. Our findings suggest that loss of even a single allele of caspase-2 resulted in accelerated tumorigenesis, and this was further enhanced in caspase-2 ؊/؊ mice. The caspase-2 ؊/؊ cells showed resistance to apoptosis induced by chemotherapeutic drugs and DNA damage. Furthermore, caspase-2 ؊/؊ MEFs had a defective apoptotic response to cell-cycle checkpoint regulation and showed abnormal cycling following ␥-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumor suppressor protein.cell survival ͉ tumorigenesis ͉ cell cycle ͉ proliferation ͉ DNA damage

The methyltransferase, Enhancer of Zeste homology 2 (EZH2), trimethylates histone 3 lysine 27 (H3K27me3) on chromatin and this repressive mark is removed by lysine demethylase 6A (KDM6A). Loss of these epigenetic modifiers results in developmental defects. We demonstrate that Ezh2 and Kdm6a transcript levels change during differentiation of multipotential human bone marrow-derived mesenchymal stem cells (MSC). Enforced expression of Ezh2 in MSC promoted adipogenic in vitro and inhibited osteogenic differentiation potential in vitro and in vivo, whereas Kdm6a inhibited adipogenesis in vitro and promoted osteogenic differentiation in vitro and in vivo. Inhibition of EZH2 activity and knockdown of Ezh2 gene expression in human MSC resulted in decreased adipogenesis and increased osteogenesis. Conversely, knockdown of Kdm6a gene expression in MSC leads to increased adipogenesis and decreased osteogenesis. Both Ezh2 and Kdm6a were shown to affect expression of master regulatory genes involved in adipogenesis and osteogenesis and H3K27me3 on the promoters of master regulatory genes. These findings demonstrate an important epigenetic switch centered on H3K27me3 which dictates MSC lineage determination. STEM CELLS 2014;32:802-815

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.

he steroid hormone ecdysone regulates both cell differentiation and cell death during insect metamorphosis, by hierarchical transcriptional regulation of a number of genes, including the Broad-Complex ( BR-C ), the zinc finger family of transcription factors. These genes in turn regulate the transcription of a number of downstream genes. DRONC, a key apical caspase in Drosophila , is the only known caspase that is transcriptionally regulated by ecdysone during development. We demonstrate that dronc T gene expression is ablated or reduced in BR-C mutant flies. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that DRONC is essential for ecdysone-mediated cell death, and that dronc upregulation in these cells is controlled by BR-C. Finally, we show that the dronc promoter has BR-C interaction sites, and that it can be transactivated by a specific isoform of BR-C. These results indicate that BR-C plays a key role in ecdysonemediated caspase regulation.

eThe main impairment to tissue maintenance during aging is the reduced capacity for stem cell self-renewal over time due to senescence, the irreversible block in proliferation. We have previously described that the basic helix-loop-helix (bHLH) transcription factor Twist-1 can greatly enhance the life span of bone marrow-derived mesenchymal stem/stromal cells (MSCs). In the present study, we show that Twist-1 potently suppresses senescence and the Ink4A/Arf locus with a dramatic decrease in the expression of p16 and to some extent a decrease in p14. Furthermore, the polycomb group protein and histone methyltransferase Ezh2, which suppresses the Ink4A/Arf locus, was found to be induced by Twist-1, resulting in an increase in H3K27me3 along the Ink4A/Arf locus, repressing transcription of both p16/p14 and senescence of human MSCs. Furthermore, Twist-1 inhibits the expression of the bHLH transcription factor E47, which is normally expressed in senescent MSCs and induces transcription of the p16 promoter. Reduced Twist-1 wild-type expression and function in bone cells derived from SaethreChotzen patients also revealed an increase in senescence. These studies for the first time link Twist-1 to histone methylation of the Ink4A/Arf locus by controlling the expression of histone methyltransferases as well as the expression of other bHLH factors. C lonogenic bone marrow (BM)-derived mesenchymal stem/ stromal cells (BMSCs) are a heterogeneous mix of stem cells and committed progenitors that vary in their morphology, proliferation, and differentiation potential (9-12, 18, 23). This is attributed to the existence of a developmental hierarchy of stromal cellular differentiation, comprised largely of committed progenitor cells and a minor population of self-renewing multipotent stem cells capable of differentiating into adipocytes, osteoblasts (OB), chondrocytes, and myocytes (6, 21).Adult somatic stem cells including BMSCs exhibit an increased propensity for cellular senescence during ex vivo expansion, which is accompanied by a reduction in self-renewal and multidifferentiation potential. Senescence is a "fail-safe" mechanism which is activated in response to various stresses such as DNA damage, oxidative damage, and oncogene activation (13, 24). During senescence, cells fail to respond to mitogenic stimuli, undergo dramatic changes in chromatin structure and gene expression, become enlarged and flattened, and remain viable yet nondividing. The Ink4A/Arf locus (the locus of p14Arf and p16) is central to cellular senescence as it signals via p53 and retinoblastoma (Rb) to inhibit proliferation. p14ARF activates p53 by sequestering Mdm2, an E3 ubiquitin ligase, to the nucleolus, thereby preventing the Mdm2-mediated targeting of p53 to proteolytic degradation (16). p53 subsequently activates p21 CIP1/WAF1, which inhibits the cell cycle. Moreover, when activated, p16 inhibits the cyclin-dependent kinases CDK4/6 and leads to the hypophosphorylation of Rb, which in turn represses E2F-mediated cell cycle progression (2,22).Transcr...

The steroid hormone ecdysone regulates moulting, cell death, and differentiation during insect development. Ecdysone mediates its biological effects by either direct activation of gene transcription after binding to its receptor EcR–Usp or via hierarchical transcriptional regulation of several primary transcription factors. In turn, these transcription factors regulate the expression of several downstream genes responsible for specific biological outcomes. DRONC, the Drosophila initiator caspase, is transcriptionally regulated by ecdysone during development. We demonstrate here that the dronc promoter directly binds EcR–Usp. We further show that mutation of the EcR–Usp binding element (EcRBE) reduces transcription of a reporter and abolishes transactivation by an EcR isoform. We demonstrate that EcRBE is required for temporal regulation of dronc expression in response to ecdysone in specific tissues. We also uncover the participation of a putative repressor whose function appears to be coupled with EcR–Usp. These results indicate that direct binding of EcR–Usp is crucial for controlling the timing of dronc expression in specific tissues.

BackgroundThe 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation.ResultsWe used siRNA knockdown and retroviral mediated enforced expression of TET molecules and discovered TET1 to be a repressor of both osteogenesis and adipogenesis. TET1 was found to recruit the co-repressor proteins, SIN3A and the histone lysine methyltransferase, EZH2 to osteogenic genes. Conversely, TET2 was found to be a promoter of both osteogenesis and adipogenesis. The data showed that TET2 was directly responsible for 5hmC levels on osteogenic and adipogenic lineage-associated genes, whereas TET1 also played a role in this process. Interestingly, TET3 showed no functional effect in BMSC osteo-/adipogenic differentiation. Finally, in a mouse model of ovariectomy-induced osteoporosis, the numbers of clonogenic BMSC were dramatically diminished corresponding to lower trabecular bone volume and reduced levels of TET1, TET2 and 5hmC.ConclusionThe present study has discovered an epigenetic mechanism mediated through changes in DNA hydroxymethylation status regulating the activation of key genes involved in the lineage determination of skeletal stem cells, which may have implications in BMSC function during normal bone regulation. Targeting TET molecules or their downstream targets may offer new therapeutic strategies to help prevent bone loss and repair following trauma or disease.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0247-4) contains supplementary material, which is available to authorized users.

No abstract