X inactivation involves the stable silencing of one of the two X chromosomes in XX female mammals. Initiation of this process occurs during early development and involves Xist (X-inactivespecific transcript) RNA coating and the recruitment of Polycomb repressive complex (PRC) 2 and PRC1 proteins. This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group protein BMI1 and the variant histone MACROH2A. We find that in addition to MACROH2A, PRC1 is recruited to the inactivated X chromosome in somatic cells in a highly dynamic, cell cycle-regulated manner. Importantly, RNAimediated knock-down of CULLIN3 or SPOP results in loss of MACROH2A1 from the inactivated X chromosome (Xi), leading to reactivation of the Xi in the presence of inhibitors of DNA methylation and histone deacetylation. Likewise, Xi reactivation is also seen on MacroH2A1 RNAi under these conditions. Hence, we propose that the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3͞ SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing.Bmi1 ͉ SPOP͞CULLIN3 E3 ligase ͉ X inactivation ͉ Polycomb silencing

Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized. Polycomb repressive complex 2 (PRC2) is thought to be involved in the initiation of gene silencing, whereas Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression. Here, we investigate the kinetic properties of the binding of one of the PRC1 core components, BMI1, with PcG bodies. PcG bodies are unique nuclear structures located on regions of pericentric heterochromatin, found to be the site of accumulation of PcG complexes in different cell lines. We report the presence of at least two kinetically different pools of BMI1, a highly dynamic and a less dynamic fraction, which may reflect BMI1 pools with different binding capacities to these stable heterochromatin domains. Interestingly, PRC2 members EED and EZH2 appear to be essential for BMI1 recruitment to the PcG bodies. Furthermore, we demonstrate that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity. Together, these results provide new insights in the mechanism for regulation of chromatin silencing by PcG proteins and suggest a highly regulated recruitment of PRC1 to chromatin.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin.In eukaryotic cells DNA is packaged into chromatin, and this packaging impacts all DNA-templated processes, including transcription. Regulated changes in chromatin structure are crucial to establish and maintain the diverse expression profiles that characterize the hundreds of cell types in multicellular organisms (1). The nucleosome is the structural unit of chromatin and comprises 147 bp of DNA wrapped around a histone octamer, which is composed of two copies each of the four core histones H2A, H2B, H3, and H4.Chromatin structure can be affected through the action of ATP-dependent chromatin remodeling enzymes or by the covalent modification of histone proteins, creating binding sites for additional regulatory proteins (1, 2). In addition, chromatin structure can be modulated through the binding of effector proteins to nucleosomes. Poly(ADP-ribose) polymerase 1 (PARP-1) 5 is an example of a nucleosome binding protein that can affect chromatin structure (3, 4). PARP-1 is the prototypical member of a family of PARP proteins, which catalyze the transfer of ADP-ribose units from donor nicotinamide adenine dinucleotide (NAD ϩ ) molecules to target proteins (5). PARP-1 functions as a structural component of chromatin, modulator of chromatin structure, and a sensor of DNA damage through its intrinsic enzymatic activity (4, 6, 7). In the absence of NAD ϩ , PARP-1 binds to nucleosomes, compacts chromatin, and inhibits transcription in vitro (3). Furthermore, catalytically inactive PARP-1 maintains silencing of heterochromatic retrotransposons in Drosophila (8). However, at physiological concentrations of NAD ϩ , PARP-1 is enzymatically active and does not bind nucleosomes (3). Despite this, PARP-1 binds chromatin in vivo and is implicated in transcriptional silencing, suggesting that modulation of PARP-1 activity in vivo may be a mechanism that is employed to direct changes in chromatin structure.Chromatin structure can also be regulated by rep...

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The mechanism of tumor necrosis factor ␣ (TNF␣)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNF␣ on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNF␣ and cycloheximide (TNF␣/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc 1 and aa 3 . TNF␣/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNF␣/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death. Tumor necrosis factor ␣ (TNF␣)1 is a cytokine that is cytotoxic against certain tumor cells (1), and this effect is enhanced by cycloheximide (CHX) (2). In the presence of CHX not only is less TNF␣ required, but also cell death occurs in a shorter period of time (3). Although these effects have been described extensively, the molecular mechanisms of action are not well understood (4, 5). The reported ability of antioxidants to protect cells against TNF␣-induced cytotoxicity suggests that mitochondrial dysfunction and generation of reactive oxygen species (ROS) in the mitochondria may play an essential role (6 -11). Oxidative stress can result in severe metabolic dysfunction, including the peroxidation of lipid membranes (12), an increase in cytosolic Ca 2ϩ (13), induction of the mitochondrial permeability transition (14), and DNA damage (reviewed in Refs. 15 and 16).Previous investigations have shown contradictory results regarding whether TNF␣ induces cytotoxicity through necrosis or apoptosis (17). Recent results indicate that TNF␣ induces necrosis in L929 cells, although apoptosis-like features have also been observed (18,19). Regardless of the mode of cell death, however, it has been shown that TNF␣-induced cytotoxicity of L929 cells is mediated by mitochondrial formation of ROS (20,21). In intact mitochondria, three components of the respiratory chain have been found to be involved in the generation of ROS (22, 23); one is located in complex I, and the other two are ubisemiquinone (24) and reduced cytochrome b (25, 26), which are both located in complex III. Furthermore, substantial evidence in other sy...

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up-regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K-Ras(G12V) conditional knock-in and caerulein-treated K-Ras(G12V) mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells-but not acinar cells-in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar-ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up-regulated in high-grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development.

BackgroundCutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and frequently progresses from an actinic keratosis (AK), a sun-induced keratinocyte intraepithelial neoplasia (KIN). Epigenetic mechanisms involved in the phenomenon of progression from AK to cSCC remain to be elicited.MethodsExpression of microRNAs in sun-exposed skin, AK and cSCC was analysed by Agilent microarrays. DNA methylation of miR-204 promoter was determined by bisulphite treatment and pyrosequencing. Identification of miR-204 targets and pathways was accomplished in HaCat cells. Immunofluorescence and immunohistochemistry were used to analyze STAT3 activation and PTPN11 expression in human biopsies.ResultscSCCs display a marked downregulation of miR-204 expression when compared to AK. DNA methylation of miR-204 promoter was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC. In HaCaT cells miR-204 inhibits STAT3 and favours the MAPK signaling pathway, likely acting through PTPN11, a nuclear tyrosine phosphatase that is a direct miR-204 target. In non-peritumoral AK lesions, activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartments, whereas AK lesions adjacent to cSCCs display activated STAT3 in the nuclei.ConclusionsOur data suggest that miR-204 may act as a “rheostat” that controls the signalling towards the MAPK pathway or the STAT3 pathway in the progression from AK to cSCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0537-z) contains supplementary material, which is available to authorized users.

Programmed cell death ligand 1 (PD-L1) expression by tumor cells plays an important role in the inhibition of T cell-mediated immune response in cancer. PD-L1 expression by tumor cells has been linked to poor prognosis in a wide variety of cancers. However, PD-L1 expression in cutaneous squamous cell carcinoma (cSCC) has been scarcely studied, and its role as a prognosis biomarker remains controversial. The association of PD-L1 expression and the metastatic risk in a series of cSCC was assessed. PD-L1 and CD8 immunostainings of full excision sections of 99 primary tumors and 24 lymphatic metastases were semiquantitatively evaluated. Primary cSCCs were grouped according to the development of lymphatic metastatic spread [metastasizing squamous cell carcinoma (MSCC)] (n = 48) or the absence of progression [nonmetastasizing squamous cell carcinoma (NMSCC)] (n = 51). PD-L1-positive expression (cut off ≥1%) was found in 26% NMSCCs and in 50% MSCCs (P = 0.02). PD-L1 association with an increased metastatic risk was confirmed in the multivariate analysis (P < 0.05), along with the following features: recurrence, poor differentiation, and perineural invasion. Ninety percent of the metastases of PD-L1-positive tumors were also positive for PD-L1, displaying a trend toward a higher PD-L1 expression when compared with their primary tumors (P = 0.058). No significant differences in the peritumoral inflammatory infiltrate or in the expression of CD8 were found between metastasizing and nonmetastasizing primary tumors. Our results suggest that PD-L1 may play a relevant role in metastatic spread and may be a candidate prognostic biomarker in cSCC.