The ESCRT-III complex is implicated in the reformation of the nuclear envelope; the CHMP2A component of ESCRT-III is directed to the forming nuclear envelope through classical ESCRT-assembly mechanisms, with the help of the p97 complex component UFD1, and provides an activity essential for nuclear envelope reformation. At an early stage of cell division in animal cells the nuclear envelope breaks down, and once cell division draws to a close at anaphase, the envelope must reform around the nuclei of the two daughter cells. It is known that the nuclear envelope is derived from the membranes of another organelle, the endoplasmic reticulum, and that it is sealed through a membrane fusion step, but the mechanism of fusion remains partially understood. Two groups report this week [in this issue of Nature] that this process is regulated by ESCRT-III, a protein complex best known for membrane constriction and sealing during various cellular processes. Harald Stenmark and colleagues show that ESCRT-III and two other proteins, VPS4 and spastin, cooperatively coordinate envelope fusion and spindle disassembly in a process mechanistically similar to the cytokinesis that ensures physical separation of the two daughter cells. Jeremy Carlton and colleagues find that the CHMP2A component of ESCRT-III is directed to the forming nuclear envelope where it localizes to sites of fusion through binding to CHMP4B, and with the help of UFD1 — a component of the p97 complex previously implicated in nuclear envelope fusion. During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents1,2,3,4. NE reformation requires the coating of chromatin by membrane derived from the endoplasmic reticulum, and a subsequent annular fusion step to ensure that the formed envelope is sealed1,2,4,5. How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis1,6. Here we show that the endosomal sorting complex required for transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the forming NE in human cells, and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged multivesicular body protein 2A (CHMP2A) is directed to the forming NE through binding to CHMP4B, and provides an activity essential for NE reformation. Localization also requires the p97 complex member ubiquitin fusion and degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodelling events.

SirT1 is a class III histone deacetylase that has been implicated in metabolic and reactive oxygen species control. In the vasculature it has been shown to decrease endothelial superoxide production, prevent endothelial dysfunction and atherosclerosis. However, the mechanisms that mediate SirT1 antioxidant functions remain to be characterized. The transcription factor FoxO3a and the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) have been shown to induce the expression of antioxidant genes and to be deacetylated by SirT1. Aims: Here we investigated SirT1 regulation of antioxidant genes and the roles played by FoxO3a and PGC-1α in this regulation. Results: We found that SirT1 regulates the expression of several antioxidant genes in bovine aortic endothelial cells, including Mn superoxide dismutase (MnSOD), catalase, peroxiredoxins 3 and 5 (Prx3, Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TR2), and uncoupling protein 2 (UCP-2) and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1α prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1α complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H₂O₂-dependent FoxO3a and PGC-1α acetylation. Data showing that FoxO3a knockdown increases PGC-1α acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1α may be dependent of the formation of a FoxO3a/PGC-1α complex. Innovation: A unifying mechanism for SirT1 activities is suggested. Conclusion: We show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends on the formation of a FoxO3a/PGC-1α complex. Antioxid. Redox Signal. 19, 1507–1521.

Peroxisome proliferator activated receptor α (PPARα) is a distinctive marker of the brown fat phenotype that has been proposed to coordinate the transcriptional activation of genes for lipid oxidation and for thermogenic uncoupling protein 1 in brown adipose tissue. Here, we investigated the involvement of PPARα in the transcriptional control of the PPARγ coactivator (PGC)-1α gene. Treatment with PPARα agonists induced PGC-1α mRNA expression in brown fat in vivo and in primary brown adipocytes. This enhancement of PGC-1α transcription was mediated by PPARα binding to a PPAR-responsive element in the distal PGC-1α gene promoter. PGC-1α gene expression was decreased in PPARα-null brown fat, both under basal conditions and in response to thermogenic activation. Moreover, PPARα- and cAMP-mediated pathways interacted to control PGC-1α transcription. PRDM16 (PRD1-BF1-RIZ1 homologous domain-containing 16) promoted PPARα induction of PGC-1α gene transcription, especially under conditions in which protein kinase A pathways were activated. This enhancement was associated with the interaction of PRDM16 with the PGC-1α promoter at the PPARα-binding site. In addition, PPARα promoted the expression of the PRDM16 gene in brown adipocytes, and activation of PPARα in human white adipocytes led to the appearance of a brown adipocyte pattern of gene expression, including induction of PGC-1α and PRDM16. Collectively, these results suggest that PPARα acts as a key component of brown fat thermogenesis by coordinately regulating lipid catabolism and thermogenic gene expression via induction of PGC-1α and PRDM16.

Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here we show that Foxo3a is a direct transcriptional regulator of a group of oxidative stress protection genes in vascular endothelial cells. Importantly, Foxo3a activity requires the transcriptional co-activator PGC-1α, because it is severely curtailed in PGC-1α-deficient endothelial cells. Foxo3a and PGC-1α appear to interact directly, as shown by co-immunoprecipitation and in vitro interaction assays, and are recruited to the same promoter regions. The notion that Foxo3a and PGC-1α interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1α transcriptional activity at the sod2 (manganese superoxide dismutase) promoter requires a functional FoxO site. We also demonstrate that Foxo3a is a direct transcriptional regulator of PGC-1α, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1α control of the oxidative stress response.

In this study, we report the identification of a novel role of SIRT6 in both epirubicin and paclitaxel resistance in breast cancer. We found that SIRT6 protein levels are elevated in paclitaxel- and epirubicin-resistant MCF-7 cells compared with the parental sensitive cells. SIRT6 knockout and depletion sensitized cells to both paclitaxel and epirubicin treatment, whereas SIRT6 ectopic overexpression led to increased resistance to paclitaxel and epirubicin. Moreover, our data suggest that SIRT6 could be mediating epirubicin resistance through enhancing the DNA repair response to epirubicin-induced DNA damage. Clonogenic assays also revealed that mouse embryonic fibroblasts (MEFs) lacking SIRT6 have decreased long-term viability in response to epirubicin. The tumour suppressor FOXO3a increases its levels of acetylation in MEFs depleted of SIRT6, whereas its induction by epirubicin is attenuated in breast cancer cells overexpressing SIRT6. Further cell viability studies demonstrate that deletion of FOXO1/3/4 in MEFs can confer sensitivity to both paclitaxel and epirubicin, suggesting that SIRT6 reduces paclitaxel and epirubicin sensitivity, at least in part, through modulating FOXO acetylation and expression. Consistently, immunohistochemical analysis of 118 breast cancer patient samples revealed that high SIRT6 nuclear staining is significantly associated with poorer overall survival (P = 0.018; Kaplan–Meier analysis). Multivariate Cox analysis demonstrated that nuclear SIRT6 staining remained associated with death after correcting for tumour stage and lymph-node involvement (P = 0.033). Collectively, our data suggest that SIRT6 has a role in paclitaxel and epirubicin sensitivity via targeting FOXO proteins and that SIRT6 could be a useful biomarker and therapeutic target for paclitaxel- and epirubicin-resistant cancer.

In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2, 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4, 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7's N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.