Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)–dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.

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Murine embryonic stem (ES) cells have unusually long telomeres, much longer than those in embryonic tissues. Here we address whether hyper-long telomeres are a natural property of pluripotent stem cells, such as those present at the blastocyst inner cell mass (ICM), or whether it is a characteristic acquired by the in vitro expansion of ES cells. We find that ICM cells undergo telomere elongation during the in vitro derivation of ES-cell lines. In vivo analysis shows that the hyper-long telomeres of morula-injected ES cells remain hyper-long at the blastocyst stage and longer than telomeres of the blastocyst ICM. Telomere lengthening during derivation of ES-cell lines is concomitant with a decrease in heterochromatic marks at telomeres. We also found increased levels of the telomere repeat binding factor 1 (TRF1) telomere-capping protein in cultured ICM cells before telomere elongation occurs, coinciding with expression of pluripotency markers. These results suggest that high TRF1 levels are present in pluripotent cells, most likely to ensure proficient capping of the newly synthesized telomeres. These results highlight a previously unnoticed difference between ICM cells at the blastocyst and ES cells, and suggest that abnormally long telomeres in ES cells are likely to result from continuous telomere lengthening of proliferating ICM cells locked at an epigenetic state associated to pluripotency.ouse embryonic stem (ES) cells are pluripotent, proliferate indefinitely, and bear very long telomeres (1-3). ES cells emerge from preimplantation blastocyst-stage embryos (4), but how this process takes place is largely unknown. In previous studies, we observed that telomeres of mouse ES cells were much longer than those of mouse embryonic fibroblasts (MEFs) of the same genetic background (5), which are typically obtained at embryonic day 13.5 (E13.5). This observation raised the issue of whether blastocyst inner cell mass (ICM) cells, which are the natural equivalents of ES cells, also have hyper-long telomeres. If this is the case, then telomeres must shorten during fetal development, despite high telomerase activity (6-8). An alternative explanation emerges, however, that hyper-long telomeres in ES cell are aberrant and may result from the in vitro establishment and expansion of ES cells.ES-like pluripotent stem cells can be generated from differentiated cells (i.e., MEFs) by using defined factors, giving rise the so-called induced pluripotent stem (iPS) cells, which are considered functional equivalents of ES cells (9-16). We recently showed that iPS telomeres increase in length during and after nuclear reprogramming until reaching ES cell hyper-long telomeres. This elongation process occurs concomitantly to lower density of trimethylated histones H3K9 and H4K20 at the telomeric chromatin (5). Furthermore, hyper-long telomeres were not observed in iPS cells derived from first-generation telomerasedeficient MEFs, indicating that they do not originate from a selective reprogramming of a subset of parental cells with v...

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe 2؉ and H 2 O 2 in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe 3؉ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. . Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 M), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.A prerequisite to gaining access to the cellulose and hemicellulose components of woody biomass is the circumvention of the lignin barrier. Filamentous fungi, the predominant degraders of wood, have evolved at least two mechanisms to circumvent this barrier. White rot fungi circumvent the lignin barrier by degrading it with extracellular peroxidases (14, 47), with eventual degradation to the level of CO 2 (28). In contrast, brown rot fungi cannot degrade the lignin component to CO 2 . However, these fungi can access the cellulose components with minimal modification of the lignin. These modifications include demethylation of aryl methoxy groups and ring hydroxylation (for a more extensive review, see reference 29).Due to the limited size of the wood pores and the nonspecific nature of wood degradation, Cowling and Brown (12) suggested that low-molecular-weight oxidants are the initial agents in wood decay. Koenigs (33) showed that a number of wood-decomposing fungi produce H 2 O 2 and noted the similarities between wood treated with the hydroxyl radical and with brown rot fungi (34). Illman et al. (23) subsequently detected the hydroxyl radical in incubations with the brown rot fungus Poria placenta by use of electron spin resonance and spin trapping agents. Further supporting the involvement of the hydroxyl radical is the formation of 3-hydroxy derivatives (the expected products from a hydroxyl radical attack) of phthalic hydrazide in incubations with brown rot fungi (5).The most likely nonphotochemical source of the hydroxyl radical is Fenton's reagent, defined by the following chemistry: The quinone undergoes cyclic oxidation-reduction reactions, serving as a shuttle for electrons from intracellular donors to extracellular acceptors. Although a similar mechanism has been proposed for white rot fungi (4, 17, 18) for h...

Although telomere length is genetically determined, mouse embryonic stem (ES) cells with telomeres of twice the normal size have been generated. Here, we use such ES cells with ‘hyper-long' telomeres, which also express green fluorescent protein (GFP), to generate chimaeric mice containing cells with both hyper-long and normal telomeres. We show that chimaeric mice contain GFP-positive cells in all mouse tissues, display normal tissue histology and normal survival. Both hyper-long and normal telomeres shorten with age, but GFP-positive cells retain longer telomeres as mice age. Chimaeric mice with hyper-long telomeres also accumulate fewer cells with short telomeres and less DNA damage with age, and express lower levels of p53. In highly renewing compartments, such as the blood, cells with hyper-long telomeres are longitudinally maintained or enriched with age. We further show that wound-healing rates in the skin are increased in chimaeric mice. Our work demonstrates that mice with functional, longer and better preserved telomeres can be generated without the need for genetic manipulations, such as TERT overexpression.

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KLF6 (Krüppel-like factor 6) is a transcription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-β1 (transforming growth factor-β1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-β auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-β on KLF6 expression and splicing, and to define the contribution of TGF-β on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-β1 through at least two mechanisms. First, in specific cell types, TGF-β1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6–Sp1 co-operation is further enhanced by the TGF-β–Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6–Sp1–Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-β to regulate target genes.