Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.DOI: http://dx.doi.org/10.7554/eLife.02482.001

Histone H3 of nucleosomes positioned on active genes is trimethylated at Lys36 (H3K36me3) by the SETD2 (also termed KMT3A/SET2 or HYPB) methyltransferase. Previous studies in yeast indicated that H3K36me3 prevents spurious intragenic transcription initiation through recruitment of a histone deacetylase complex, a mechanism that is not conserved in mammals. Here, we report that downregulation of SETD2 in human cells leads to intragenic transcription initiation in at least 11% of active genes. Reduction of SETD2 prevents normal loading of the FACT (FAcilitates Chromatin Transcription) complex subunits SPT16 and SSRP1, and decreases nucleosome occupancy in active genes. Moreover, co-immunoprecipitation experiments suggest that SPT16 is recruited to active chromatin templates, which contain H3K36me3-modified nucleosomes. Our results further show that within minutes after transcriptional activation, there is a SETD2-dependent reduction in gene body occupancy of histone H2B, but not of histone H3, suggesting that SETD2 coordinates FACT-mediated exchange of histone H2B during transcription-coupled nucleosome displacement. After inhibition of transcription, we observe a SETD2-dependent recruitment of FACT and increased histone H2B occupancy. These data suggest that SETD2 activity modulates FACT recruitment and nucleosome dynamics, thereby repressing cryptic transcription initiation.

Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer.DOI: http://dx.doi.org/10.7554/eLife.09214.001

No abstract

Human ATP-binding cassette (ABC) transporters mediate a critical function in the cell, namely the transport of molecules across lipid membranes. Associated to their ubiquitous tissue distribution, they are key players in cellular homeostasis but also potential causative or contributing factors for many pathologies, including Alzheimer's disease (AD). In the central nervous system (CNS), numerous ABC transporters are present throughout the brain parenchyma and especially at the blood-brain barrier (BBB). AD is a neurodegenerative disorder mainly characterized by extracellular deposition of amyloid-β (Aβ) peptides and intracellular accumulation of hyperphosphorylated forms of tau protein. Besides being degraded via proteolytic and phagocytic processes mediated by brain parenchymal cells, a major mechanism for eliminating cerebral Aβ is through its transport across the BBB into the peripheral blood. In fact, many AD cases are associated with impaired Aβ clearance. Consistently, several studies have recently uncovered important roles for ABC transporters in AD pathophysiology. Hence, this review focuses on the relevance of ABC transporters in CNS homeostasis by highlighting AD as a strong example of the deleterious consequences that might result from the former's altered expression and/or activity in the brain. The potentiality of human ABC transporters as novel pharmacological targets for both the diagnosis and therapeutics of AD is emphasized.

Zinc Oxide Nanoparticles (ZnO NPs) are a type of metal oxide nanoparticle with an extensive use in biomedicine. Several studies have focused on the biosafety of ZnO NPs, since their size and surface area favor entrance and accumulation in the body, which can induce toxic effects. In previous studies, ZnO NPs have been identified as a dose- and time-dependent cytotoxic inducer in testis and male germ cells. However, the consequences for the first cell stage of spermatogenesis, spermatogonia, have never been evaluated. Therefore, the aim of the present work is to evaluate in vitro the cytotoxic effects of ZnO NPs in spermatogonia cells, focusing on changes in cytoskeleton and nucleoskeleton. For that purpose, GC-1 cell line derived from mouse testes was selected as a model of spermatogenesis. These cells were treated with different doses of ZnO NPs for 6 h and 12 h. The impact of GC-1 cells exposure to ZnO NPs on cell viability, cell damage, and cytoskeleton and nucleoskeleton dynamics was assessed. Our results clearly indicate that higher concentrations of ZnO NPs have a cytotoxic effect in GC-1 cells, leading to an increase of intracellular Reactive Oxygen Species (ROS) levels, DNA damage, cytoskeleton and nucleoskeleton dynamics alterations, and consequently cell death. In conclusion, it is here reported for the first time that ZnO NPs induce cytotoxic effects, including changes in cytoskeleton and nucleoskeleton in mouse spermatogonia cells, which may compromise the progression of spermatogenesis in a time- and dose-dependent manner.

The nuclear envelope (NE) is the central organizing unit of the eukaryotic cell serving as a genome protective barrier and mechanotransduction interface between the cytoplasm and the nucleus. The NE is mainly composed of a nuclear lamina and a double membrane connected at specific points where the nuclear pore complexes (NPCs) form. Physiological aging might be generically defined as a functional decline across lifespan observed from the cellular to organismal level. Therefore, during aging and premature aging, several cellular alterations occur, including nuclear‐specific changes, particularly, altered nuclear transport, increased genomic instability induced by DNA damage, and telomere attrition. Here, we highlight and discuss proteins associated with nuclear transport dysfunction induced by aging, particularly nucleoporins, nuclear transport factors, and lamins. Moreover, changes in the structure of chromatin and consequent heterochromatin rearrangement upon aging are discussed. These alterations correlate with NE dysfunction, particularly lamins’ alterations. Finally, telomere attrition is addressed and correlated with altered levels of nuclear lamins and nuclear lamina‐associated proteins. Overall, the identification of molecular mechanisms underlying NE dysfunction, including upstream and downstream events, which have yet to be unraveled, will be determinant not only to our understanding of several pathologies, but as here discussed, in the aging process.

The production of highly specialized spermatozoa from undifferentiated spermatogonia is a strictly organized and programmed process requiring extensive restructuring of the entire cell. One of the most remarkable cellular transformations accompanying the various phases of spermatogenesis is the profound remodelling of the nuclear architecture, in which the nuclear envelope (NE) seems to be crucially involved. In recent years, several proteins from the distinct layers forming the NE (i.e. the inner and outer nuclear membranes as well as the nuclear lamina) have been associated with meiosis and/or spermiogenesis in different mammalian species. Among these are A-and B-type lamins, Dpy-19-like protein 2 (DPY19L2), lamin B receptor (LBR), lamina-associated polypeptide 1 (LAP1), LAP2/emerin/MAN1 (LEM) domain-containing proteins, spermatogenesis-associated 46 (SPATA46) and diverse elements of the linker of nucleoskeleton and cytoskeleton (LINC) complex, namely Sad-1/UNC-84 homology (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain-containing proteins. Herein, we summarize the current state of the art on the cellular and subcellular distribution of NE proteins expressed during mammalian spermatogenesis, and discuss the latest research developments regarding their testis-specific functions. This review provides a comprehensive and innovative overview of the NE network as a regulatory platform and as an essential determinant of efficient meiotic chromosome recombination as well as spermiogenesis-associated nuclear remodelling and differentiation in mammalian male germline cells. Thus, this review provides important novel insights on the biological relevance of NE proteins for male fertility.