Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca 2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca 2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1-or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.A rticular cartilage is a hydrated connective tissue that supports loads and minimizes friction in the diarthrodial joints. It has a highly differentiated extracellular matrix (ECM) composed primarily of type II collagen, the large aggregating proteoglycan, aggrecan, and water. Chondrocytes are the only cells in cartilage and are responsible for maintaining and remodeling cartilage through a homeostatic balance of anabolic and catabolic activities. Under normal physiologic conditions, chondrocytes are exposed to millions of cycles of mechanical loading per year (1). These mechanical signals play an important role in regulating chondrocyte anabolic and biosynthetic activity, as evidenced by cartilage atrophy following periods of disuse or immobilization (2-7). However, under abnormal loading conditions (e.g., due to obesity, trauma, or joint instability), mechanical factors play a critical role in the onset and progression of osteoarthritis (1). Such "injurious" loading has been modeled in vitro using explant culture systems that replicate many of the early cellular and molecular events characteristic of osteoarthritis (8). Osteoarthritis is a painful and debilitating disease of weight-bearing joints that affects over 26 million people in the United States (9) with posttraumatic arthritis being responsible for ∼12% of the incidence of osteoarthritis (10).Despite the critical importance of mechanical loading in health and disease of synovial joints, the mechanisms of mechanotransduction of chondrocytes are not fully understood and are likely to differ under physiologic and pathologic conditions (11)(12)(13)(14). Although many different mechanisms have been shown to be involved in chondrocyte mechanotransduction (13,(15)(16)(17), recent st...

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase͞nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.DNA repair ͉ histone methylation

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event.Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.double-strand break | I-Sce-I | chromatin immunoprecipitation | MRN complex | mathematical modeling H istone methylation is highly regulated by a family of proteins termed histone methylases, which usually share a SET domain (1-3). Histone methylation plays a key role in chromatin remodeling and as such regulates transcription, replication, cell differentiation, genome stability, and apoptosis (1-3). Because of its role in replication and genome stability, histone methylation has been hypothesized to play an important role in DNA repair. DNA double-strand breaks (DSBs) are a cytotoxic form of DNA damage that disrupts many of the cellular functions regulated by histone methylation described above (4-6). Previous reports indicate that histone methylation may be important in DNA DSB repair by homologous recombination: The DSB repair component 53BP1, which is required for proper homologous recombination, is recruited to sites of damage by methylated histone H3 lysine 79 (H3K79) and histone H4 lysine 20 (H4K20) (7-9). However, neither H3K79 nor H4K20 methylation is induced by DNA damage (9), so other histone methylation events at sites of DNA damage have been sought. In addition, a mechanism by which histone methylation might regulate NHEJ DSB repair has yet to be defined. In this study, a survey of histone methylation events after DSB induction revealed that the major immediate H3 methylation event is H3K36me2.Metnase is a DNA DSB repair component that is a fusion of a SET histone methylase domain with a nuclease domain and a domain from a member of the transposase/integrase family (10-14). We showed previously that Metnase enhances nonhomologous end-joining (NHEJ) repair of, and survival after, DNA DSBs, and that its SET dom...