Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcriptional activities. The SS18 protein functions as a transcriptional coactivator and is associated with the SWI/SNF complex, whereas the SSX proteins function as transcriptional corepressors and are associated with the polycomb complex. The domains involved in these opposite transcriptional activities are retained in the SS18-SSX fusion proteins. Here, we set out to determine the direct transcriptional consequences of conditional SS18-SSX2 fusion protein expression using complementary DNA microarray-based profiling. By doing so, we identified several clusters of SS18-SSX2-responsive genes, including a group of genes involved in cholesterol synthesis, which is a general characteristic of malignancy. In addition, we identified a group of SS18-SSX2-responsive genes known to be specifically deregulated in primary synovial sarcomas, including IGF2 and CD44. Furthermore, we observed an uncoupling of EGR1, JUNB, and WNT signaling in response to SS18-SSX2 expression, suggesting that the SWI/ SNF-associated coactivation functions of the SS18 moiety are impaired. Finally, we found that SS18-SSX2 expression affects histone modifications in the CD44 and IGF2 promoters and DNA methylation levels in the IGF2 imprinting control region. Together, we conclude that the SS18-SSX2 fusion protein may act as a so-called transcriptional ''activator-repressor,'' which induces downstream target gene deregulation through epigenetic mechanisms. Our results may have implications for both the development and clinical management of synovial sarcomas. (Cancer Res 2006; 66(19): 9474-82)

Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu for signs of microbial infection or damage, or after activation when they are phagocytosing cellular debris or foreign material. These morphofunctional alterations require active actin cytoskeleton remodeling and metabolic adaptation. Here we analyzed RAW 264.7 and Maf-DKO macrophages as models to study whether there is a specific association between aspects of carbohydrate metabolism and actin-based processes in LPS-stimulated macrophages. We demonstrate that the capacity to undergo LPS-induced cell shape changes and to phagocytose complement-opsonized zymosan (COZ) particles does not depend on oxidative phosphorylation activity but is fueled by glycolysis. Different macrophage activities like spreading, formation of cell protrusions, as well as phagocytosis of COZ, were thereby strongly reliant on the presence of low levels of extracellular glucose. Since global ATP production was not affected by rewiring of glucose catabolism and inhibition of glycolysis by 2-deoxy-D-glucose and glucose deprivation had differential effects, our observations suggest a non-metabolic role for glucose in actin cytoskeletal remodeling in macrophages, e.g. via posttranslational modification of receptors or signaling molecules, or other effects on the machinery that drives actin cytoskeletal changes. Our findings impute a decisive role for the nutrient state of the tissue microenvironment in macrophage morphodynamics.

Background: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression.