To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG). When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG). Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.

In both animal and yeast cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate polarized organization of the actin cytoskeleton. In the budding yeast Saccharomyces cerevisiae, the Ras-like GTPase Bud1/Rsr1 and its guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor Bud5 are involved in the selection of a specific site for growth, thus determining cell polarity. We found that Bud5 is localized at the cell division site and the presumptive bud site.

bMicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1␣ (HIF-1␣) protein levels and decreased cell growth during hypoxia. HIF-1␣ protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1␣ protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations-a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.