Background- m6A methylation is the most prevalent internal post-transcriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known. Methods- To determine the role of m6A methylation in the heart we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase METTL3 both in culture and in vivo. We generated cardiac-restricted gain and loss of function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function. Results- We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, while increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for maintenance of cardiac homeostasis. Conclusions- Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Background-MicroRNAs (miRs) participate in many cardiac pathophysiological processes, including ischemia/reperfusion (I/R)-induced cardiac injury. Recently, we and others observed that miR-494 was downregulated in murine I/R-injured and human infarcted hearts. However, the functional consequence of miR-494 in response to I/R remains unknown. Methods and Results-We generated a mouse model with cardiac-specific overexpression of miR-494. Transgenic hearts and wild-type hearts from multiple lines were subjected to global no-flow I/R with the Langendorff system. Transgenic hearts exhibited improved recovery of contractile performance over the reperfusion period. This improvement was accompanied by remarkable decreases in both lactate dehydrogenase release and the extent of apoptosis in transgenic hearts compared with wild-type hearts. In addition, myocardial infarction size was significantly reduced in transgenic hearts on I/R in vivo compared with wild-type hearts. Similarly, short-term overexpression of miR-494 in cultured adult cardiomyocytes demonstrated an inhibition of caspase-3 activity and reduced cell death on simulated I/R. In vivo treatment with antisense oligonucleotide miR-494 increased I/R-triggered cardiac injury relative to the administration of mutant antisense oligonucleotide miR-494 and saline controls. We further identified that 3 proapoptotic proteins (PTEN, ROCK1, and CaMKII␦) and 2 antiapoptotic proteins (FGFR2 and LIF) were authentic targets for miR-494. Importantly, the Akt-mitochondrial signaling pathway was activated in miR-494 -overexpressing myocytes. Conclusions-Our findings suggest that although miR-494 targets both proapoptotic and antiapoptotic proteins, the ultimate consequence is activation of the Akt pathway, leading to cardioprotective effects against I/R-induced injury. Thus, miR-494 may constitute a new therapeutic agent for the treatment of ischemic heart disease. (Circulation. 2010; 122:1308-1318.)Key Words: myocardial infarction Ⅲ apoptosis Ⅲ cardiomyocyte Ⅲ microRNA Ⅲ reperfusion injury I schemic heart disease, a leading cause of death worldwide, is the most common consequence of coronary artery disease. 1,2 Although reperfusion of an occluded human coronary is effective for reducing overall mortality, it is now recognized that restoration of the blood flow through the previously ischemic myocardium can yield additional reperfusion injury, including cardiomyocyte dysfunction and cell death. 3 The cellular mechanisms underlying ischemia/reperfusion (I/R)-induced injury are complex and involve a multitude of signaling pathways and molecular players. 4 Therefore, it would be rational to develop an effective pharmacological or genetic agent aimed at multiple molecular targets. MicroRNAs (miRs), a new class of Ϸ22-nt non-protein-coding single-strand RNAs, have emerged as regulators that control the expression of hundreds of proteins. 5 As a consequence, they may widely influence the signaling networks leading to pathological/physiological responses such as myocardial I/R inju...

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

medvedm@email.uc.edu

We present a logistic regression-based method (LRpath) for identifying predefined sets of biologically related genes enriched with (or depleted of) differentially expressed transcripts in microarray experiments. We functionally relate the odds of gene set membership with the significance of differential expression, and calculate adjusted P-values as a measure of statistical significance. The new approach is compared with Fisher's exact test and other relevant methods in a simulation study and in the analysis of two breast cancer datasets. Overall results were concordant between the simulation study and the experimental data analysis, and provide useful information to investigators seeking to choose the appropriate method. LRpath displayed robust behavior and improved statistical power compared with tested alternatives. It is applicable in experiments involving two or more sample types, and accepts significance statistics of the investigator's choice as input.

SUMMARY Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.

The MS Windows based program named Gaussian Infinite Mixture Modeling (GIMM) implementing the Gibbs sampler and corresponding C++ code are available at http://homepages.uc.edu/~medvedm/GIMM.htm SUPPLEMENTAL INFORMATION: http://expression.microslu.washington.edu/expression/kayee/medvedovic2003/medvedovic_bioinf2003.html