Type III CHD chromatin remodelers are only in metazoa and Dictyostelium, required for development, and mutated in several congenital human disorders. Although Type III CHDs mediate nucleosome translocation in vitro, in vivo actions on chromatin are not established. We have analyzed genome-wide chromatin structure and transcription profiles during growth and development of WT Dictyostelium and cells lacking ChdC, a Type III CHD. During WT development, chromatin is reorganized in 2,500 genes (20% of the genome), which are significantly enriched for differential expression. Loss of chdC has a very specific effect on nucleosome organization that is restricted to 15% of the genome. Remarkably, 50% of the genes remodeled during WT development, lose both remodeling and regulated gene expression in chdC-nulls. This demonstrates a requirement for active nucleosomal re-positioning during multicell development, and provides insight for CHD function in nucleosomal organization, chromatin remodeling, and regulated gene expression. TORC1 and TORC2 function antagonistically in Dictyostelium to mediate, respectively, growth or development. To investigate this, we determined media requirements that differentially activate or inhibit TORC1, TORC2, and the low-energy sensor AMP kinase (AMPK), and evaluated genetic pathway enhancers and suppressors. We developed cell culture conditions optimized for growth, but highly sensitive to the TORC1 inhibitor rapamycin. Rapamycin arrests growth and induces early developmental markers, chemotaxis, and aggregation in growth media; untreated cells remained unicellular and undifferentiated. The experimental conditions will allow us to define essential TORC1 substrates that inhibit GDT, and early gene targets that may be induced by TORC1 inhibition, independently of nutrient depletion. Chemotaxis directs embryogenesis, immunity, cell renewal, wound healing, and pathogenesis of cancer metastasis and chronic inflammation. Dictyostelium chemotaxis is most often studied in developed cells, but it can be difficult to discriminate chemotaxis-specific defects from developmental deficiencies. We have begun focus on folate response in growing cells. Growing Dictyostelium have complex mechanisms to detect folate gradients and ensure positive movement during chemotaxis. But, folate receptors (FolRs) have not been identified and folate is rapidly de-activated, making it difficult to examine responses to non-varying concentrations. We have developed new assays for folate and shown conversion of active folate to an inactive 2-deaminated (deA-F) form. By precisely modulating folate, we re-examined adaptive and non-adaptive pathways and G protein dependence and independence, and compared them to cAMP responses. Importantly, preparative amounts of deA-F improve folate specific binding assays for identifying FolRs. Dictyostelium has highly efficient homologous recombination for gene disruption and knock-in targeting, but site-specific mutagenesis is difficult. We are adapting CRISPR26 for Dictyostelium. We have designed Dicty-specific CRISPR vectors for universal gene-targeted mutagenesis. Efficiency of CRISPR site mutagensis is 1-3%, but we are modifying parameters for improvement. Excessive cellular triacylglyceride (TAG) storage within intracellular neutral lipid droplets is a well-known risk factor for many metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets (LDs) are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. LD surfaces are targeted by Perilipin (Plin) family proteins with specificity to different cells. Plin2 is the most highly expressed LSD protein in liver. Livers of fasted or high-fat fed WT mice have increased triacylglycerol (TAG) and Plin2-LSDs; plin2-/- mice are protected from hepatic steatosis during fasting or when fed high-fat diets, but mechanistic processes had not been determined. We have shown that LSDs of plin2-/- liver cells are coated with Plin3 and Plin5 and have elevated levels of associated lipolytic enzymes and regulatory proteins. Accordingly, rates of lipolysis and FA oxidation are increased, thus, reducing lipid stores in liver. RNA-seq and LSD proteomic profiles of livers of WT and plin2-/- mice under differing conditions are being analyzed. Plin2 is also an abundant LD protein in skeletal muscle, but its function is unclear. We show that myotubes established from Plin2-/- mice contain reduced content of LDs and accumulate less oleic acid (OA) in triacylglycerol (TAG) and diacylglycerol, due to a constantly elevated LD hydrolysis compared to corresponding Plin2+/+ myotubes. The reduced ability to store TAG in LDs in Plin2-/- myotubes alters energy metabolism from utilization of glucose towards fatty acids (FAs). Plin2-/- myotubes are characterized by higher oxidation of OA, lower glycogen synthesis, and reduced glucose oxidation compared to Plin2+/+ myotubes. In accord with these metabolic changes, ablation of Plin2 resulted in higher expression of transcription factors that stimulate expression of genes important for FA oxidation, and of pyruvate dehydrogenase kinase isozyme 4, a key enzyme switching fuel source from glucose towards FA. Ablation of Plin2 also resulted in higher expression of oxidative fiber markers but lower expression of glycolytic fiber markers. Our results suggest that Plin2 is essential for balancing the pool of skeletal muscle LDs to avoid an uncontrolled hydrolysis of the intracellular TAG pool. The consequences of an increased release of FAs due to lack of Plin2 have a major impact on skeletal muscle energy metabolism. Myocardial ischemia is associated with alterations in cardiac metabolism,resulting in decreased fatty acid oxidation and increased lipid accumulation. However, little is known about how myocardial lipid content and dynamics affect the function of the ischemic heart. In this study, we investigated the role of the lipid droplet protein perilipin 5 (Plin5) in the pathophysiology of myocardial ischemia. In a human cohort, we found that a common noncoding polymorphism, rs884164, decreases the cardiac expression of PLIN5 and is associated with reduced heart function following myocardial ischemia. In mice, Plin5 deficiency dramatically reduced the triglyceride content in the heart. Under normal conditions, Plin5-/- mice maintained a close to normal heart function by decreasing fatty acid uptake and increasing substrate utilization from glucose, thus preserving the energy balance. However, during stress or myocardial ischemia, Plin5 deficiency resulted in myocardial reduced substrate availability, severely reduced heart function and increased mortality. Our findings indicate that Plin5 function and maintained lipid utilization are protective for cardiac function following ischemia. Adipose mass of plin1-/- mice is <30% of WT, due to elevated basal lipolysis. plin1-/- mice are susceptible to liver steatosis and glucose intolerance, but develop mild insulin resistance. The lack of more severe complications may result from protection to insulin resistance in muscle, and increases in adipose and muscle fatty acid (FA) oxidation and circulating leptin and adiponectin.
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