Nucleosome placement can direct transcription, but precise interactions are complex and largely unresolved at the whole genome level. Type III CHD proteins, a subfamily of SWI2 chromatin remodelers, are only present in metazoa and Dictyostelium, required for multicellular development, and mutated in several complex congenital human disorders (eg CHARGE syndrome). Type III CHDs mediate nucleosome translocation in vitro, but in vivo actions on chromatin organization and developmental regulation are not established. We investigated nucleosome re-positioning and gene expression in vivo, using deep sequence analyses of genome-wide chromatin structure and transcription profiles during growth and development of wild-type (WT) Dictyostelium and cells lacking ChdC, a Type III CHD. We demonstrate major WT chromatin reorganization of a gene subset enriched for differential expression during development. Loss of chdC has a very specific effect on chromatin structure relative to WT. Although altered nucleosomal organization in chdC-nulls is restricted to only 15% of the genome, it occurs in 50% of genes that are remodeled in WT. Association of altered chromatin remodeling and mis-regulated gene expression in chdC-nulls demonstrates the requirement for active nucleosomal re-positioning during multicellular development. Our data provide new mechanistic insight for related human diseases, showing in vivo function of Type III CHD chromatin remodeling proteins, for developmentally regulated nucleosome re-positioning, and gene expression. Chemotaxis directs embryogenesis, immunity, cell renewal, wound healing, and pathogenesis of cancer metastasis and chronic inflammation. Identification of novel small molecule probes of chemotaxis is important to dissect mechanisms and develop therapeutics. Current screens that identified compounds that inhibit chemotaxis have had poor activity, with few advanced to clinical trials. To identify novel chemotactic inhibitors, we developed a unique, unbiased assay for high-throughput screening using rapid, laser-scanning cytometry and Dictyostelium, a system with similar chemotactic mechanisms as mammalian migratory cells. We quantify chemotaxis in a miniaturized system using a GFP-marker only expressed in chemotaxing cells;a viability counter-screen is incorporated to eliminate toxic compounds. We screened 1,280 small molecule compounds in 1536-well formats and identified two that specifically inhibited GFP expression and chemotaxis. Both were confirmed to inhibit chemotaxis of Dictyostelium and human neutrophils in EZ-TAXIscan assays. This screening approach can rapidly identify potential new lead compounds for drug development and research tools universal to humans and other systems. Mutations in two presenilin genes in humans cause familial Alzheimers disease. Presenilins (PS) have both proteolytic-dependent and -independent. Analyzing the cellular role of presenilins in mammals has been complicated by embryonic lethality following deletion of both genes. We previously showed that PS has a role in the development of Dictyostelium. Here we show that development of PS-nulls can be rescued by expression of human PS1. We also show that aberrant development can be improved with PS proteins mutated at sites essential for proteolytic activity. Proteolytic- independent signaling by PS during Dictyostelium development is confirmed, as assayed by loss of substrate cleavage. Our data suggest an ancient non-proteolytic role for PS proteins in regulating intracellular signaling and development, and provide a novel model system for the analysis of human PS function. 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;Plin1 is the major adipocyte form, while Plin2 is the predominant form in liver. We studied Plin2 function in lipid storage in mice during induced hepatic steatosis. WT and plin2-/- mice were placed on chow or high fat diets or fasted overnight for each diet. WT mice that had been fasted overnight or placed on a high-fat diet accumulate abnormal levels of liver LDs that are primarily coated by Plin2. plin2-/- mice showed very significantly reduced hepatic lipid levels compared to WT under all conditions. LDs were isolated from livers of WT and plin2-/- mice and Plin protein compositions analyzed;the absence of Plin2 was compensated by the presence of Plin3 and Plin5 in the normal fed state, and by more dramatic increases in Plin5 during the fasted states or the high fat diet. Hepatic LDs of plin2-/- mice showed significantly increased recruitment of ATGL (lipase) and the ATGL regulatory protein CGI58 during conditions of induced hepatic steatosis as compared to WT. To better understand the protection of plin2-/- hepatocytes to steatosis, we studied primary aspects of TAG storage and hydrolyses in isolated primary hepatocytes from plin2-/- and WT mice. Lipolytic rates are significantly enhanced in plin2-/- primary hepatocytes, with significantly elevated levels of non-mitochondrial fatty acid oxidation and upregulated effects on hepatic gene expressions involved in fatty acid oxidation compared to WT. The increased levels of Plin3 and Plin5 in hepatocytes appear more permissive for access of LDs to lipolytic enzymes than are hepatic LDs primarily coated with Plin2. Lipid droplet proteins are also implicated in regulation of energy dynamics in the cell. Cells from oxidative tissue have high and fluctuating energy demands that require efficient coupling between energy storage in lipid droplets and utilization in mitochondria. Plin5 is highly expressed in heart and other oxidative tissues, and can stabilize lipid droplets and facilitate association between lipid droplets and mitochondria in cell culture. As myocardial lipids are stored in droplets that are closely associated with mitochondria, there is recent interest in Plin5 and its role for cardiac function. Indeed, increased expression of Plin5 in mouse leads to myocardial lipid accumulation. Studies in humans showed that a polymorphism in a noncoding region of PLIN5 is associated with decreased heart function after myocardial ischemia. Here, we investigated Plin5 and its role for cardiac dysfunction and outcome after myocardial ischemia. We show that deficiency in Plin5 in mice results in diminished metabolic flexibility of the heart and reduced heart function and outcome after myocardial ischemia, suggesting that Plin5 plays a role for cardioprotection during ischemia in the heart, and providing functional evidence that Plin5 is essential for cardiac physiology.
Showing the most recent 10 out of 28 publications
