A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics.

Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

Comparison of normalization and differential expression analyses using RNA-Seq data from 726 individual Drosophila melanogaster.

BACKGROUND: A generally accepted approach to the analysis of RNA-Seq read count data does not yet exist. We sequenced the mRNA of 726 individuals from the Drosophila Genetic Reference Panel in order to quantify differences in gene expression among single flies. One of our experimental goals was to identify the optimal analysis approach for the detection of differential gene expression among the factors we varied in the experiment: genotype, environment, sex, and their interactions. Here we evaluate three different filtering strategies, eight normalization methods, and two statistical approaches using our data set. We assessed differential gene expression among factors and performed a statistical power analysis using the eight biological replicates per genotype, environment, and sex in our data set. RESULTS: We found that the most critical considerations for the analysis of RNA-Seq read count data were the normalization method, underlying data distribution assumption, and numbers of biological replicates, an observation consistent with previous RNA-Seq and microarray analysis comparisons. Some common normalization methods, such as Total Count, Quantile, and RPKM normalization, did not align the data across samples. Furthermore, analyses using the Median, Quantile, and Trimmed Mean of M-values normalization methods were sensitive to the removal of low-expressed genes from the data set. Although it is robust in many types of analysis, the normal data distribution assumption produced results vastly different than the negative binomial distribution. In addition, at least three biological replicates per condition were required in order to have sufficient statistical power to detect expression differences among the three-way interaction of genotype, environment, and sex. CONCLUSIONS: The best analysis approach to our data was to normalize the read counts using the DESeq method and apply a generalized linear model assuming a negative binomial distribution using either edgeR or DESeq software. Genes having very low read counts were removed after normalizing the data and fitting it to the negative binomial distribution. We describe the results of this evaluation and include recommended analysis strategies for RNA-Seq read count data.

Transcriptional effects of gene dose reduction.

Large-scale gene dose reductions usually lead to abnormal phenotypes or death. However, male mammals, Drosophila, and Caenorhabditis elegans have only one X chromosome and thus can be considered as monosomic for a major chromosome. Despite the deleterious effects brought about by such gene dose reduction in the case of an autosome, X chromosome monosomy in males is natural and innocuous. This is because of the nearly full transcriptional compensation for X chromosome genes in males, as opposed to no or partial transcriptional compensation for autosomal one-dose genes arising due to deletions. Buffering, the passive absorption of disturbance due to enzyme kinetics, and feedback responses triggered by expression change contribute to partial compensation. Feed-forward mechanisms, which are active responses to genes being located on the X, rather than actual gene dose are important contributors to full X chromosome compensation. In the last decade, high-throughput techniques have provided us with the tools to effectively and quantitatively measure the small-fold transcriptional effects of dose reduction. This is leading to a better understanding of compensatory mechanisms.

Molecular characterization of vernalization loci VRN1 in wild and cultivated wheats.

BACKGROUND: Variability of the VRN1 promoter region of the unique collection of spring polyploid and wild diploid wheat species together with diploid goatgrasses (donor of B and D genomes of polyploid wheats) were investigated. Accessions of wild diploid (T. boeoticum, T. urartu) and tetraploid (T. araraticum, T. timopheevii) species were studied for the first time. RESULTS: Sequence analysis indicated great variability in the region from -62 to -221 nucleotide positions of the VRN1 promoter region. Different indels were found within this region in spring wheats. It was shown that VRN1 promoter region of B and G genome can also contain damages such as the insertion of the transposable element.Some transcription factor recognition sites including hybrid C/G-box for TaFDL2 protein known as the VRN1 gene upregulator were predicted inside the variable region. It was shown that deletions leading to promoter damage occurred in diploid and polyploid species independently. DNA transposon insertions first occurred in polyploid species. At the same time, the duplication of the promoter region was observed in A genomes of polyploid species. CONCLUSIONS: We can conclude that supposed molecular mechanism of the VRN1 gene activating in cultivated diploid wheat species T. monococcum is common also for wild T. boeoticum and was inherited by T. monococcum. The spring polyploids are not related in their origin to spring diploids. The spring T. urartu and goatgrass accessions have another mechanism of flowering activation that is not connected with indels in VRN1 promoter region. All obtained data may be useful for detailed insight into origin of spring wheat forms in evolution and domestication process.

vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala).

vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.

Evolution and origin of merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene.

BACKGROUND: Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor suppressor gene, belongs to the ezrin-radixin-moesin (ERM) subgroup of the protein 4.1 superfamily, which links cell surface glycoproteins to the actin cytoskeleton. While merlin's functional activity has been examined in mammalian and Drosophila models, little is understood about its evolution, diversity, and overall distribution among different taxa. RESULTS: By combining bioinformatic and phylogenetic approaches, we demonstrate that merlin homologs are present across a wide range of metazoan lineages. While the phylogenetic tree shows a monophyletic origin of the ERM family, the origin of the merlin proteins is robustly separated from that of the ERM proteins. The derivation of merlin is thought to be in early metazoa. We have also observed the expansion of the ERM-like proteins within the vertebrate clade, which occurred after its separation from Urochordata (Ciona intestinalis). Amino acid sequence alignment reveals the absence of an actin-binding site in the C-terminal region of all merlin proteins from various species but the presence of a conserved internal binding site in the N-terminal domain of the merlin and ERM proteins. In addition, a more conserved pattern of amino acid residues is found in the region containing the so-called "Blue Box," although some amino acid substitutions in this region exist in the merlin sequences of worms, fish, and Ciona. Examination of sequence variability at functionally significant sites, including the serine-518 residue, the phosphorylation of which modulates merlin's intra-molecular association and function as a tumor suppressor, identifies several potentially important sites that are conserved among all merlin proteins but divergent in the ERM proteins. Secondary structure prediction reveals the presence of a conserved alpha-helical domain in the central to C-terminal region of the merlin proteins of various species. The conserved residues and structures identified correspond to the important sites highlighted by the available crystal structures of the merlin and ERM proteins. Furthermore, analysis of the merlin gene structures from various organisms reveals the increase of gene length during evolution due to the expansion of introns; however, a reduction of intron number and length appears to occur in the merlin gene of the insect group. CONCLUSION: Our results demonstrate a monophyletic origin of the merlin proteins with their root in the early metazoa. The overall similarity among the primary and secondary structures of all merlin proteins and the conservation of several functionally important residues suggest a universal role for merlin in a wide range of metazoa.