Large-scale analysis of Drosophila core promoter function using synthetic promoters

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it “computes” expression remains poorly understood. To dissect its function, we carried out a comprehensive structure–function analysis in Drosophila. First, we performed a genome-wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters’ activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans

Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage.

BackgroundLipids are stored within cells in lipid droplets (LDs). They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, predominantly phosphatidylcholine (PC). LDs are very dynamic and can rapidly change in size upon lipid uptake or release. These dynamics require a fast adaptation of LD surface. We have recently shown that two Lands cycle PC synthesizing enyzmes, LPCAT1 and LPCAT2 can localize to the LD surface.ResultsHere, we show that knock-down of both enzymes leads to an increase in LD size without changes in the total amount of neutral lipids, while interference with the de-novo Kennedy pathway PC biosynthesis is associated with changes in triacylglyceride synthesis. We show that function of LPCAT1 and 2 is conserved in Drosophila melanogaster by the ortholog CG32699. Furthermore we demonstrate that modulation of the LD pool by LPCAT1 influences the release of lipoprotein from liver cells.ConclusionActivity of the Kennedy pathway regulates the balance between phospholipids and neutral lipids, while the Lands cycle regulates lipid droplet size by regulating surface availability and influencing surface to volume ratio. Differences in lipid droplet size may account for differences in lipid dynamics and be relevant to understand lipid overload diseases

A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol.

Haploid genetic screening of cells under different types of mitochondrial perturbation shows that a pathway involving OMA1, DELE1 and the eIF2 alpha kinase HRI communicates mitochondrial stress to the cytosol to trigger the integrated stress response.Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies(1-3). Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 alpha) in mammalian cells(4,5). eIF2 alpha phosphorylation is mediated by the four eIF2 alpha kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress(6). However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown(1,2,7). Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease

Differential effects of insulin-deficient diabetes mellitus on visceral vs. subcutaneous adipose tissue-multi-omics insights from the Munich MIDY pig model.

Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and β-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas β-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus.

Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically.Methods: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics.Results: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples.Conclusions: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets. (C) 2019 The Authors. Published by Elsevier GmbH

Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia.

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukemia remain largely unexplored. In this study, we characterized mutations in SRSF2, U2AF1, and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile which is highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline, we accurately detected aberrant splicing in splicing factor mutated samples. A large proportion of differentially used junctions were novel, including several junctions in leukemia-associated genes. In SRSF2(P95H) mutants, we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Furthermore, we observed a validated impact on overall survival for two junctions overused in SRSF2(P95H) mutants. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes

Circadian clocks guide dendritic cells into skin lymphatics

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses 1,2 . Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood 3,4 . Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies