Project description:Total RNA was extracted at different stages (TV, T0, T11 and T20) during autogamy of strain d4-2. Genes specifically induced during autogamy were identified following analysis of microarray hybridization data 3 technical replicates of the time course were hybridized Subset one and three were labelled with Cy3 Subset two was labelled with Cy5
Project description:Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown that requires a decisive shift in the balance of Cdk1 and PP2A:B55 activity. Beyond this point Cyclin B/Cdk1 is essential to phosphorylate a distinct subset mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how Cyclin A, B and Greatwall coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation. The phosphoproteomics dataset aims to identify substrates that are affected specifically by acute depletion of Cyclin Bprotein.
Project description:Clear cell renal cell carcinoma (ccRCC) is the most common variant of kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and the mechanisms underlying ccRCC growth and progression will provide clues to treat this aggressive malignant tumor. Here, we report that the RING ligase praja2 is a novel component of the endocytic system that supports clathrin-mediated receptor endocytosis. At the molecular level, we identify the adaptor protein AP2 as a binding partner and substrate of praja2. Functionally, we demonstrate that praja2 is required for AP2-mediated receptor endocytosis and clearance. Downregulation of praja2 in RCC cells and tissues is associated with a marked upregulation of membrane receptors, as EGFR, VEGFR and TfR. A negative feedback loop links EGF signaling to proteolysis of praja2 and sustains downstream mitogenic and proliferative pathways. Restoring praja2 expression in RCC cells remarkably decreases EGFR levels, rewires cancer cell metabolism and inhibits RCC growth and metastatic diffusion. In praja2 knockout mice, upregulation of RTKs levels associates with profound histopathological renal alterations. Our findings identify praja2 as a component of the endocytic pathway that supports receptor endocytosis and clearance. Downregulation of praja2 in RCC cells, thus, sustains RTK signaling and promotes kidney cancer growth and diffusion.
Project description:A series of valdiazen chemical probes were developed and applied in affinity-based pulldown experiments to enrich potential valdiazen-binding proteins.
Project description:We have investigated the genome regulatory network that controls responses to the hormone jasmonate in seedlings. There is much interest currently in understanding the systems-level actions of transcription factors. However, such studies can provide much greater biological insight if set in the context of the signal transduction pathway that initiates gene expression changes and the downstream organismal phenotypes they control. We have generated an integrated framework of the response to jasmonate that spans from signal perception and transduction, through activity of master and secondary-regulatory transcription factors, to gene expression outputs and chromatin remodelling. We make use of genome-wide target maps for hundreds of transcription factors, dynamic histone modification analysis, global proteome and phosphoproteome analyses, massively multiplexed protein-protein interactome data and time series transcriptome analysis. This enables us to predict previously unknown components of the jasmonate regulatory mechanism and validate them through targeted mutant studies. The result is a comprehensive understanding of how a plant hormone remodels cellular function and plant behaviour.
Project description:Cyclin-dependent kinases (CDKs) are key players in cell cycle regulation and transcription. The CDK-family member Cdk10 is important for neural development and can act as a tumor suppressor but the underlying molecular mechanisms are largely unknown. Here, we provide an in depth analysis of Cdk10 substrate specificity and function. Using recombinant Cdk10/CycQ protein complexes we characterize RNA pol II CTD, c-MYC, and RB1 as in vitro protein substrates. Using an analog-sensitive mutant kinase, we identify 66 proteins as Cdk10 substrates from HEK cells. Among these, proteins involved in cell cycle, translation, stress response, growth signalling, as well as rRNA, and mRNA transcriptional regulation are found. Cdk10 is able to auto-phosphorylate in trans within the T-loop, offering the possibility of a positive feedback loop for activation. We also identify Cdk10 as an in vitro substrate of Cdk1 and Cdk5 at multiple sites, allowing for a potential cross-talk between these CDKs. With this functional characterization, Cdk10 adopts a hybrid position in both cell cycle and transcriptional regulation
Project description:The goal of this study is to identify the targets of RVE8, a MYB-like transcription factor involved in the circadian clock in Arabidopsis. Analysis of 7 days old rve8-1 RVE8::RVE8:GR and rve8-1 seedlings treated with dexamethasone or mock identified genes responsive to RVE8 induction.The RVE8 up-regulated genes are enriched for evening-phased genes while the down-regulated genes are enriched for a morning phase. This study reveals that RVE8 is a master regulator of circadian gene expression in Arabidopsis. Transgenic line rve8-1 RVE8::RVE8:GR and rve8-1 treated with DEX or mock with three biological replicates each, 12 samples in total.
Project description:MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide (nt) RNAs that regulate gene expression at transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress responses or pathogen responses. miRNAs with their related target genes have been widely studied in model plants?and increasing studies have been performed on some crops?however, the number of identified miRNAs in cotton was limited, and global identification of related targets through degradome sequencing has not been developed previously. In this study, we globally identified small RNAs and their related target genes during cotton somatic embryogenesis by the high throughput small RNA and degradome sequencing technology using fresh hypocotyls and EC (embryogenic calli) of Gossypium hirsutum YZ1. A total of 36 differentially expressed conserved miRNA families of which 19 miRNA families represented by 29 precursors and 25 novel miRNAs were identified, with star sequences of 20 known miRNAs and 2 novel miRNAs discovered. 234 genes in EC and 322 genes in CK were identified as targets of 23 and 30 known miRNA families, and 16 genes were found as targets of 8 novel miRNAs. The expression profiles of several miRNAs and their targets were verified by qRT-PCR and 5’RACE were further used to validate the sliced sites of the targets. Interestingly, four TAS3 D6 and D7 were also found in both degradome libaries which can perfectly match their precursors. The profiling of the miRNAs and their target genes provides more information about the regulatory network of miRNAs during somatic embryogenesis in cotton. small RNA and degradome sequencing of CK(fresh hypocotyls) and EC (embryogenic calli) of Gossypium hirsutum YZ1
Project description:Choline (reference 1) and glucose (reference 2) was studied using HR MAS MR spectroscopy data and 105 and 38 gene transcripts were selected respectively from the microarray data to study differences between two xenograft models. Reference 1: Moestue SA et al, Distinct choline metabolic profiles are associated with differences in gene expression for basal-like and luminal-like breast cancer xenograft models, BMC Cancer 2010 Aug 17;10:433 (PMID: 20716336). Reference 2: Grinde et al., submitted The microarray data from the luminal-like and basal-like xenograft models were compared for selected genes, using Limma Bioconducotr package
Project description:Disturbances in lipid homeostasis can cause mitochondrial dysfunction and lipotoxicity requiring tight regulation of intracellular lipid metabolism and fatty acid (FA) flux. Perilipin 5 (PLIN5) decorates intracellular lipid droplets (LD) in oxidative tissues and controls triacylglycerol (TG) turnover via its interactions with Adipose triglyceride lipase (ATGL) and the ATGL co-activator Comparative gene identification-58 (CGI-58). Furthermore, PLIN5 anchors mitochondria to the LD membrane via its carboxyl-terminal domain. However, the role of this LD-mitochondria coupling (LDMC) in cellular FA flux and energy catabolism is less established. In this study, we investigated the impact of abolished LDMC on cellular FA flux, mitochondrial respiration and protein interaction. To do so, we established novel PLIN5 truncation variants lacking LDMC while maintaining normal interactions with lipolytic key players. Radiotracer studies with transgenic cell lines stably overexpressing either wild type or truncated PLIN5 revealed that LDMC has no significant impact on FA esterification upon lipid loading or TG catabolism during stimulated lipolysis. Moreover, we found that LDMC is not essential for FA shuttling into mitochondria, which was in line with only minor effects of disrupted LDMC on FA oxidation. In contrast, LDMC significantly improved the mitochondrial respiratory capacity and metabolic flexibility of lipid-challenged cardiomyocytes, which was corroborated by LDMC-dependent interactions of PLIN5 with mitochondrial proteins involved in mitochondrial respiration, dynamics and cristae organization. Taken together, this study suggests that PLIN5 preserves mitochondrial function by adjusting FA supply via the regulation of TG hydrolysis and that LDMC is a vital part of mitochondrial integrity.