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Additional file 3:Gene Ontology analysis of the predicted targets of selected sub-clusters of miRNAs with comparable expression profiles. Schematic representation of selected GO/KEGG categories relevant to eye development and function that were enriched among the targets of at least 70% of the miRNAs present within each sub-cluster. The miRNAs within each sub-cluster considered for functional analysis of their predicted targets are listed on the last column of the table. The tissues in which each cluster was predominantly expressed are listed in the column labeled "Expression". For each GO/KEGG category the percentage of representation of this term among the miRNAs analyzed is reported within the cell. The terms of BP Biological Processes and KEGG pathways that relate to similar cellular functions are arbitrarily coloured for readers' clarity. The exact reference numbers that correspond to the GO Biological Processes and KEGG pathways of the table are the following: cell adhesion (GO:0007155); axonogenesis (GO:0007409); morphogenesis of a branching structure (GO:0001763); modification-dependent macromolecule catabolic process (GO:0043632); epithelium development (GO:0060429); transmission of nerve impulse (GO:0019226); cell migration (GO:0016477); cell motion (GO:0006928); neuron development (GO:0048666); neuron differentiation (GO:0030182); neuron projection development (GO:0031175); pattern specification process (GO:0007389); cell-cell signaling (GO:0007267); synaptic transmission (GO:0007268); vesicle-mediated transport (GO:0016192); vasculature development (GO:0001944); Wnt signaling pathway (GO:0016055); regulation of actin cytoskeleton (KEGG: mmu04810); Adherens junction (KEGG: mmu04520); Axon guidance (KEGG: mmu04360); Focal adhesion (KEGG: mmu04510); Glioma (KEGG: mmu05214); GnRH signaling pathway (KEGG: mmu04912); Melanogenesis (KEGG: mmu04916); Melanoma (KEGG: mmu05218); Neurotrophin signaling pathway (KEGG: mmu04722); Wnt signaling pathway (KEGG: mmu04310); MAPK signaling pathway (KEGG: mmu04010); TGF-beta signaling pathway (KEGG: mmu04350). Abbreviations are, cor: cornea; ret: retina; RPE: retinal pigment epithelium. (PDF 37 KB)

Autophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.

Background Transcription factors (TFs) act downstream of the major signalling pathways functioning as master regulators of cell fate. Their activity is tightly regulated at the transcriptional, post-transcriptional and post-translational level. Proteins modifying TF activity are not easily identified by experimental high-throughput methods. Results We developed a computational strategy, called Differential Multi-Information (DMI), to infer post-translational modulators of a transcription factor from a compendium of gene expression profiles (GEPs). DMI is built on the hypothesis that the modulator of a TF (i.e. kinase/phosphatases), when expressed in the cell, will cause the TF target genes to be co-expressed. On the contrary, when the modulator is not expressed, the TF will be inactive resulting in a loss of co-regulation across its target genes. DMI detects the occurrence of changes in target gene co-regulation for each candidate modulator, using a measure called Multi-Information. We validated the DMI approach on a compendium of 5,372 GEPs showing its predictive ability in correctly identifying kinases regulating the activity of 14 different transcription factors. Conclusions DMI can be used in combination with experimental approaches as high-throughput screening to efficiently improve both pathway and target discovery. An on-line web-tool enabling the user to use DMI to identify post-transcriptional modulators of a transcription factor of interest che be found at http://dmi.tigem.it. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0700-3) contains supplementary material, which is available to authorized users.

Autophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.

Supplementary Data. Supplementary analysis and supplementary figures and tables. (DOCX 2030 kb)

Additional file of miRNeye: a microRNA expression atlas of the mouse eye

Summary The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models., Graphical Abstract, Highlights • c-Abl is a TFEB regulator that mediates its tyr phosphorylation • c-Abl inhibition promotes TFEB activity independently of mTORC1 • c-Abl inhibition reduces cholesterol accumulation in NPC1 models, Biological Sciences; Molecular Biology; Cell Biology

Discriminating transcriptional changes that drive disease pathogenesis from nonpathogenic and compensatory responses is a daunting challenge. This is particularly true for neurodegenerative diseases, which affect the expression of thousands of genes in different brain regions at different disease stages. Here we integrate functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington’s Disease (HD) patients. We selected 312 genes whose expression is dysregulated both in HD patients and in HD mice, and then replicated and/ or antagonized each alteration in a Drosophila HD model. High-throughput behavioral testing in this model and controls revealed that transcriptional changes in synaptic biology and calcium signaling are compensatory, whereas alterations involving the actin cytoskeleton and inflammation drive disease. Knockdown of disease-driving genes in HD patient-derived cells lowered mutant Huntingtin levels and activated macroauthophagy, suggesting a mechanism for mitigating pathogenesis. Our multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points.

Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease., Author Summary Shwachman Diamond syndrome (SDS) is an inherited disease. SDS presents, as hallmarks, exocrine pancreatic insufficiency, increased rate of infections, and higher incidence of leukemia. Most cases are due to mutations in the SBDS gene. SBDS encodes for a ribosome maturation factor. In this study, we immortalized mouse fibroblasts carrying one of the most common mutation of SDS patients and performed a thorough analysis of their properties. We show that the loss of SBDS activity causes a rewiring of gene expression and cellular metabolism. Overall we find a reduction of protein synthesis capability, a lower energy status, and increased lysosomal capability. SBDS mutant cells have an increased susceptibility to various forms of stress, but are strikingly resistant to oncogene-induced transformation. We propose a model that explains the complex phenotype of SDS patients and suggests roads for a rationale treatment.

Contains fulltext : 108716.pdf (Publisher’s version ) (Open Access) Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25x, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.

Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and the ability to differentiate into specific cell types. We performed the first genome-wide analysis of the mouse ES cell transcriptome using ∼250,000 gene trap sequence tags deposited in public databases. We unveiled >8000 novel transcripts, mostly non-coding, and >1000 novel alternative and often tissue-specific exons of known genes. Experimental verification of the expression of these genes and exons by RT-PCR yielded a 70% validation rate. A novel non-coding transcript within the set studied showed a highly specific pattern of expression by in situ hybridization. Our analysis also shows that the genome presents gene trapping hotspots, which correspond to 383 known and 87 novel genes. These “hypertrapped” genes show minimal overlap with previously published expression profiles of ES cells; however, we prove by real-time PCR that they are highly expressed in this cell type, thus potentially contributing to the phenotype of ES cells. Although gene trapping was initially devised as an insertional mutagenesis technique, our study demonstrates its impact on the discovery of a substantial and unprecedented portion of the transcriptome.

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate ​TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through ​mucolipin 1 (​MCOLN1) activates calcineurin, which binds and dephosphorylates ​TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed ​TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through ​TFEB required ​MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.