The integration of positive and negative intra- and extra-cellular signals dictates whether a cell will proliferate or differentiate. While it is intuitive to speculate that nutrients availability may influence this alternative, a comprehensive complement of the molecular determinants involved in this process has not been elucidated yet. In this study, we will investigate how nutrients (glucose) affect skeletal myogenesis. C2C12 cells will be cultured in high glucose and low glucose conditions, and their differenciation will be studied.
Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt.
No sample metadata fields
View SamplesSelective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in satellite cells Overall design: To establish the role of the deacetylase SIRT1 in skeletal muscle we examined the genome wide distribution of H4K16ac in quiescent (FI) and proliferating (Cul) satellite cells isolated from WT mice (C57Bl/6 background) and SIRT1mKO (generated via breeding of Pax7cre/+ knock-in mice with mice containing the floxed exon 4 SIRT1 allele). We also analyzed the distribution of SIRT1 in quiescent and proliferating FACS isolated WT satellite cells (two replicates). We generated the mRNA profiles (at least two replicate for each experiment) of FACS isolated quiescent, proliferating and differentiating (1 day in differentiation medium) satellite cells of WT mice and SIRT1mKO. The selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program.
The NAD(+)-dependent SIRT1 deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells.
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View SamplesThousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen lncRNAs. One specific lncRNA, Non-coding RNA Activated by DNA Damage (NORAD), has recently been shown by genetic deletion to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here, we combine RNA antisense purification (RAP) and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX (an emerging component of the DNA-damage response) and encodes the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex, which we term NORAD-Activated Ribonucleoprotein Complex 1 (NARC1), containing known suppressors of genomic instability: topoisomerase I (TOP1), ALYREF and the PRPF19/CDC5L complex. Cells depleted of NORAD or RBMX display an increased frequency of chromosome segregation errors, reduced replication-fork velocity and altered cell cycle progression phenotypes that are mechanistically linked to TOP1 and PRPF19/CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function and that NORAD is required for the assembly of a previously unknown topoisomerase complex (NARC1) that contributes to maintaining genomic stability. Moreover, we uncover a novel function for lncRNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex. Overall design: We examined gene expression changes and alternative splicing events in wildtype and NORAD depleted cells using RNA sequencing.
The NORAD lncRNA assembles a topoisomerase complex critical for genome stability.
Cell line, Subject, Time
View SamplesThe mammalian genome contains thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to play critical roles in diverse cellular processes through a variety of mechanisms. While some lncRNA loci encode RNAs that act non-locally (in trans), emerging evidence indicates that many lncRNA loci act locally (in cis) to regulate expression of nearby genes—for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. To address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen targeting more than 10,000 lncRNA transcriptional start sites (TSSs) to identify noncoding loci that influence a phenotype of interest. We found 11 novel lncRNA loci that, upon recruitment of an activator, each mediate BRAF inhibitor resistance in melanoma. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation results in dosage-dependent activation of four neighboring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit to systematically discover functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function. Overall design: RNA-seq on A375 cells overexpressing candidate lncRNA or protein-coding gene.
Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood.
Specimen part, Cell line, Subject
View SamplesRNA-seq and ATAC-seq data to understand how gene regulation and chromatin accessibility correlates with function enrichment in CRISPR screen for melanoma drug resistance
Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood.
No sample metadata fields
View SamplesHuman monocyte derived dendritic cells matured via galectin-1 or LPS.
Galectin-1-matured human monocyte-derived dendritic cells have enhanced migration through extracellular matrix.
No sample metadata fields
View SamplesAcute lymphoblastic leukemia (ALL) is an heterogeneous disease comprising several subentities that differ for both immunophenotypic and molecular characteristics. Over the years, the biologic understanding of this neoplasm has largely increased. Gene expression profiling has recently allowed to identify specific signatures for the different ALL subsets and permitted identification of pathways deregulated by a given lesion. MicroRNAs (miRNAs) are small non-coding RNAs which play a pivotal role in several cellular functions. In this study, we investigated miRNA and gene expression profiles in a series of adult ALL cases by microarray analysis and combined them by bioinformatic analysis. Interestingly, those miRNAs which are differentially expressed between the ALL classes accounted for a large proportion of miRNA/mRNA expression pairs identified by the above analysis. Moreover, the analysis highlighted several putative miRNA targets involved in apoptosis and cell-cycle regulation.
Characterization of B- and T-lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles.
Sex, Age, Specimen part
View SamplesGoals/objectives: to identify various gene expression in B cell subsets derived from human PBMC and cord blood
Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells.
Specimen part
View SamplesIn order to identify miR-21 targets by a biochemical high-throughput method, we immunopurified RISC Complex and associated mRNAs in both control and miR-21 overexpressing Jurkat cells.
miR-21 is a negative modulator of T-cell activation.
Cell line
View SamplesT-lymphocyte activation is efficiently mimicked in vitro by treatment with anti CD3 / anti CD28 antibodies. We report miR-21 induction upon CD3/CD28 stimulation of primary T-lymphocytes. In order to assess the function of miR-21 in T-lymphocytes we interfered with miR-21 function by lentiviral transduction of a miR-21 sponge construct. MRNA profile of miR-21 sponge and control transduced T-lymphocytes 48hrs after stimulation.
miR-21 is a negative modulator of T-cell activation.
No sample metadata fields
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