Project description:We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG island–containing promoters but enriched in predicted enhancers. Classification of the DM sites into those undermethylated in neurons (neuronal type) and those undermethylated in nonneuronal cells (glial type), combined with findings of others that methylation within control elements typically negatively correlates with gene expression, yielded large sets of predicted neuron-specific and non– neuron-specific genes. These sets of predicted genes were in excellent agreement with the available direct measurements of gene expression in human and mouse. We also found a distinct set of DNA methylation patterns that were unique for neuronal cells. In particular, neuronal-type differential methylation was overrepresented in CpG island shores, enriched within gene bodies but not in intergenic regions, and preferentially harbored binding motifs for a distinct set of transcription factors, including neuron-specific activity-dependent factors. Finally, non-CpG methylation was substantially more prevalent in neurons than in nonneuronal cells. Genomic DNA was isolated from FACS-sorted human brain neuronal and nonneuronal nuclei. DNA was bisulfite converted and hybridised to the Illumina Infinium 450K Human Methylation Beadchip array. Six subjects in two technical replicate expriments were analyzed.
Project description:We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG island–containing promoters but enriched in predicted enhancers. Classification of the DM sites into those undermethylated in neurons (neuronal type) and those undermethylated in nonneuronal cells (glial type), combined with findings of others that methylation within control elements typically negatively correlates with gene expression, yielded large sets of predicted neuron-specific and nonneuron-specific genes. These sets of predicted genes were in excellent agreement with the available direct measurements of gene expression in human and mouse. We also found a distinct set of DNA methylation patterns that were unique for neuronal cells. In particular, neuronal-type differential methylation was overrepresented in CpG island shores, enriched within gene bodies but not in intergenic regions, and preferentially harbored binding motifs for a distinct set of transcription factors, including neuron-specific activity-dependent factors. Finally, non-CpG methylation was substantially more prevalent in neurons than in nonneuronal cells. Extended Reduced Representation Bisulfite Sequencing (ERRBS) was performed on genomic DNA to validate the Infinium HM450K DNA methylation data (Kozlenkov et. al., 2013, Nucleic Acids Research, accepted for publication).
Project description:Oncogenic stress induces a global disturbance of cellular homeostasis and subsequently triggers the activation of tumour suppressor pathways. Energy capacity loss, redox state imbalance, and biosynthetic deficiency constitute the primary signals activating safeguard mechanisms, culminating in levels of stress that should not be compatible with infinite proliferative capacities of tumour initiating cells (TIC)s or cancer stem cells (CSC)s. Here, we show that the control of the glucocorticoid response by embryonic factors involved in mammary stemness5, 6 enables basal breast CSCs to not be faced with this oncogenic stress. The glucocorticoid response is thus correlated with stemness properties in basal breast cancers and is required for neoplastic transformation of mammary progenitors. This effect does not rely on hijacking tumour suppressor pathways, but on homeostatic buffering capacity. Through extensive analysis of homeostatic parameters and metabolic profiling, we show that the glucocorticoid response in mammary CSCs limits the “Warburg effect”, the ability of an oncogene to drive glucose fermentation in the presence of oxygen. The glucocorticoid response dampens glycolytic flow and maintains respiratory capacity resulting in reduction of mitochondria energisation. This enables CSCs to sustain carbon and nitrogen flows generated via glycolysis and the tricarboxylic acid cycle (TCA), required for homeostasis and anabolism maintenance. Overall, our results reveal the role of hormonal control of metabolic reprogramming in a CSC in rendering the cell permissive to oncogenic activity. Immortalized human mammary epithelial cells (HMEC-hTERT cells) were infected with SNAIL retroviral expression constructs and an inducible form of RAS G12V. The gene expression profiles of the resulting cell lines, cultured in standard conditions, in limited nutrient supply or after plating them 16h in low-adherent (LA) conditions, were performed.
Project description:Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics (the study of the whole protein complement of a microbial community) can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Here we present a systematic investigation of variables concerning database construction and annotation, and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. Taxonomic and functional results were revealed to be strongly database-dependent, especially when dealing with mouse samples. As a striking example, in mouse the Firmicutes/Bacteroidetes ratio varied up to 10-fold depending on the database used. Finally, we provide recommendations regarding metagenomic sequence processing aimed at maximizing gut metaproteome characterization, and contribute to identify an optimized pipeline for metaproteomic data analysis.
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics. virus-derived siRNA (vsiRNA) expression comparison between control and 4 different virus-infected cells in control as well as 5 different RNAi pathway protein knock-downs in Drosophila dl1 cells
Project description:To identify potentially regulated target genes of MeCP2 we used celltype-specific models for transgenic overexpression and ablation of MeCP2. Gene expression was analyzed in cardiac biopsies of adult male mice. The study contains three different treatment groups: 1. healthy mice (Sham), 2. mice suffering from 4-6 weeks of cardiac pressure overload (TAC) and 3. with a reversible induction of TAC (TAC followed by 2 weeks without TAC) In all experiments we added the corresponding WT for the muted strains. The three different disease conditions were performed individually and thus the results are not directly comparable.
Project description:Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3′ untranslated region (3′ UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stem–loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3′ UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases. 1) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in poly(A) RNA-seq data derived from endothelial cell transcriptome after ADAR1 or ADAR2 knockdown (n=2 biological replicates per condition, total n=8 biological samples). 2) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from peripheral blood mononuclear cells (n=12 total biological samples; n=4 replicates per condition). 3) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions (n=2 biological replicates per condition, total n=4 biological samples). 4) Evaluation of RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions after ADAR1 knockdown (n=3 replicates per condition, total n=12 biological samples). 5) HuR iCLIP RNA-sequencing data derived from HUVEC HuR iCLIP after ADAR1 knockdown (scrambled control and siADAR1, n=1 per condition, total n=2 biological samples).
Project description:We exploited the phosphorylation state of the RNAPII CTD to assess the transcriptional status of most P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide recruitment and activity of RNAPII. We raised and used 3 different anti-RNAPII antibodies in ChIP-on-chip analysis to map the position and status of RNAPII across the entire P. falciparum genome over the 48 h of the IDC at 8 h intervals resulting in TP1-6. Total 4 antibodies were used for ChIP-chip analysis for six time points each done in triplicates, except one antibody 8WG16 which was done in duplicate. We also used 8WG16, commercial RNAPII antibody in similar study done in duplicates.
Project description:Elucidating cytosine modification difference between human populations can enhance our understanding of ethnic specificity in complex traits such as disease predisposition and drug response. In this study, cytosine modification levels in 133 HapMap lymphoblastoid cell lines (LCLs) derived from individuals of European or African ancestry were profiled using the Illumina HumanMethylation450 BeadChip. Approximately 13% of the analyzed CpG sites showed differential modification between the two populations at false discovery rate (FDR) of 1%. CpG sites with greater modification levels in European descents were enriched in the proximal regulatory regions, while those greater in African descents were biased toward gene bodies. More than half of the detected population-specific cytosine modifications could be explained by genetic variation. A substantial proportion of local modification quantitative trait loci (mQTL) exhibited population-specific effects, suggesting that genetic epistasis and/or genotype × environment interaction could be common. Distinct inter-individual correlations were observed between gene expression and cytosine modifications in both proximal promoters and gene bodies, demonstrating a regulatory role of inter-individual variation in cytosine modification. Furthermore, a number of SNPs (single nucleotide polymorphisms) previously identified for complex traits with known racial disparities could be annotated as mQTLs for population-specific CpGs. Our findings revealed abundant population-specific cytosine modifications and the underlying genetic basis, as well as the relatively independent contribution of genetic and epigenetic variations to population differences in gene expression. 60 HapMap CEU and 73 HapMap YRI samples from Coriell Insitute were profiled for cytosine modification levels.
Project description:Trisomy 21 (T21) is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in T21, and to eliminate the noise of the genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for T21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either up- or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins’ fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of DS and wild-type, also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall LADs position was not altered in trisomic cells. However, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results suggest that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome and that GEDDs may therefore contribute to some T21 phenotypes. DNaseI HS mapping in monozygotic twins discordant for trisomy 21 (2 replicates of each).