We used a high-throughput technology, DNA microarray, to screen the entire genome for the changes in gene expression in diseased tissue to characterize Dupuytren's contracture at a molecular level and find genes that are involved in development of the disease.
Microarray analysis of Dupuytren's disease cells: the profibrogenic role of the TGF-β inducible p38 MAPK pathway.
Sex, Specimen part
View SamplesNF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB (NFYB) and NF-YC (NFYC)) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5,000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (non-modified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively co-localizes with FOS in all genomic contexts, and at promoters and enhancers this often occurs in the absence of JUN and the AP-1 motif. NF-Y also co-associates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve co-association with FOS.
NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors.
Cell line, Treatment
View SamplesThe activity of enhancers and promoters fine-tunes the transcriptional program of mammalian cells through the recruitment and interplay between cell type-specific and ubiquitous transcription factors. Despite their key role in modulating transcription, the identification of enhancers is challenged by their limited sequence conservation and highly variable distance from target genes. Although enhancers are characterised by the strong enrichment of mono-methylation at lysine 4 of histone H3, mirrored by low tri-methylation at the same residue, a comprehensive list of enhancers-associated histone post-translational modifications (PTMs) is still lacking. We undertook a proteomics investigation, based on chromatin immunoprecipitation combined with mass spectrometry (MS), to identify histone marks specifically associated to cis-regulatory elements in macrophages, focusing on enhancers. We also profiled their plasticity during the transcriptional activation induced by an inflammatory stimulus. The proteomic analysis suggested novel PTM associations, which were validated by analysis of ChIP- and RNA-seq data, whose intersection revealed the existence of novel sub-populations of enhancers marked by specific signatures: the dual mark H3K4me1/K36me2 labels transcription at enhancers, whereas H3K4me1/K36me3 and H3K4me1/K79me2 tag distinct intronic enhancers. While demonstrating that analyzing restricted genomic regions can disclose the combinatorial language of histone modifications, this study highlights the potential of MS-based proteomics in addressing fundamental questions in epigenetics. Overall design: Total RNA was extracted from 5x10^6 untreated RAW 264.7 cells using RNAeasy kit (Qiagen). Libraries were then prepared using TruSeq RNA sample preparation Kit (Illumina) after depleting ribosomal RNA
Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers.
Specimen part, Cell line, Treatment, Subject
View SamplesUpstream of N-ras (UNR) is a conserved RNA-binding protein that regulates mRNA translation and stability by binding to sites generally located in untranslated regions (UTRs). In Drosophila, sex-specific binding of UNR to msl2 mRNA and the non-coding RNA roX plays key roles in the control of X-chromosome dosage compensation in both sexes. In order to investigate broader sex-specific functions of UNR, we have identified its RNA targets in adult male and female flies by high-throughput RNA binding and transcriptome analysis. Here we show that UNR binds to a large set of protein-coding transcripts and to a smaller set of non-coding RNAs in a sex-specific fashion. Overall design: Two replicates of UNR IP were performed in D.melanogaster adult males and females, and enrichment in either sex was compared with IgG IP as control. To correlate sex-specific UNR binding with sex-specific transcription and splicing we performed RNA-Seq experiments in males and females.
Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR.
Specimen part, Subject
View SamplesThe human nm23-H1 was discovered as a tumor metastasis suppressor based on its reduced expression in melanoma cell lines with low versus high metastatic potential. It encodes for one of two subunits of the nucleoside-diphosphate kinase. Besides its role in the maintenance of the cells NTP pool, nm23 plays a key role in different cellular processes. The role of nm23-H1 in these processes still has to be elucidated. Our goal was to identify Nm23-H1 downstream targets by subjecting Nm23-H1 overexpressing CAL 27 cells oral squamous cell carcinoma (OSSC) to microarray analysis. The genes with changed expression patterns could be clustered into several groups: transforming growth factor (TGF) signaling pathway, cell adhesion, invasion and motility, proteasome machinery, cell-cycle, epithelial structural and related molecules and others. Based on the expression patterns observed we presume that nm23-H1 might have a role in OSSCs, which should be confirmed by future experiments.
Downstream targets of Nm23-H1: gene expression profiling of CAL 27 cells using DNA microarray.
Specimen part, Disease, Disease stage, Cell line
View SamplesCockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43 Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. We show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome cells (UVSS1KO) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.
The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells.
Specimen part, Cell line
View SamplesWe transfected PC3 cells with 100nM of a scrambled antisense oligonucleotide (AON) and an AON directed against MBNL1 exon 7 (36 basepairs) in order to skip the latter. Cells were harvested at 72h post-transfection and RNAseq was performed with ribozero depletion. Overall design: For RNA-Seq library preparation we followed the Illumina TruSeq RNA Sample Preparation Kit v2 manual. At least 70 million, 75bp long paired end reads were mapped to the GRCh37/hg19 version of the human genome per replicate using STAR 2.4.2a (doi: 10.1093/bioinformatics/bts635) using the default parameters.
MBNL1 alternative splicing isoforms play opposing roles in cancer.
Specimen part, Cell line, Subject, Time
View Samples63 melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 oligo arrays. The aim of this study was to identify potential downstream targets of key oncogenes and TSGs in melanoma (including p14ARF, p16INK4A, BRAF etc).
Confirmation of a BRAF mutation-associated gene expression signature in melanoma.
No sample metadata fields
View SamplesWe report the application of single-molecule-based sequencing technology for high-throughput profiling of NSC transcriptome. Overall design: Wild type and Sox2-deleted NSC were sequenced; three independent samples from wild type, and three from Sox2-deleted brains (different individual mice).
Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance.
Specimen part, Subject
View SamplesAlthough recent evidence suggests that overlapping sense/antisense transcription is a common feature in higher eukaryotes, the possibility that overlapping transcripts could interact to each other and bear a specific biological function has not been explored. Here we show that a plethora of sense/antisense transcript pairs are co-expressed from 8q24.21 within the same cell and acquire a stable double-stranded RNA conformation. Interestingly, these molecules display predominantly nuclear localization and establish specific interactions with nuclear components. A detailed characterization of a particular sense/antisense pair (ndsRNA-2a) revealed that this molecule displays differential localization throughout the cell cycle, interacts with RCC1 and RAN and through the latter with the mitotic RANGAP1-SUMO1/RANBP2 complex. Notably, an increased number of bi/multi-nucleated cells and chromatin bridges were observed upon ndsRNA-2a overexpression, whereas strand-specific ndsRNA-2a knockdown leads to mitotic catastrophe and cell death. This suggests a functional role of ndsRNA-2a in cell cycle progression that critically requires its double stranded nature. Finally, the identification of hundreds of sense/antisense transcripts pairs harboring ndsRNA profile signatures and their regulation by cellular cues suggests that ndsRNAs constitute a novel class of regulatory molecules that are likely to be involved in a plethora of biological processes. Overall design: PLB985 long (3x datasets) and small (3x datasets) strand specific RNA-Seq for captured RNAs. Global PLB985 for long (2x datasets) and small RNAs (2x datasets). Global libraries for EtOH (vehicle) treated (1x dataset) or retinoic acid induced differentiated PLB985 cells (1x dataset).
Human cells contain natural double-stranded RNAs with potential regulatory functions.
No sample metadata fields
View Samples