Retinoids induce rapid dynamic changes in the non-coding RNAs and epigenetic profiles of murine Hox clusters.
Ontology highlight
ABSTRACT: Physiologically relevant concentrations of retinoic acid are added to Mouse ES cells and a time course (0-72 hours) is examined with expression tiling arrays and RNA-seq to characterize the early dynamics of expression of coding and non-coding RNAs in and around the Hox clusters. Gene expression is examined at various timepoints (0-72 hrs) after retinoic acid induced neuronal differentiation
Project description:Based on the association of TP53 mutation and upregulated TP63 expression in the squamous subtype, we used cell lines derived from genetically engineered mouse models of PDAC (KRAS Trp53fl/+ and KRAS Trp53fl/+ Trp63fl/fl) to begin to unravel the functional consequences of these events in defining squamous tumours. RNA-Seq libraries were generated using TruSeq Stranded Total RNA (Part no. 15031048 Rev. D April 2013) kits, using on a Perkin ElmeraTMs Sciclone G3 NGS Workstation (Product no. SG3-31020-0300). Ribosomal depletion step was performed on 1 ug of total RNA using Ribo-Zero Gold prior to a heat fragmentation step aimed at producing libraries with an insert size between 120-200 bp. cDNA was then synthesized from the enriched and fragmented RNA using InvitrogenaTMs SuperScript II Reverse Transcriptase (Catalog no. 18064) and random primers. The resulting cDNA was further converted into double stranded DNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintain the strandedness of the library. Following 3aTM adenylation and adaptor ligation libraries were subjected to 15 cycles of PCR to produce RNA-Seq libraries ready for sequencing. Prior to sequencing, exome and RNA-Seq libraries were qualified and quantified via CaliperaTMs LabChip GX (Part no. 122000) instrument using the DNA High Sensitivity Reagent kit (Product no. CLS760672). Quantification of libraries for clustering was performed using the KAPA Library Quantification Kits For Illumina sequencing platforms (Kit code KK4824) in combination with Life Technologies Viia 7 real time PCR instrument. All libraries were sequenced using the Illumina HiSeq 2000/2500 system with TruSeq SBS Kit v3 - HS.
Project description:AIM: We performed RNA-sequencing experiments seeking genes whose expression changed due to nerve injury. In addition, we wanted to test whether inhibition of the methyl transferase G9a/GLP, that methylates H3K9me2, could reverse those expression changes due to nerve ligation. G9a/GLP methylase was pharmacologically inhibited using UNC0638. METHOD: We generated cDNA libraries from RNA purified from DRGs obtained from Sham operated (4), SNL (4), and SNL plus UNC0638 (3) rats. We sequenced the cDNA libraries generating single end 50 bp reads on the illumina HiSeq 2500 platform. Sequencing reads were aligned to the rat genome rn4 using TopHat RESULTS: We were able to map 16876 genes, from which 2035 changed their expression values at least two fold when we compared SNL to Sham operated (pâ?¤ 0.01). There were 1205 upregulated and 832 down-regulated genes. Next, we focused on genes whose expression was either up or down-regulated 2 fold due to nerve ligation, and the values would be normalized to control level after G9a/GLP inhibito (UNC0638)r treatment. We identified 639 genes in our data set that behave this way We generated cDNA libraries from RNA purified from DRGs obtained from Sham operated (4), SNL (4), and SNL plus UNC0638 (3) rats. We sequenced the cDNA libraries generating single end 50 bp reads on the illumina HiSeq 2500 platform. Sequencing reads were aligned to the rat genome rn4 using TopHat
Project description:The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here, we describe a whole-genome transcriptome analysis of human benign prostatic basal and luminal populations by using deep RNA sequencing. Combined with comprehensive molecular and biological characterizations, we show that the differential gene expression profiles account for their distinct functional phenotypes. Strikingly, in contrast to luminal cells, basal cells preferentially express gene categories associated with stem cells, neural and neuronal development and RNA processing. Consistent with their expression profiles, basal cells functionally exhibit intrinsic stem-like and proneural properties with enhanced ribosome RNA (rRNA) transcription activity. Of clinical relevance, the treatment failed castration-resistant and anaplastic prostate cancers molecularly resemble a basal-like phenotype. Therefore, we link the cell-type specific gene signatures to subtypes of prostate cancer development, and identify genes associated with patient clinical outcome. Human total RNA profiles of 3 pairs of benign prostatic basal and luminal populations freshly purified from prostate tissues of three prostate cancer patients by deep RNA-seq.
Project description:Shotgun sequencing of sleeping sickness patient blood. WARNING: these results cannot be compared with those from trypanosome poly(A)+ mRNA, because the poly(A) selection introduces substantial bias, including loss of long mRNAs. Details will appear in the publication. These are additional sequencing runs that match some from E-MTAB-5293
Project description:Objective: Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and have overlaps in their transcriptome and epigenome profiles. Notably, destruction of one of these cell populations can stimulate repopulation via transdifferentiation of the other cell type, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity. Methods: We sorted human a- and b-cells and performed the â??Assay for Transposase-Accessible Chromatin with high throughput sequencingâ?? (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. Results: We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The â??group specific proteinâ?? (GC; or vitamin D binding protein) was restricted to a-cells, while CHODL (chondrolectin) immunoreactivity was only present in b-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with common single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. Conclusions: We have determined the genetic landscape of human α- and β-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel α- and β-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion. ATAC-seq on 3 human alpha cell samples, 3 human beta cell samples, and 2 human acinar cell samples. RNA-seq on 7 human alpha cell samples and 8 human beta cell samples.
Project description:The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that results in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was overrepresented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways. RNA-Seq of cell lines MCF10A, MCF7 and MDA-MB-231.
Project description:Rats (four per isolate) were infected with Trypansooma rhodesiense taken from the CNS of two different patients from Lwala. Blood was taken at parasitaemias ranging from 3-20 E7. The results can be compared with those from human patient samples.
Project description:Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information. RNA-Seq analysis to investigate transcriptomes of hPGC-like cells (hPGCLCs), fetal hPGCs, TCam-2 and hESCs
Project description:We measured heart gene expression in 192 heterogeneous stock rats. These animals were part of a larger cohort that were extensively phenotyped and genotyped and originally published in PMID: 23708188, although the gene expression data here were not included in that study.
Project description:Pineal function follows a 24-hour schedule, dedicated to the conversion of night and day into a hormonal signal, melatonin. In mammals, 24-hour changes in pineal activity are controlled by a neural pathway that includes the central circadian oscillator in the suprachiasmatic nucleus and the superior cervical ganglia (SCG), which innervate the pineal gland. In this study, we have generated the first next-generation RNA sequencing evidence of neural control of the daily changes in the pineal transcriptome. We found over 3000 pineal transcripts that are differentially expressed (p <0.001) on a night/day basis (70% of these genes increase at night, 376 with fold change >4 or <1/4), the majority of which had not been previously identified as such. Nearly all night/day differences were eliminated by neonatal removal or decentralization of the SCG, confirming the importance of neural input for differential night/day changes in transcript abundance. In contrast, very few non-rhythmic genes showed evidence of changes in expression due to the surgical procedure itself, which is consistent with the hypothesis that post neonatal neural stimulation is not required for cell fate determination and maintenance of phenotype. Many of the transcripts that exhibit marked differential night/day expression exhibited similar changes in response to in vitro treatment with norepinephrine, the SCG neurotransmitter which mediates pineal regulation. Similar changes were also seen following treatment with an analog of the norepinephrine second messenger, cyclic AMP. For the in vivo data, there were 8 biological conditions: day and night time points for each of four surgical groups: Control (Ctrl) Sham-surgery (Sham), Decentralized (DCN), and Ganglionectomized (SCGX). Samples were pooled into three biological replicates for each biological condition. For the in vitro data there were 3 biological conditions: Untreated control (CN), DBcAMP-treated (DB), and Norepinephrine-treated (NE). For the pineal enrichment comparison, three samples (i.e. no biological replicates) were used: pineal-day, pineal-night and mixed-tissue. For the mixed tissues sample, the following tissues from three rats sacrificed at ZT7 were used: cortex, cerebellum, midbrain, hypothalamus, hindbrain, spinal cord, retina, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, adrenal gland. Total RNA was extracted from each tissue, and then equal amounts of each of the 15 tissues were combined for the final pooled sample.