In the urinary tract, smooth muscle (SM) is present in the renal pelvis, the ureter, the bladder and the urethra and plays a crucial role in the functional and structural integrity of these organs. In Tshz3 mutant ureters the myogenic program is not activated in the proximal region due to the absence of expression of myocardin (Myocd), a key regulator of SM differentiation. We set out to characterize TSHZ3-dependent mechanisms that participate to the process of ureteric smooth muscle cells (SMC) differentiation.
The tiptop/teashirt genes regulate cell differentiation and renal physiology in Drosophila.
Specimen part
View SamplesTSHZ3, which encodes a zinc-finger transcription factor, was recently positioned as a hub gene in a module of genes with the highest expression in the developing human neocortex, but its functions remained unknown. Here, we identify TSHZ3 as the critical region for a syndrome associated with heterozygous deletions at 19q12q13.11, which includes autism spectrum disorder (ASD). In Tshz3 null mice, differentially expressed genes include layer-specific markers of cerebral cortical projection neurons (CPNs) and their human orthologues are strongly associated with ASD. Furthermore, heterozygous Tshz3-deficient mice show functional changes at synapses established by CPNs and exhibit core ASD-like behavioral abnormalities. These findings reveal essential roles for Tshz3 in CPN development and function, whose alterations can account for ASD in the newly-defined TSHZ3 deletion syndrome. Overall design: Three independent replicates, each containing cortices from 3-4 embryos from multiple litters, were prepared from wild-type and Tshz3 mutant neocortex at E18.5. Caubit et al., TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons. Nat. Genet ###, xxx-yyy (2016).
TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons.
Specimen part, Subject
View SamplesEBF1 is essential for B cell specification and commitment. To explore the dynamics of EBF1 initiated B cell programming, we performed EBF1 ChIP-seq, ATAC-seq, bisulfite-seq, RNA-seq and several histone ChIP-seq analyses at different stages of the transition from Ebf1-/- pre-pro-B to pro-B triggered by EBF1 restoration. We also performed Pax5 ChIP-seq in Ebf1-/- pre-pro-B cell and EBF1-restored pro-B cell to study the pioneering function of EBF1 that allows other transcription factors to access certain chromatin sites. Overall design: Time series RNA-Seq analysis during the differentiation from Ebf1-deficient pre-pro-B cell to EBF1-restored pro-B cell.
Dynamic EBF1 occupancy directs sequential epigenetic and transcriptional events in B-cell programming.
Subject
View SamplesEed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation.
Polycomb protein EED is required for silencing of pluripotency genes upon ESC differentiation.
Specimen part, Cell line
View SamplesWe determined genome-wide nucleosome occupancy in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell type and protein specific binding preferences of transcription factors to sites with either low (e.g. Myc, Klf4, Zfx) or high (e.g. Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome depleted regions around transcription start and termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to their CpG-content or histone methylation marks. Throughout the genome nucleosome occupancy was dependent on the presence of certain histone methylation or acetylation modifications. In addition, the average nucleosome-repeat length increased during differentiation by 5-7 base pairs, with local variations for specific genomic regions. Our results reveal regulatory mechanisms of cell differentiation acting through nucleosome repositioning. Overall design: The Total RNA from ESCs, NPCs and MEFs was extracted by guanidinisothiocyanat/phenol extraction with the Trifast kit (Peqlab). Total RNA preparations were treated with DNase I, phenol/chloroform extracted and precipitated before further processing. RNAs were depleted of 5S, 5.8S, 18S and 28S rRNAs using the Human/Mouse/Rat Ribo-Zero rRNA Removal Kit (Epicentre) according to the manufacturer’s protocol. After rRNA depletion, RNAs were fragmented with a kit from Ambion. Libraries for Solexa sequencing were generated according to the standard Illumina protocol that comprised first strand cDNA synthesis, second strand cDNA synthesis, end repair, addition of a single A base, and adapter ligation. Sequencing was performed on the Illumina GAIIx (replicate 1) and Illumina HiSeq 2000 (replicate 2) platforms at the sequencing core facilities of the BioQuant in Heidelberg, Germany. RNA reads were aligned with TopHat. Further expression analysis was with the Genomatix software suite (Genomatix, Munich, Germany) and the Eldorado gene annotation. For each transcript a normalized expression value was calculated from the read distribution that accounts for the length differences using the program DEseq for the analysis of differential expression.
Genome-wide nucleosome positioning during embryonic stem cell development.
Specimen part, Cell line, Subject
View SamplesRNA expression in WT and jhd2? cells in various nutritional sources Overall design: Strand-specific total RNA was sequenced (Illumina stranded TruSeq, with dUTP second strand-incorporation) from wildtype and mutants cells, in biological replicates, normalized by RNA spike-in controls
Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.
Cell line, Subject
View SamplesGerstmann Strausller Scheinker (GSS) human prion disease homogenate is i.c. inoculated into mice exhibiting a proline to leucine alteration at codon 101 of the murine prion protein gene (101LL). This results in a disease with an incubation period of approximately 291 days. Normal brain homogenate i.c. inoculated into 101LL mice which are aged matched are used as controls.
Distribution of Misfolded Prion Protein Seeding Activity Alone Does Not Predict Regions of Neurodegeneration.
Sex, Specimen part
View SamplesLong terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported that a member of the THE1B class of LTR elements in classical Hodgkin Lymphoma (cHL) acted as a promoter for the growth factor receptor gene CSF1R and that expression of this gene is required for tumor survival. However, to which extent and how such elements participate in globally shaping the unique cHL gene expression program is unknown. To address this question we mapped the genome-wide activation of THE1-LTRs in cHL cells using a targeted next generation sequencing approach (RACE-Seq). Integration of these data with global gene expression data from cHL and control B cell lines showed a unique pattern of LTR activation impacting on gene expression, including genes associated with the cHL phenotype. We also show that global LTR activation is induced by strong inflammatory stimuli. Together these results demonstrate that LTR activation provides an additional layer of gene deregulation in classical Hodgkin lymphoma and highlight the potential impact of genome-wide LTR activation in other inflammatory diseases. Overall design: RNA-Seq in laser capture microdissected (LCM) tumour (TU) and non tumour cells (NTC) primary HL material from patient samples
Global long terminal repeat activation participates in establishing the unique gene expression programme of classical Hodgkin lymphoma.
Specimen part, Subject
View SamplesPresently, there is a deficiency of effective therapies designed to target clear cell renal cell carcinoma (ccRCC), with poor prognosis resulting in patients with advanced disease. Additionally, there is a lack of molecular factors which can be remedially targeted resulting in tumor specific inhibition, and therefore current therapeutic approaches often produce adverse side effects in patients. We identified that Stearoyl-CoA desaturase 1 (SCD1) was consistently overexpressed in patient ccRCC samples, and further investigation of SCD1 as a potential molecular target for ccRCC intervention utilizing a SCD1 inhibitor (A939572) resulted in tumor specific growth inhibition and induction of cell death. In order to understand the mechanism by which the SCD1 inhibitor mediated its anti-tumor effects, we performed gene array analysis and compared expression patterns between treated and untreated samples.
Stearoyl-CoA desaturase 1 is a novel molecular therapeutic target for clear cell renal cell carcinoma.
Cell line, Treatment
View SamplesWe developed a computational framework that integrates chromosomal copy number and gene expression data for detecting aberrations that promote cancer progression. We demonstrate the utility of this framework using a melanoma dataset. Our analysis correctly identified known drivers of melanoma and predicted multiple novel tumor dependencies. Two dependencies, TBC1D16 and RAB27A, confirmed empirically, suggest that abnormal regulation of protein trafficking contributes to proliferation in melanoma. Together, these results demonstrate the ability of integrative Bayesian approaches to identify novel candidate drivers with biological, and possibly therapeutic, importance in cancer.
An integrated approach to uncover drivers of cancer.
Cell line
View Samples