PolyA RNAseq from HCT116 cells in normoxia and hypoxia
ABSTRACT: To determine the effects of depleting TIP60, CDK8, or HIF1A on the transcriptional response to hypoxia, we performed RNAseq analysis of four HCT116 colorectal carcinoma cell lines (shNT, HIF1A-/-, shTIP60 and shCDK8) in normoxic and hypoxic (24hrs, 1% O2) conditions. PolyA RNA for two independent biological replicates was purified from HCT116 cells stably expressing an shRNA against a non-targeting control (shNT), TIP60 (shTIP60) or CDK8 (shCDK8), or genetically deleted HIF1A (HIF1A-/-) subjected to 24hrs 1% O2 (hypoxia) or maintained under ambient oxygen (21%; normoxia) was sequenced on the Ion Torrent platform. Reads were aligned to the human genome and gene-level counts were used for differential expression analysis.
Project description:To test if CDK8 acts directly at HIF1A target genes, we performed ChIP-seq experiments in HCT116 cells under normoxic and hypoxic conditions. ChIP-seq for CDK8 versus Input under normoxia and 24hrs hypoxia (1% O2).
Project description:Subtypes of innate lymphoid cells (ILC), defined by effector function and transcription factor expression, have recently been identified. In the adult, ILC derive from common lymphoid progenitors in bone marrow, although transcriptional regulation of the developmental pathways involved remains poorly defined. TOX is required for development of lymphoid tissue inducer cells, a type of ILC3 required for lymph node organogenesis, and NK cells, a type of ILC1. We show here that production of multiple ILC lineages requires TOX, as a result of TOX-dependent development of common ILC progenitors. Comparative transcriptome analysis demonstrated failure to induce various aspects of the ILC gene program in the absence of TOX, implicating this nuclear factor as a key early determinant of ILC lineage specification. TOX KO vs. wild tyype
Project description:The neuromuscular junction (NMJ) is a specialized tripartite synapse composed of the motor axon terminal, covered by perisynaptic Schwann cells (PSCs), and the muscle fibre, separated by a basal lamina. It is exposed to different kind of injures such as mechanical traumas, pathogens including neurotoxins, and neuromuscular diseases such as amyotrophic lateral sclerosis and immune-mediated disorders, and has retained throughout vertebrate evolution an intrinsic ability for repair and regeneration, at variance from central synapses1. Following peripheral nerve injury, an intense but poorly defined crosstalk takes place at the NMJ among its components, functional to nerve terminal regeneration. To identify crucial factors released by PSCs and the muscle to induce nerve regrowth, we performed a transcriptome analysis of the NMJ at different time points after injection of -latrotoxin, a presynaptic neurotoxin isolated from the venom of the black widow spider. This toxin is a simple and controlled method to induce an acute, localized and reversible nerve terminal degeneration not blurred by inflammation, and can help to identify molecules involved in the intra- and inter-cellular signalling governing NMJ regeneration.
Project description:Identifying miRNA-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical seed base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic (this dataset). Within these targets, mostly non-canonical MREs were identified by sequencing RNase-resistant fragments.
Project description:To define the contribution of CDK8 versus CDK19 to gene expression control, we performed a series of microarray assays for cells where each kinase was stably knocked down. Toward this end, we subjected HCT116 cells to three different stress stimuli: 5-fluorouracil (5FU), glucose deprivation, and hypoxia. We found that CDK8, but not CDK19, functions as a widespread coactivator of HIF1A target genes in hypoxia. Total RNA from HCT116 cells harvested using an RNeasy kit (Qiagen) was used for gene expression analysis on Affymetrix HuGene 1.0 ST arrays following the manufacturer’s instructions. Differential gene expression was determined with Partek software using one-way ANOVA.
Project description:microRNAs (miRNAs) accomplish a remarkable variety of biological functions. Their expression is tightly controlled, and the final production of a miRNA is dependent on the cooperation of multiple mechanisms and their net effect. Here we show that miR-124-1 is transcriptionally activated during erythroid differentiation by GATA-1, however its post-transcriptional processing is attenuated. We found that QKI5 binds to a distal QKI response element (QRE) embedded in the primary transcript of miR-124-1 (pri-124-1) and modulates Microprocessor function by direct association with DGCR8. Strikingly, Microprocessor recruitment to pri-124-1 is disrupted upon RNAi-mediated depletion of QKI5, consistent with the decrease in mature miR-124. Moreover, addition of QKI5 increases the conversion efficiency of pri-124-1 in cell-free extracts. For erythropoiesis, the decreased QKI5 leads to attenuated Microprocessor-mediated processing of pri-124-1, which confers the exquisite miRNA abundance necessary for development. This regulation also gives rise to a unique miRNA signature required for normal erythropoiesis. Thus, this QKI5-regulated miRNA processing may represent a common paradigm for erythroid development, and specifically, it may serve as a post-transcriptional fault security to prevent misexpression of certain miRNAs, that is essential for the establishment of particular gene expression patterns during development. Two samples are analyzed: K562 cells transduced with GFP lentivirus; and K562 cells transduced with QKI5-overexpressing lentivirus.
Project description:The aim of this work was to elucidate the role of LKB1/STK11 and PARD3 in glioblastoma multiforme cells. For this we silenced the expression of these proteins using specific siRNA in two different patient-derived glioblastoma stem cells, U3031MG and U3034MG, then we analysed how the knock-down of these genes affects gene expression using Ion torrent AmpliseqTM.
Project description:TGFB2-AS1 is a long non-coding RNA which is induced by ΤGFβ signaling. In order to assess the importance of TGFB2-AS1 on the regulation of gene expression, we performed an AmpliSeq transcriptomic array in human keratinocytes (HaCaT), which stably over-express TGFB2-AS1 or control pcDNA3 empty vector. In addition, cells were stimulated with TGFβ1 for 24 hours, in order to observe the effects of TGFB2-AS1 on gene expression, downstream of TGFβ signaling. RNA from the following four conditions was used in this experiment: 1) pcDNA3, 2) pcDNA3+TGFβ1, 3) pcDNA3-TGFB2-AS1, 4) pcDNA3-TGFB2-AS1+TGFβ1. Biological triplicates were used per condition.