MRNA-seq for HCT116 Parental and ARID1A knockdown cells, mRNA-seq for DLD1 Parental, ARID1A knockdown and ARID1A knockout cells, mRNA-seq for COLO320DM Parental and ARID1A knockdown and ARID1A knockout cells
Ontology highlight
ABSTRACT: The study identifies genes that are regulated by the loss of the chromatin remodeller subunit ARID1A in colorectal cancer cell lines. This gene is frequently mutated in colorectal cancer.
Project description:Glioblastoma (GBM) is an aggressive and incurable brain tumor in nearly all instances, whose disease progression is driven in part by the glioma stem cell (GSC) subpopulation. Here, we explored the effects of Schlafen family member 11 (SLFN11) in the molecular, cellular and tumor biology of GBM.
Project description:Lambda interferons IFNL1-3 mediate antiviral immunity by inducing interferon sensitive genes (ISGs) in epithelial tissues. Contrarily, a variant creating the functional gene IFNL4 is associated with impaired clearance of hepatitis C virus (HCV) despite of higher liver expression of ISGs in untreated HCV patients. We aimed to explore IFNL4 signaling mechanism by comparing expression profiles from human hepatic cell line clones with genetic modifications influencing the ISG signaling pathway (IFNLR1/IL10R2 knockouts, IFNL4/IFNL3 expression stimulation by transfection).
Project description:The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, and in particular in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, that was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.
Project description:Mature mRNAs undergo quality control during translation that may lead to RNA degradation by triggering the nonsense mediated decay (NMD) pathway. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction or an intron in the 3'UTR activates NMD. However, many of the features that lead to the activation of this pathway are unclear. UPF1, an RNA helicase, is the core NMD factor. UPF1 forms a multi-protein complex by recruiting a series of factors and other protein complexes in a process that depends on the UPF1 phosphorylation-dephosphorylation cycle. Among the factors recruited by UPF1, SMG5-SMG7 and SMG6 have critical importance in executing NMD. The SMG5-SMG7 heterodimer induces the exonucleolytic degradation of the mRNA, which depends on the recruitment of deadenylation factors. SMG6 has endonucleolytic activity and cleaves the targeted transcript close to the stop codon. The redundancy between the exonucleolytic and endonucleolytic paths to achieve degradation during NMD has been previously reported in the literature. To investigate the apparent redundancy between SMG5-SMG7 and SMG6 activity and to further understand the features that lead to the activation of NMD, we have generated two clones of SMG7 knockout human cells using CRISPR-Cas9. We generated mRNA-Sequencing data for control and both SMG7 KO clones with additional siRNA-mediated knockdown of Luciferase (Luc) as control, SMG5 or SMG6.
Project description:The goal of the experiment was to identify which genes are differentially expressed between the unedited and SPI1-edited populations. The RS4:11 cell line was edited both mono and biallelicaly via electroporation with guides and Cas9. Following editing, RNA from unedited (SPI1 +/+), mono (SPI1 +/-) and biallellicaly edited (SPI1 -/-) cells were extracted through the Direct-zol RNA Microprep kit. cDNA libraries for sequencing were then prepared using the TruSeq Stranded mRNA Library Prep Kit and the IDT for Illumina-TruSeq RNA UD Indexes (Illumina). Samples were then sequenced on the Illumina NovaSeq platform.
Project description:The synthetic lethal relationship between MYC and SAE2 has been previously described in FC-MYC cells using RNAi (Littler et al., 2019). Building on this work, we conducted transcriptomic analysis on MYC-low and MYC-high cells following inhibition of SUMOylation with the SAE1/2 inhibitor, ML-792 (SAEi). FC-MYC cells were grown for twenty-four hours in the absence (MYC-low) and presence (MYC-high) of 500ng/mL tetracycline and treated with either DMSO (vehicle control) or 25 nM SAEi for 24 hours. Three biological replicates were performed for RNAseq analysis, with additional subsets of cells reserved for complementary analyses, including DNA content profiling by flow cytometry and MYC protein expression analysis through western blotting. DNA content analysis showed that SAEi treatment had minimal affect on MYC-low cells but induced polyploidy and apoptosis in MYC-high cells. Western blotting confirmed MYC expression at the protein level as expected. This dataset provides transcriptomic profiles of MYC-low and MYC-high cells under SUMOylation inhibition, offering insights into the differential impact of SAEi on different MYC backgrounds. The dataset also includes analysis of parental RKO cells exposed to the SAEi.
Project description:mRNA-seq for HCT116 Parental and ARID1A knockdown cells, mRNA-seq for DLD1 Parental, ARID1A knockdown and ARID1A knockout cells, mRNA-seq for COLO320DM Parental and ARID1A knockdown and ARID1A knockout cells
Project description:ALKBH4 is a versatile demethylase that catalyzes the removal of methyl group from monomethylated lysine-84 on actin and N6-methyladenine in DNA. We conducted a quantitative proteomic experiment to reveal the differential expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results revealed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes in ALKBH4-deficient cells. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein. Yeast two-hybrid screening identified several proteins that are associated with chromatin and/or involved with transcription, as interaction partners of human ALKBH4, though global gene expression was only marginally changed upon overexpression of ALKBH4. We reason that a comprehensive assessment of how genetic depletion of ALKBH4 modulates protein expression at the global proteome scale may provide new insights into the biological function of ALKBH4 protein. In the present study, we conducted such an analysis and we found that CRISPR-mediated ablation of ALKBH4 led to substantial changes in expression of a large number of proteins, including the markedly diminished expression of GSTP1 and HSPB1 proteins. Mechanistically, these changes arise from elevated expression of DNMT1 and the ensuing epigenetic silencing of these two genes.
Project description:To address how CSF3R mutations affect hematopoiesis in a severe congenital neutropenia (SCN) background we used SCN patient, or control, derived induced pluripotent stem cells (iPSC). HPCs were generated from CRISPR-Cas9 genome edited CSF3R-d715 SCN or control-derived iPSCs with the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). Floating cells were harvested at Day 12 of the hematopoietic induction protocol. CD34+CD45+ HPCs were FACS sorted in TRIzol and RNA was isolated according to the manufacturer’s protocol. SMARTer Ultra Low Input RNA kit for sequencing (Clontech, v4 Cat# 634891) was used to generate cDNA. Sequencing libraries were generated using TruSeq Nano DNA Sample Preparation kits (Illumina, Cat# 20015964), according to the low sample protocol and paired-end sequenced on a HiSeq 2500 or Novaseq 6000 (both Illumina).