IMEF SDHC-loss RNA-seq time course
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103662 | GEO | 2017/11/21
SECONDARY ACCESSION(S): PRJNA403858
REPOSITORIES: GEO
Similar Datasets
Project description:We have developed a tet-inducible immortalized mouse embryonic fibroblast (iMEF) cell culture model of SDH-loss paraganglioma/pheochromocytoma in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). Using this model and an isogenic Sdhc wt control line (R26M2rtTA/+;TetOcre;Sdhcfl/wt), we have characterized the time-course of Sdhc gene rearrangement, protein loss, and succinate accumulation following induction with doxycycline. Using reduced representation bisulfite sequencing (RRBS), we have quantified changes in the genome-wide distribution of cytosine/5’-methyl-cytosine due to succinate accumulation using our Sdhc -/- cell line. Through a time-course experimental design, we have grown R26M2rtTA/+;TetOcre;Sdhcfl/fl (experimental) and R26M2rtTA/+;TetOcre;Sdhcfl/wt (control) iMEFs in standard DMEM media containing 10% FBS and quantified DNA methylation changes in these cells as a function of time after triggering SDHC gene rearrangement via doxycycline exposure.
2017-11-21 | GSE103609 | GEO
Project description:We have developed a tet-inducible SV-40 large T antigen-expressing lentivirus-immortalized mouse embryonic fibroblast (iMEF) cell culture model of mitochondrial electron transport chain (ETC) dysfunction involving complex II (succinate dehydrogenase; SDH) in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). For comparison, we include an isogenic Sdhc wt control line (R26M2rtTA/+;TetOcre;Sdhcfl/wt) that retains one intact copy of Sdhc upon doxycycline exposure. Following doxycycline induction of both cell lines, dilutional subcloning was employed to obtain stable Sdhc -/- (SDHC KO) and Sdhc +/- (Control) cell lines. Sdhc gene rearrangement status for each clonally-derived cell line was confirmed by PCR. Cell lines were grown in standard DMEM containing penicillin/streptomycin antibiotics (0.5 mg/mL), non-essential amino acids (100 micromolar each of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine), sodium pyruvate (1 mM), and HEPES buffer (10 mM) at 21% O2 and 5% CO2. Following cell line derivation and verification of Sdhc genetic status, long-range genomic contacts were profiled using eHi-C.
2020-10-22 | GSE129955 | GEO
Project description:We have developed a tet-inducible SV-40 large T antigen-expressing lentivirus-immortalized mouse embryonic fibroblast (iMEF) cell culture model of mitochondrial electron transport chain (ETC) dysfunction involving complex II (succinate dehydrogenase; SDH) in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). For comparison, we include an isogenic Sdhc wt control line (R26M2rtTA/+;TetOcre;Sdhcfl/wt) that retains one intact copy of Sdhc upon doxycycline exposure. Following doxycycline induction of both cell lines, dilutional subcloning was employed to obtain stable Sdhc -/- (SDHC KO) and Sdhc +/- (Control) cell lines. Sdhc gene rearrangement status for each clonally-derived cell line was confirmed by PCR. Cell lines were grown in standard DMEM containing penicillin/streptomycin antibiotics (0.5 mg/mL), non-essential amino acids (100 micromolar each of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine), sodium pyruvate (1 mM), and HEPES buffer (10 mM) at 21% O2 and 5% CO2. Following cell line derivation and verification of Sdhc genetic status, chromatin epigenomic marks including H3K4me3, H3K27me2, H3K27ac, CTCF, acetyllysine, propionyllysine, and butyryllysine were characterized by CUT&RUN.
2020-10-22 | GSE129954 | GEO
Project description:We have developed a tet-inducible SV-40 large T antigen-expressing lentivirus-immortalized mouse embryonic fibroblast (iMEF) cell culture model of mitochondrial electron transport chain (ETC) dysfunction involving complex II (succinate dehydrogenase; SDH) in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). For comparison, we include an isogenic Sdhc wt control line (R26M2rtTA/+;TetOcre;Sdhcfl/wt) that retains one intact copy of Sdhc upon doxycycline exposure. Following doxycycline induction of both cell lines, dilutional subcloning was employed to obtain stable Sdhc -/- (SDHC KO) and Sdhc +/- (Control) cell lines. Sdhc gene rearrangement status for each clonally-derived cell line was confirmed by PCR. Cell lines were grown in standard DMEM containing penicillin/streptomycin antibiotics (0.5 mg/mL), non-essential amino acids (100 micromolar each of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine), sodium pyruvate (1 mM), and HEPES buffer (10 mM) at 21% O2 and 5% CO2. Following cell line derivation and verification of Sdhc genetic status, accessible chromatin regions in each cell line were interrogated by ATAC-seq.
2020-10-22 | GSE129953 | GEO
Project description:We have developed a tet-inducible immortalized mouse embryonic fibroblast (iMEF) cell culture system for studying the cross-talk between TCA cycle metabolism and the epigenome, in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). Using this model and an isogenic Sdhc wt control line (R26M2rtTA/+;TetOcre;Sdhcfl/wt), we have characterized differences in H3K4me3, H3K27me3, and chromatin succinylation that occur genome-wide in response to SDHC loss. For these experiments, both cell lines were grown in standard DMEM media containing 10% FBS at 37˚C and 5% CO2. 16 days after induction of Sdhc gene rearrangement by doxycycline induction, chromatin from the respective lines was prepared and patterns of H3K4me3, H3K27me3, and chromatin succinylation were studied via ChIP-seq.
2018-02-15 | GSE104362 | GEO
Project description:We have developed a tet-inducible SV-40 lentivirus-immortalized mouse embryonic fibroblast (iMEF) cell culture model of SDH-loss paraganglioma/pheochromocytoma in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). For comparison, we include an isogenic Sdhc wt control line (R26M2rtTA/+;TetOcre;Sdhcfl/wt) that retains one intact copy of Sdhc upon doxycycline exposure. Following doxycycline induction of both cell lines, dilutional subcloning was employed to obtain stable Sdhc -/- (experimental) and Sdhc +/- (control) cell lines. Sdhc gene rearrangement status for each clonally-derived cell line was confirmed by PCR. Cell lines were grown in standard DMEM containing penicillin/streptomycin antibiotics (0.5 mg/mL), non-essential amino acids (100 micromolar each of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine), sodium pyruvate (1 mM), and HEPES buffer (10 mM) at 21% O2 and 5% CO2. Following cell line derivation and verification of Sdhc genetic status, transcriptomic profiling for each cell line was performed by RNA-seq.
2018-05-10 | GSE114244 | GEO
Project description:We have developed a tet-inducible SV-40 large T antigen-expressing lentivirus-immortalized mouse embryonic fibroblast (iMEF) cell culture model of mitochondrial electron transport chain (ETC) dysfunction involving complex II (succinate dehydrogenase; SDH) in which silencing gene rearrangement of the Sdhc floxed allele is driven by doxycycline-dependent expression of cre-recombinase from a tet-inducible promoter (R26M2rtTA/+;TetOcre;Sdhcfl/fl). Following doxycycline induction, dilutional subcloning was employed to obtain stable Sdhc -/- (SDHC KO) cell lines. Sdhc gene rearrangement status for each clonally-derived cell line was confirmed by PCR. Cell lines were grown in standard DMEM containing penicillin/streptomycin antibiotics (0.5 mg/mL), non-essential amino acids (100 micromolar each of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine), sodium pyruvate (1 mM), and HEPES buffer (10 mM) at 21% O2 and 5% CO2. Following cell line derivation and verification of Sdhc genetic status, cells were treated with histone acetyltransferase inhibitors C646 (10 µM) and MB-3 (100 µM) or vehicle for 3 days. Accessible chromatin regions were subsequently interrogated by ATAC-seq.
2020-10-22 | GSE145754 | GEO
Project description:Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumours affecting a range of body sites and have the highest degree of heritability of any cancer type. SDHA, SDHB, SDHC, SDHD and SDHAF2 (collectively SDHx) code for subunits of succinate dehydrogenase, an enzyme complex in the tricarboxylic cycle that converts succinate to fumarate. Mutations in all SDHx genes play a role in PPGL pathogenesis and completely abolish succinate dehydrogenase enzymatic activity causing intracellular accumulation of oncometabolites which competitively inhibit 2-oxoglutarate-dependent oxygenases. This is a family of enzymes includes TET DNA demethylases and Jumonji C (JmjC) domain-containing histone demethylates. We and others have found that the inhibition of DNA demethylation leads to profound global DNA hypermethylation which influences expression of genes responsible for important clinical characteristics of these tumours but cannot explain the full spectrum of transcriptomic changes. We profiled histone PMTs to complement DNA methylation data and fully characterise the epigenome of these tumours.
2024-01-26 | PXD025689 | Pride
Project description:Pediatric GIST commonly harbors a disabled succinate dehydrogenase complex (SDH), which yields tumors with highly conserved genomes but characteristic epigenomic signatures. Mysteriously, nearly half of such SDH-deficient GIST, including tumors from Carney Triad patients, lack identifiable mutations in SDH component genes and genes required for complex assembly (SDHA, SDHB, SDHC, SDHD, SDHAF, termed SDHx). Genomic sequencing coupled with DNA methylation and transcriptional profiling have exposed SDHC promoter-specific CpG island epimutation and concomitant gene silencing in the majority of SDHx-WT GIST.
2015-01-02 | GSE56670 | GEO
Project description:Pediatric GIST commonly harbors a disabled succinate dehydrogenase complex (SDH), which yields tumors with highly conserved genomes but characteristic epigenomic signatures. Mysteriously, nearly half of such SDH-deficient GIST, including tumors from Carney Triad patients, lack identifiable mutations in SDH component genes and genes required for complex assembly (SDHA, SDHB, SDHC, SDHD, SDHAF, termed SDHx). Genomic sequencing coupled with DNA methylation and transcriptional profiling have exposed SDHC promoter-specific CpG island epimutation and concomitant gene silencing in the majority of SDHx-WT GIST. We performed whole genome expression profiling on 20 FFPE dSDH GIST tumors using Affymetrix U133P2 arrays which contain >54K gene target probesets. Included here were data from 7 SDHx-w.t. and 13 SDHx mutants that passed array QC.
2015-01-02 | E-GEOD-56670 | biostudies-arrayexpress