Reporter Gene Silencing in Targeted Mouse Mutants is Associated with Promoter CpG Island Methylation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE70317 | GEO | 2015/07/28
SECONDARY ACCESSION(S): PRJNA288306
REPOSITORIES: GEO
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Project description:We report the generation and analysis of high-throughput DNA methylation profiles at nucleotide resolution in a subset of targeted gene trap mouse mutants. Using high-throughput sequencing of bisulfite treated DNA, we generated DNA methylation percentage for CpG islands, and LacZ (reporter) gene in mice with the apparent silencing of the targeted gene promoter reflected by reduced reporter mRNA level. These results were contrasted with findings for a set of mutants with no silencing or CpG methylation following targeted mutagenesis using the same gene trap vector. Our findings supports the hypothesis that presence of the exogenous DNA in the targeting vector may influence the expression of genes in close proximity or may lead to promoter silencing of the target where the promoter is marked by CpG methylation. Examination of CpG methylation profiles in Knock-out and wild type mice We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. CpG Islands (samples labeled as CpG) and trans gene (samples labeled as LacZ) were amplified after Bisulfite treatment. Please note that the same gDNA was used to amplify CpG Island (Gene_CpG_KO ) and LacZ (Gene_LacZ_KO) reporter for the same gene. PCR product of amplification was gel separated, isolated and pooled. After libraries were prepared and sequenced, the alignment was performed. CpG island and LacZ alignments were done separately resulting in three different Processed Data files per gene investigated: Gene_CpG_KO, Gene_LacZ_KO and Gene_CpG_WT. LacZ reference is included in the submission, but is also available for download from KOMP Phenotype website (www.kompphenotype.org). Also please note that the libraries were prepared using Illumina TruSeq RNA Sample Prep Kit starting from adapter binding step as samples were double stranded Bisulfite treated DNA amplicons. So the library preparation was done as with RNASeq, but samples investigated were bisulfite treated.
2015-07-28 | E-GEOD-70317 | biostudies-arrayexpress
Project description:Reporter Gene Silencing in Targeted Mouse Mutants is Associated with Promoter CpG Island Methylation
| PRJNA288306 | ENA
Project description:In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes, however the molecular mechanisms of this specificity remain unclear. Here we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in vivo, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long term epigenetic silencing during mammalian development.
2021-04-22 | PXD023974 | Pride
Project description:The enhancer/promoter of the vitellogenin II (VTG) gene has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity- and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE, but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo, but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltranferase Eco72IM was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.
2020-05-27 | PXD015594 | Pride
Project description:Effect of how CpG density on the promoter affects the gene silencing and DNA methylation rate of a promoter
2023-03-01 | GSE224403 | GEO
Project description:Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4 (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin, and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.
2015-09-30 | GSE73586 | GEO
Project description:DNA methylation is a chromatin modification that is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation have been identified, relatively little is understood about how DNA methylation influences gene expression. Using complementary genetic and biochemical approaches we identified a protein complex that antagonizes the transcriptional gene silencing of two LUCIFERASE (LUC) reporters in a manner that requires DNA methylation. At its core, this complex contains LOW IN LUCIFERASE EXPRESSION (LIL), an α-crystallin domain protein, and METHYL-CpG-BINDING DOMAIN 7 (MBD7), a protein previously associated with DNA methylation. At the LUC reporters, loss of MBD7 or LIL resulted in decreased LUC expression concomitant with modest, but reproducible increases in DNA methylation that can be phenocopied by DNA demethylase mutants. These findings are consistent with other reports and reveal a genetic connection between MBD7, LIL and DNA demethylation. However, we found that the hyper-methylation and gene expression phenotypes at a LUC reporter can be genetically uncoupled, demonstrating that changes in DNA methylation alone are not sufficient to silence LUC expression, and suggesting a role for the MBD7-LIL complex downstream of DNA methylation. Consistent with this hypothesis, our more extensive analysis of DNA methylation in mbd7 and lil mutants revealed only a small number of hyper-methylated loci genome wide. Furthermore, these loci displayed minimal overlap with demethylase targets, suggesting that, in general, the DNA demethylation machinery does not function in a manner dependent on the MBD7-LIL complex. Taken together, our findings place the MBD7-LIL complex amongst a small number of factors that regulate gene expression without causing significant changes in DNA methylation. This complex, however, is unique in that it functions to suppress, rather than enforce the silencing effects of DNA methylation, enabling gene expression of several transgene reporters despite high levels of promoter methylation.
2017-04-26 | GSE83355 | GEO
Project description:DNA methylation is a chromatin modification that is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation have been identified, relatively little is understood about how DNA methylation influences gene expression. Using complementary genetic and biochemical approaches we identified a protein complex that antagonizes the transcriptional gene silencing of two LUCIFERASE (LUC) reporters in a manner that requires DNA methylation. At its core, this complex contains LOW IN LUCIFERASE EXPRESSION (LIL), an α-crystallin domain protein, and METHYL-CpG-BINDING DOMAIN 7 (MBD7), a protein previously associated with DNA methylation. At the LUC reporters, loss of MBD7 or LIL resulted in decreased LUC expression concomitant with modest, but reproducible increases in DNA methylation that can be phenocopied by DNA demethylase mutants. These findings are consistent with other reports and reveal a genetic connection between MBD7, LIL and DNA demethylation. However, we found that the hyper-methylation and gene expression phenotypes at a LUC reporter can be genetically uncoupled, demonstrating that changes in DNA methylation alone are not sufficient to silence LUC expression, and suggesting a role for the MBD7-LIL complex downstream of DNA methylation. Consistent with this hypothesis, our more extensive analysis of DNA methylation in mbd7 and lil mutants revealed only a small number of hyper-methylated loci genome wide. Furthermore, these loci displayed minimal overlap with demethylase targets, suggesting that, in general, the DNA demethylation machinery does not function in a manner dependent on the MBD7-LIL complex. Taken together, our findings place the MBD7-LIL complex amongst a small number of factors that regulate gene expression without causing significant changes in DNA methylation. This complex, however, is unique in that it functions to suppress, rather than enforce the silencing effects of DNA methylation, enabling gene expression of several transgene reporters despite high levels of promoter methylation.
2017-05-05 | GSE59639 | GEO
Project description:DNA methylation is a chromatin modification that is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation have been identified, relatively little is understood about how DNA methylation influences gene expression. Using complementary genetic and biochemical approaches we identified a protein complex that antagonizes the transcriptional gene silencing of two LUCIFERASE (LUC) reporters in a manner that requires DNA methylation. At its core, this complex contains LOW IN LUCIFERASE EXPRESSION (LIL), an α-crystallin domain protein, and METHYL-CpG-BINDING DOMAIN 7 (MBD7), a protein previously associated with DNA methylation. At the LUC reporters, loss of MBD7 or LIL resulted in decreased LUC expression concomitant with modest, but reproducible increases in DNA methylation that can be phenocopied by DNA demethylase mutants. These findings are consistent with other reports and reveal a genetic connection between MBD7, LIL and DNA demethylation. However, we found that the hyper-methylation and gene expression phenotypes at a LUC reporter can be genetically uncoupled, demonstrating that changes in DNA methylation alone are not sufficient to silence LUC expression, and suggesting a role for the MBD7-LIL complex downstream of DNA methylation. Consistent with this hypothesis, our more extensive analysis of DNA methylation in mbd7 and lil mutants revealed only a small number of hyper-methylated loci genome wide. Furthermore, these loci displayed minimal overlap with demethylase targets, suggesting that, in general, the DNA demethylation machinery does not function in a manner dependent on the MBD7-LIL complex. Taken together, our findings place the MBD7-LIL complex amongst a small number of factors that regulate gene expression without causing significant changes in DNA methylation. This complex, however, is unique in that it functions to suppress, rather than enforce the silencing effects of DNA methylation, enabling gene expression of several transgene reporters despite high levels of promoter methylation.
2017-05-05 | GSE83557 | GEO
Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SM-NM-^T35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SM-NM-^T35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SM-NM-^T35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter. Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant
2013-05-30 | E-GEOD-47453 | biostudies-arrayexpress