Dicer promotes genome stability via the bromodomain transcriptional co-activator BRD4
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE172282 | GEO | 2021/11/13
REPOSITORIES: GEO
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Project description:The RNase III enzyme dicer is essential for the processing of microRNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability and translation in a wide range of organisms. To provide a model system to explore the role of dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional dicer allele. Deletion of dicer at an early stage of T cell development compromised the survival of αβ lineage cells, while the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of cytosine DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin) and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one-third of mammalian mRNAs are putative miRNA targets, dicer appears to be dispensable for CD4/8 lineage commitment, a process where epigenetic regulation of lineage choice has been well documented. Thus, although dicer appears critical for the development of the early embryo, it may have limited impact on the implementation of lineage-specific gene expression programs.
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Project description:The RNase III enzyme dicer is essential for the processing of microRNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability and translation in a wide range of organisms. To provide a model system to explore the role of dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional dicer allele. Deletion of dicer at an early stage of T cell development compromised the survival of αβ lineage cells, while the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of cytosine DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin) and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one-third of mammalian mRNAs are putative miRNA targets, dicer appears to be dispensable for CD4/8 lineage commitment, a process where epigenetic regulation of lineage choice has been well documented. Thus, although dicer appears critical for the development of the early embryo, it may have limited impact on the implementation of lineage-specific gene expression programs. LckCre was used to delete a floxed Dicer1 allele from developing thymocytes (Cobb BS, Nesterova TB, Thompson E, Hertweck A, O'Connor E, Godwin J, Wilson CB, Brockdorff N, Fisher AG, Smale ST and Merkenschlager M. T cell lineage choice and differentiation in the absence of the RNAse III enzyme dicer. J. Exp. Med. 2005 201: 1367-1373). Thymocytes were stained for CD4 and CD8, and double-positive (DP) thymocytes were sorted by flow cytometry. Three biological replicates of Dicer lox/lox control samples and LckCre Dicer lox/lox KO samples were analysed.
2015-01-28 | E-GEOD-57511 | biostudies-arrayexpress
Project description:Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in chromatin-dependent gene silencing and maintenance of genome stability by repressing repetitive DNA elements and recruiting downstream factors essential for faithful chromosome segregation. Fundamental principles of these processes have been elucidated in the fission yeast Schizosaccharomyces pombe (S. pombe), in which Clr4, the homologue of mammalian SUV39H, is the sole H3K9 methyltransferase. Although H3K9 methylation has been intensely studied in mitotic cells, occurrence and regulation during sexual differentiation remain largely unknown. Whether distinct H3 methylation states are relevant for gametogenesis is also not known. Here, using diploid S. pombe cells, we map H3K9 methylation genome-wide during the meiotic program and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells exit mitosis and commit to the meiotic life cycle. Cells lacking the ability to tri-methylate H3K9 exhibit severe meiotic chromosome segregation defects, which does not occur in mitotic cells. Artificially increasing the affinity of the fission yeast heterochromatin protein 1 (HP1) homologue Swi6 towards H3K9me2 rescues chromosome segregation in the absence of H3K9me3. Thus, the H3K9me2 to H3K9me3 switch during early meiosis serves to retain Swi6 at centromeres, which is necessary for cohesin recruitment and kinetochore assembly. Finally, we show that the H3K9 methylation switch is regulated by differential phosphorylation of Clr4 by the cyclin-dependent kinase (CDK) Cdk1. Our results reveal how a highly conserved master regulator of the cell cycle regulates the specificity of the H3K9 methyltransferase and thereby controls the ratio of H3K9me2 to H3K9me3 to prevent unwanted gene silencing and to ensure faithful meiotic chromosome segregation. High degree of conservation of the proteins involved in our study and CDK-dependent phosphorylation of the mammalian SUV39H1 homologue suggest broad conservation of the mechanisms described here.
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Project description:Heterochromatin plays a key role in gene repression, maintaining genome integrity and chromosome segregation. Fission yeast, Schizosaccharomyces pombe, utilizes conserved components to direct heterochromatin formation using siRNA generated by RNA interference (RNAi) to guide a histone H3 lysine 9 methyltransferase to cognate chromatin. To identify compounds that inhibit heterochromatin formation, an in vivo phenotypic screen for loss of silencing was performed. A tester strain harbouring a silent dominant selectable kanMX reporter gene within fission yeast centromeric heterochromatin was used to screen a diverse library of chemicals. HMS-I1 and HMS-I2 were identified as compounds that reproducibly increased G418 resistance due to loss of kanMX silencing, and decreased the level of repressive H3K9 methylation on centromeric repeats. The pattern of changes induced by HMS-I1 and HMS-I2 were consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic interactions and expression profiling indicated that both HMS-I1 and HMS-I2 affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). Exposure to HMS-I1 was also found to alleviate silencing of reporter genes in an Arabidopsis transgenic plant line and a mouse cell line. HMS-I2 also disrupted reporter gene silencing in Arabidopsis. In vitro assays indicate that HMS-I1 impairs the activity of human HDAC6 and HDAC10. As HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities they represent potentially novel and valuable reagents for experimental and therapeutic purposes. Our findings highlight the use of in vivo chemical screening conducted in fission yeast to identify compounds that disrupt heterochromatin across plant, fungi and animal kingdoms.
2014-12-17 | GSE57207 | GEO
Project description:Heterochromatin plays a key role in gene repression, maintaining genome integrity and chromosome segregation. Fission yeast, Schizosaccharomyces pombe, utilizes conserved components to direct heterochromatin formation using siRNA generated by RNA interference (RNAi) to guide a histone H3 lysine 9 methyltransferase to cognate chromatin. To identify compounds that inhibit heterochromatin formation, an in vivo phenotypic screen for loss of silencing was performed. A tester strain harbouring a silent dominant selectable kanMX reporter gene within fission yeast centromeric heterochromatin was used to screen a diverse library of chemicals. HMS-I1 and HMS-I2 were identified as compounds that reproducibly increased G418 resistance due to loss of kanMX silencing, and decreased the level of repressive H3K9 methylation on centromeric repeats. The pattern of changes induced by HMS-I1 and HMS-I2 were consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic interactions and expression profiling indicated that both HMS-I1 and HMS-I2 affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). Exposure to HMS-I1 was also found to alleviate silencing of reporter genes in an Arabidopsis transgenic plant line and a mouse cell line. HMS-I2 also disrupted reporter gene silencing in Arabidopsis. In vitro assays indicate that HMS-I1 impairs the activity of human HDAC6 and HDAC10. As HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities they represent potentially novel and valuable reagents for experimental and therapeutic purposes. Our findings highlight the use of in vivo chemical screening conducted in fission yeast to identify compounds that disrupt heterochromatin across plant, fungi and animal kingdoms. 16 RNA samples: 2 replicates of WT untreated (vehicle DMSO) (KE1-A, KE2-A), HMS-I1 treated (KE1-B, KE2-B), HMS-I2 treated (KE1-C, KE2-C), and 2 additional replicates of WT (KE05_wt_1, KE10_wt_2), 4 replicates of clr2M-bM-^HM-^F (KE01_2118_1, KE06_2118_2, KE03_0080_1, KE08_0080_2) and mit1M-bM-^HM-^F (KE02_1295_1, KE07_1295_2, KE04_1278_1, KE09_1278_2).
2014-12-17 | E-GEOD-57207 | biostudies-arrayexpress