Project description:DRM complex mutant lin-54 vs. H3K36 methyltransferase mutant mes-4 vs. lin-54; mes-4 double mutant vs. wild type C.elegans germline
Project description:Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 marks genes expressed in the germline with methylated Lys36 on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosomes. The DRM complex, which includes E2F/DP and Retinoblastoma homologs, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 or the DRM subunit lin-54 oppositely skew target transcript levels and cause sterility; a double mutant restores near wild-type transcript levels and germ cell development. Together, 'yin-yang' regulation by MES-4 and DRM ensures transcript levels appropriate for germ cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage. We compared germline gene expression profile of wild-type N2 C. elegans, lin-54(n3423) M+Z- mutant, mes-4(ok2326) M+Z- mutant, and lin-54(n3423);mes-4(ok2326) M+Z-mutant grown at 20C. 50~70 Germlines were dissected from young adults (24hours after L4 stage), and region from the tip until late pachytene stage of meiosis were collected. Three biological replicates for each strain were performed.
Project description:Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 marks genes expressed in the germline with methylated Lys36 on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosomes. The DRM complex, which includes E2F/DP and Retinoblastoma homologs, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 or the DRM subunit lin-54 oppositely skew target transcript levels and cause sterility; a double mutant restores near wild-type transcript levels and germ cell development. Together, “yin-yang” regulation by MES-4 and DRM ensures transcript levels appropriate for germ cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage.
Project description:DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we perform microarray expression profiling analysis of lin-54, a DNA-binding member of the DRM complex. To identify genes regulated by LIN-54 in soma and germline, we analyzed wild-type and lin-54 mutant C. elegans embryos and isolated germlines. We chose embryos because they consist primarily of somatic cells, at a developmental stage with both active cell divisions and dynamic developmental gene expression programs. Since lin-54 null animals are sterile, embryos were obtained from a strain carrying the partial loss-of-function allele lin-54(n2990). Germlines were dissected from lin-54(n3423) null adults that lack detectable transcript and protein. The results revealed conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, genomics and cytological analyses show that DRM binding, a DRM binding motif, and LIN-54-regulated genes are all autosome-enriched. One paradoxical exception occurs the germline, where DRM binds autosomes but genes down-regulated in DRM mutants are enriched on X chromosomes.
Project description:DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we analyze genome-wide binding and function of the C. elegans DRM subunit LIN-54. We demonstrate that LIN-54 DNA-binding activity is required for the DRM complex to efficiently bind and regulate target genes containing adjacent putative E2F/DP and LIN-54 binding sites. We show that LIN-54 binds to the promoters of genes involved in cell division, development, and reproduction, and acts differently in the germline versus the soma. The E2F/DP-LIN-54 binding motif, individual target genes, and overall DRM function are conserved among worms, flies, and humans. Despite this conservation, we discovered one striking feature of C. elegans DRM not shared in flies or humans: it is depleted from X chromosomes. We show that DRM binding, the E2F-LIN-54 hybrid motif, and LIN-54-regulated genes are all autosome-enriched. Chromatin-immunoprecipitation of mixed staged wild-type C.elegans (N2, Bristol strain) was performed using non-commercial anti-LIN-54 antibody raised in guinea pig (Harrison et at. 2006).
Project description:DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we perform microarray expression profiling analysis of lin-54, a DNA-binding member of the DRM complex. To identify genes regulated by LIN-54 in soma and germline, we analyzed wild-type and lin-54 mutant C. elegans embryos and isolated germlines. We chose embryos because they consist primarily of somatic cells, at a developmental stage with both active cell divisions and dynamic developmental gene expression programs. Since lin-54 null animals are sterile, embryos were obtained from a strain carrying the partial loss-of-function allele lin-54(n2990). Germlines were dissected from lin-54(n3423) null adults that lack detectable transcript and protein. The results revealed conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, genomics and cytological analyses show that DRM binding, a DRM binding motif, and LIN-54-regulated genes are all autosome-enriched. One paradoxical exception occurs the germline, where DRM binds autosomes but genes down-regulated in DRM mutants are enriched on X chromosomes. We compared embryonic or germline gene expression profile of lin-54 mutants with that of wild-type N2 C. elegans. Embryos were obtained from a strain carrying the partial loss-of-function allele lin-54(n2990) grown at 25C for one generation. Germlines were isolated from lin-54(n3423) null adults that lack detectable lin-54 transcript and protein. We isolated the germline region from the tip until late pachytene stage of meiosis, because nuclei in this region are morphologically similar between wild-type and mutant and are all undergoing X chromosome silencing. 3 biological replicates of each genotype/tissue were examined.
Project description:DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we analyze genome-wide binding and function of the C. elegans DRM subunit LIN-54. We demonstrate that LIN-54 DNA-binding activity is required for the DRM complex to efficiently bind and regulate target genes containing adjacent putative E2F/DP and LIN-54 binding sites. We show that LIN-54 binds to the promoters of genes involved in cell division, development, and reproduction, and acts differently in the germline versus the soma. The E2F/DP-LIN-54 binding motif, individual target genes, and overall DRM function are conserved among worms, flies, and humans. Despite this conservation, we discovered one striking feature of C. elegans DRM not shared in flies or humans: it is depleted from X chromosomes. We show that DRM binding, the E2F-LIN-54 hybrid motif, and LIN-54-regulated genes are all autosome-enriched.
Project description:Maternally synthesized products play critical roles in development of offspring. A premier example is the C. elegans H3K36 methyltransferase MES-4, which is essential for germline survival and development in offspring. How maternal MES-4 protects the germline is not well understood, but its role in H3K36 methylation hinted that it may regulate gene expression in Primordial Germ Cells (PGCs). We tested this hypothesis by profiling transcripts from nascent germlines (PGCs and their descendants) dissected from wild-type and mes-4 mutant (lacking maternal and zygotic MES-4) larvae. mes-4 nascent germlines displayed down-regulation of some germline genes, up-regulation of some somatic genes, and dramatic up-regulation of hundreds of genes on the X chromosome. We demonstrated that up-regulation of 1 or more genes on the X is the cause of germline death by generating and analyzing mes-4 mutants that inherited different endowments of X chromosome(s). Intriguingly, removal of the THAP transcription factor LIN-15B from mes-4 mutants reduced X mis-expression and prevented germline death. lin-15B is X-linked and mis-expressed in mes-4 PGCs, identifying it as a critical target for MES-4 repression. The above findings extend to the H3K27 methyltransferase MES-2/3/6, the C. elegans version of Polycomb Repressive Complex 2. We propose that maternal MES-4 and PRC2 cooperate to protect germline survival by preventing synthesis of germline-toxic products encoded by genes on the X chromosome, including the key transcription factor LIN-15B.
Project description:This SuperSeries is composed of the following subset Series: GSE28494: Germline and embryo gene expression of wild-type vs. mutants in lin-54, a component of the C. elegans DRM complex GSE28852: Chromosome-biased binding and gene reguation by the C. elegans DRM complex [ChIP-chip] Refer to individual Series
Project description:Transcriptional profiling of C. elegans first larval stage whole animals comparing lin-35(n745) mutants and lin-35(n745) treated with mes-4(RNAi) at 26 degrees. One-condition experiment, mutant (lin-35) vs. mutant plus RNAi (lin-35, mes-4RNAi). Biological replicates 4 mutant, 4 mutant plus RNAi harvested indepedently. One lin-35 replicate and one lin-35, mes-4(RNAi) replicate per array.