Chromosome-biased binding and gene regulation by the C. elegans DRM complex
ABSTRACT: 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: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:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
Project description:Most aging hypotheses revolve around the accumulation of some sort of damage resulting in gradual physiological decline and ultimately death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend lifespan. However, lowering translational efficiency extends rather than shortens lifespan in C. elegans. We studied turnover of individual proteins in the conserved Insulin/Insulin-like Growth Factor (IGF-1) receptor mutant daf-2 by combining Stable Isotope Labeling by Nitrogen-15 in Caenorhabditis elegans and LC-MS/MS. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, while others remained unchanged, signifying that longevity is not supported by high protein turnover. This slow-down of protein turnover was most prominent for components of the translation machinery and mitochondria. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged in daf-2. The slow-down of protein dynamics and decreased abundance of the translational machinery may point at the importance of anabolic attenuation in lifespan extension as suggested by the hyperfunction theory.
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 describe new aspects of DRM binding and function revealed through genome-wide analyses of the Caenorhabditis elegans DRM subunit LIN-54. We show that LIN-54 DNA-binding activity recruits DRM to promoters enriched for adjacent putative E2F/DP and LIN-54 binding sites, suggesting that these two DNA-binding moieties together direct DRM to its target genes. Chromatin immunoprecipitation and gene expression profiling reveals conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find that LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, C. elegans DRM does not act uniformly throughout the genome: the DRM recruitment motif, DRM binding, and DRM-regulated embryonic genes are all under-represented on the X chromosome. However, germline genes down-regulated in lin-54 mutants are over-represented on the X chromosome. We discuss models for how loss of autosome-bound DRM may enhance germline X chromosome silencing. We propose that autosome-enriched binding of DRM arose in C. elegans as a consequence of germline X chromosome silencing and the evolutionary redistribution of germline-expressed and essential target genes to autosomes. Sex chromosome gene regulation may thus have profound evolutionary effects on genome organization and transcriptional regulatory networks.
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:Protein turnover rates severely decline in aging organisms, including C. elegans. However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, while only few proteins show increased turnover. Decrease in protein turnover was consistent for the minority of functional protein subsets, including tubulins and vitellogenins, while for most functionally related protein pools randomly diverging turnover patterns were observed with age. Our data suggests severe dysregulation of protein turnover of the translation machinery, whereas protein turnover of UPS and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.
Project description:ChIP-chip of HPL-2 in N2 C. elegans early embryo EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: Early Embryo; Genotype: wild type; Sex: population predominantly Hermaphrodites perhaps with some Males; NUMBER OF REPLICATES: 3; EXPERIMENTAL FACTORS: temperature 20
Project description:ChIP-chip of HPL-2 in hpl-2 C. elegans early embryo EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: PFR40; Developmental Stage: Early Embryo; Genotype: hpl-2(tm1489); Sex: population predominantly Hermaphrodites perhaps with some Males; NUMBER OF REPLICATES: 1; EXPERIMENTAL FACTORS: temperature 20
Project description:ChIP-chip of HPL-2 in met-2 set-25 C. elegans mixed-stage embryo EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: GW638; Developmental Stage: Mixed-stage Embryo; Genotype: met-2(n4256) set-25(n5021); Sex: population predominantly Hermaphrodites perhaps with some Males; NUMBER OF REPLICATES: 2; EXPERIMENTAL FACTORS: temperature 20
Project description:The Retinoblastoma-like pocket proteins p130 and p107 act as gatekeepers of the cell cycle through their activity within the DREAM (Dp/Rb-like/E2F/MuvB) transcriptional repressor complex. The goal of this study was to address how the pocket protein contributes to DREAM complex assembly and function on chromatin by utilizing a protein null mutant of the only C. elegans pocket protein LIN-35. We performed ChIP-seq of C. elegans DRM subunits in wild-type and lin-35 null late embryos to assess the effect on their chromatin localization following loss of LIN-35. Overall design: Examination of 7 DRM complex subunits (DPL-1, EFL-1, LIN-35, LIN-9, LIN-37, LIN-52, and LIN-54) in lin-35(n745) (a protein null strain) and WT C. elegans late stage embryos to assess effects of loss of LIN-35 on DRM complex chromatin assembly. Study includes reanalysis of GSM1195397 and GSM1195398 (GSE49204) with an additional replicate, generating the following processed data: LIN-37.SDQ3166.N2.rep0.vsInputRep0.SES.bw LIN-37.SDQ3166.N2.IDR.0.01.narrowPeak