Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ChIPseq examination of EAP-1 binding across generations of spr-5(by101) mutant worms

ABSTRACT: How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans Histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here we identified a genetic network of chromatin-modifying factors, including the H3K4me1/me2 methyltransferases SET-17 and SET-30, the H3K9me1/me2 methyltransferase MET-2, an H3K9me3 methyltransferase, SET-26, the H3K9me3 demethylase JMJD-2, and an H3K9me reader EAP-1, which regulate the trans-generational flow of epigenetic information. Importantly, genetic ablation of set-17, set-30, jmjd-2, or eap-1 suppresses the progressive transgenerational phenotypes, while loss of SET-26 or MET-2 accelerates the infertility of spr-5 mutant worms. We further show that loss of spr-5 also causes a trans-generational increase in lifespan, which is dependent on these chromatin regulators as well as DAF-36 and DAF-12, which control a germline to soma longevity signaling pathway. These findings suggest that the balance between the euchromatic H3K4 and the heterochromatic H3K9 methylation regulates trans-generational effects on longevity and fertility. EAP-1 binding ChIPseq libraries were prepared from 50 ul of packed young adult worms which were maintained at 16 degrees until the appropriate generation and then shifted to 25 degrees after birth. Two biological repeats were generated for wildtype and generation 20 spr-5(by101) mutant worms and a single repeat for generation 10. There were four input samples and eap-1 null mutant worms were also analyzed.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Yun Zhu

PROVIDER: E-GEOD-52102 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

A histone methylation network regulates transgenerational epigenetic memory in C. elegans.

Greer Eric L EL Beese-Sims Sara E SE Brookes Emily E Spadafora Ruggero R Zhu Yun Y Rothbart Scott B SB Aristizábal-Corrales David D Chen Shuzhen S Badeaux Aimee I AI Jin Qiuye Q Wang Wei W Strahl Brian D BD Colaiácovo Monica P MP Shi Yang Y

Cell reports 20140327 1

How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here, we identified multiple chromatin-modifying factors, including H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase, and an H3K9me reader, which either suppress or accelerate the progressive transge ...[more]

PMID: 24685137

Similar Datasets

Project description:Deep sequencing was used to detect Flock House Virus (FHV)-derived small RNAs (viRNAs) that were inherited over generations. Small RNA libraries were constructed from 4 different samples: (a) FR1gfp-transgenic worms that can mount an RNAi response and should contain viRNAs (positive control); (b) rde-4(-/-) mutants (negative control as no viRNAs are expected to be produced); (c) FR1gfp; rde-4(-/-) worms that are two generations away from their rde-4(+/-) grandparents, i.e. worms that can themselves not produce viRNAs, but may have inherited viRNA from their grandparents; and (d) the F3 progeny of wild-type animals that contained the FR1gfp transgene (and therefore produced viRNAs), but have lost this transgene through outcrossing with non-transgenic wild-type worms. This tests whether the silencing reagent can exist without its template. The Small RNA libraries were constructed using a protocol that enriches for Dicer products which harbor a single phosphate at the 5M-bM-^@M-^Y end of the RNA (Zamore et al., 2000), such as primary siRNAs, and unlike RdRP products that harbor tri-phosphate ends (Parameswaran et al., 2010). This was the protocol of choice because we were interested in identifying viRNAs that are guaranteed to be derived from the original RNAi-competent parents (Alcazar et al., 2008), and rde-4 animals can not produce primary siRNAs (Grishok et al., 2000; Parrish and Fire, 2001). rde-4 animals are, however, not defective in secondary siRNA production (Blanchard et al., 2011), and can therefore continue to amplify secondary siRNAs de novo; such autonomously-produced siRNAs will not be distinguishable from inherited ones. The detection of rare primary siRNAs is important, as even a single M-bM-^@M-^\triggerM-bM-^@M-^] siRNA can induce a full-blown RNAi-response that is not proportional to the primary trigger (Groenenboom et al., 2005).In agreement with the functional assays we detected different viRNAs complementary to several regions of the viral genome in the positive control (FR1gfp), no viRNAs in the negative control (rde-4(-/-)), and a number of viRNAs in the worms that could not generate their own viRNAs and thus inherited these viRNAs from their grandparents (F3 generation FR1gfp; rde-4(-/-))(Table 2; Figure 4). Moreover, we detect viRNAs in the worms in which the FR1gfp transgene had been crossed out, confirming that the viRNA transmit in a template-independent manner. The inherited viRNA matched the two most abundant types of viRNAs detected in the positive control (Figure 4) and these viRNAs were all of the reverse orientation (negative strand, which typically exists in much lower quantities than the positive strand (Felix et al., 2011)); both observations make it highly unlikely that the detected viRNAs merely represent unspecific break-down products of the viral RNA. In regard to the low number of inherited viRNA reads it needs to be considered that our protocol enriches specifically for rare primary viRNA species. Moreover, these viRNA species are derived from a response mounted in RNAi-competent grandparents, and are therefore possibly diluted over the course of several generations. Taken together, our results support the genetic experiments that argued for the existence of trans-acting factors that are transmitted in a non-Mendelian manner to ensuing generations. 4 samples examined. Small RNA libraries generated from: C. elegans animals.

Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ÂºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ÂºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300Âµl beads and 150Âµg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ÂºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).

Dataset Information

ChIPseq examination of EAP-1 binding across generations of spr-5(by101) mutant worms

Publications

A histone methylation network regulates transgenerational epigenetic memory in C. elegans.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure