Abstract
Epigenetic information contributes to proper gene expression and development, and can be transmitted not only through mitotic divisions but also from parents to progeny. We investigated the roles in epigenetic inheritance of MES-4 and MET-1, the two Caenorhabditis elegans enzymes that methylate H3K36 (histone H3 Lys 36). Mass spectrometry analysis confirmed immunostaining results showing that both MES-4 and MET-1 catalyze H3K36me3. In the adult germline, MES-4 is enriched in the distal mitotic zone and MET-1 is enriched in the meiotic pachytene zone. Embryos inherit H3K36me3-marked chromosomes from both the oocyte and sperm, and a maternal load of MES-4 and MET-1. Maternal MES-4 quickly associates with sperm chromosomes; that association requires that the sperm chromosomes bear H3K36me3, suggesting that MES-4 is recruited to chromosomes by preexisting H3K36me3. In embryos that inherit H3K36me3-positive oocyte chromosomes and H3K36me3-negative sperm chromosomes, MES-4 and H3K36me3 are maintained on only a subset of chromosomes until at least the 32-cell stage, likely because MES-4 propagates H3K36me3 on regions of the genome with preexisting H3K36me3. In embryos lacking MES-4, H3K36me3 levels on chromosomes drop precipitously postfertilization. In contrast to the relatively high levels of MES-4 in early-stage embryos, MET-1 levels are low at early stages and start increasing by the ∼26-cell stage, consistent with expression from the zygotic genome. Our findings support the model that MET-1 mediates transcription-coupled H3K36me3 in the parental germline and transcriptionally active embryos, and that MES-4 transmits an epigenetic memory of H3K36me3 across generations and through early embryo cell divisions by maintaining inherited patterns of H3K36me3.
Highlights
MULTICELLULAR organisms must generate a wide array of cell types from a single cell, the zygote, and must ensure that cell fates are maintained during the lifetime of the Carrozza et al 2005)
We show that both MET-1 and MES-4 contribute to H3K36me3
Previous immunostaining results suggested that in C. elegans, both MET-1 and MES-4 contribute to H3K36me3: the level of H3K36me3 immunostaining in embryos is high in wild-type, reduced in met-1 mutants, reduced in mes-4 mutants, and undetectable in double met-1; mes-4 mutants (Furuhashi et al 2010; Rechtsteiner et al 2010)
Summary
C. elegans were maintained at 15 or 20° on NGM (Nematode Growth Medium) agar plates using Escherichia coli OP50 as a food source. A coverslip was placed over the sample and the slide was immersed in liquid nitrogen for at least 2 min. Slides were incubated with 1.5% ovalbumin/1.5% bovine serum albumin in PBS-T (13 PBS and 0.1% Tween 20) for 30 min at room temperature, followed by primary antibody diluted in PBS-T overnight at 4°. Images were acquired using the Solamere imaging set-up described above and used for quantification of H3K36me antibody staining, which was performed using the protocol and antibodies described above. Wild-type and met-1 heterozygous L4s were shifted from 20° to the experimental temperature (25, 25.5, or 26.5°). Fertile wild-type and homozygous met-1 mutants were chosen from each generation to produce progeny to score in the generation. To generate csr-1(RNAi) embryos, hermaphrodites were placed on RNAi feeding plates as synchronized L1s and cultured at 24° until they started producing embryos
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