A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory - PubMed (original) (raw)

A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory

David J Katz et al. Cell. 2009.

Abstract

Epigenetic information undergoes extensive reprogramming in the germline between generations. This reprogramming may be essential to establish a developmental ground state in the zygote. We show that mutants in spr-5, the Caenorhabditis elegans ortholog of the H3K4me2 demethylase LSD1/KDM1, exhibit progressive sterility over many generations. This sterility correlates with the misregulation of spermatogenesis-expressed genes and transgenerational accumulation of the histone modification dimethylation of histone H3 on lysine 4 (H3K4me2). This suggests that H3K4me2 can serve as a stable epigenetic memory, and that erasure of H3K4me2 by LSD/KDM1 in the germline prevents the inappropriate transmission of this epigenetic memory from one generation to the next. Thus, our results provide direct mechanistic insights into the processes that are required for epigenetic reprogramming between generations.

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Figures

Figure 1

Figure 1

spr-5 is a H3K4me2 demethylase. A, Alignment of the C. elegans LSD1/KDM1 orthologs with human LSD1/KDM1. The key catalytic residues (N660 and K661) are shown at their relative position within the amine oxidase domain and the location of the mutations are shown beneath each gene. B, ChIP of H3K4me2 at hop-1 in wild-type (N2), spr-5(by101) and spr-5;amx-1 mutants. The fold enrichment (of the percentage of input precipitated) in H3K4me2 Ab over no Ab, with the positions of the primer sets in the promoter and gene body indicated above. C, Western blots of wild-type (N2) and spr-5;amx-1 protein extracts probed with actin, histoneH3 and H3K4me2 (positions marked). The ratio of H3K4me2/histoneH3 normalized to actin is shown below. Immunofluorescence with SPR-5 N (D,G,H) and C terminal (E,F) Ab’s showing the adult gonad (D), oocytes (E), an early embryo (F), sperm (G), and the corresponding DAPI image of sperm (H). The inset in panels E,F and G are zoomed in on part of the panel. The identical staining pattern was observed with both N and C terminal Ab’s.

Figure 2

Figure 2

The germline mortality phenotype. A, The average brood size of spr-5, spr-5;amx-1, spr-5;amx-1;T08D10.2 (triple), wild-type (N2), amx-1 and T08D10.2 strains in progressive generations are shown. B, The percentage of sterile animals in the same experiment as (A). The complete data from each strain are shown individually in Supplemental Figure 2. C, The percentage of animals with no progeny, the percentage of animals with a sterile phenotype and the average brood size of outcrossed spr-5;amx-1 (f28) worms compared to the corresponding (f30) non-outcrossed and wild-type generations.

Figure 3

Figure 3

Oogenesis and spermatogenesis defects in spr-5;amx-1 mutants. DIC microscopic imaging of the proximal gonad from wild-type (A) and severely sterile spr-5;amx-1 (B) adult worms. Black arrowheads point to oocytes (A) and defective looking oocytes (B and C). Black bars denote mature sperm in the spermatheca (A) as compared to early spermatogenic stages in the proximal gonad (B) and spermatheca (C). DIC imaging (C) and corresponding acridine orange staining (D) showing residual bodies (*).

Figure 4

Figure 4

Increased H3K4me2 retention in Z2/ Z3 correlates with sterility. A, Embryos from the experiment in Figure 2 were assayed for PGC H3K4me2 levels by immunofluorescence. Each number represents the percentage of embryos scored positive for H3K4me2 retention out of 50 total embryos for that generation. Examples of wild-type (B) and retained (C) H3K4me2 levels in Z2/Z3. Arrowheads indicate Z2 and Z3. D, The percentage of sterile offspring in spr-5, spr-5;amx-1, nos-2(RNAi), nos-2(RNAi);spr-5 and nos-2(RNAi);spr-5;amx-1 assayed at generations 1 and 16. L4440 is the vector only RNAi control.

Figure 5

Figure 5

Increased H3K4me2 affects transcription. DIC (A,C) and GFP (B,D) imaging of H2A:GFP transgene expression in wild-type (A,B) and spr-5 (C,D) animals.

Figure 6

Figure 6

The trans-generational accumulation of H3K4me2 and expression at spermatogenesis genes. A, Genes that were changed twofold or greater in comparisons between spr-5 f1,f13 and f26 were compared against the topomap gene function list from over 500 microarray experiments (

http://elegans.uky.edu/gl/cgi-bin/gl\_mod.cgi?action=compare2

). Only over-representation P values of less than 1×10−6 are shown. The number of overlapping genes and the corresponding P value are listed. Mountains with no listed gene function have no known function. All genes that are listed under multiple regulated categories (ex. up1v13, down13v26) are not listed in the individual category (ex. down13v26). B, The chromosomal distribution of the 234 regulated genes, represented as the ratio of observed to expected. The number of genes on each chromosome is shown within the bar. (*)Indicates significant (P<0.001) deviation from the expected. C, Quantitative RT-PCR showing the relative expression of 12 spermatogenesis genes from the 88 coordinately expressed genes (Figure 6A, column 3) over 28 generations. The expression is normalized to actin (act-1). D, ChIP on 4 of the spermatogenesis genes from c, showing the percentage of input precipitated with an H3K4me2 Ab in spr-5 mutants from generations 3, 15 and 27. The primers used for ChIP are immediately downstream of the start site. The percentage precipitated with no Ab was <0.1 in all cases.

Figure 7

Figure 7

The summary of data (A) and model (B). A, Graphs comparing the percentage of H3K4me2 precipitated by ChIP in spermatogenesis-expressed genes(Figure 6D), the percentage of H3K4me2 retention in the PGCs (Figure 4), the relative expression of spermatogenesis-enriched genes (Figure 6C), the percentage of sterile animals (Figure 2B) and the brood size (Figure 2A) over generations in spr-5 mutants. The trans-generational increase in H3K4me2 in spermatogenesis-enriched genes due to the failure to demethylate results in increased H3K4me2 retention in the PGCs and the continuous misexpression of spermatogenesis-enriched genes. This leads to an increasing percentage of sterile animals and decreasing brood size, which can be reset by outcrossing the mutation (Figure 2). B, Germ cells, sperm and oocytes have high levels (black nuclei) of H3K4me2 throughout the genome. SPR-5 erases the epigenetic memory of germline transcription by demethylating H3K4me2 at germline-expressed loci in the gametes or early embryo (grey nucleus). A second mechanism removes H3K4me2 throughout the genome in the PGCs (white nuclei). In spr-5 mutants, the failure to reset germline H3K4me2 over multiple generations results in the inappropriate retention of H3K4me2 in the PGCs (black nuclei) and sterility.

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