G9a co-suppresses LINE1 elements in spermatogonia

Abstract

Background: Repression of retrotransposons is essential for genome integrity and the development of germ cells. Among retrotransposons, the establishment of CpG DNA methylation and epigenetic silencing of LINE1 (L1) elements and the intracisternal A particle (IAP) endogenous retrovirus (ERV) is dependent upon the piRNA pathway during embryonic germ cell reprogramming. Furthermore, the Piwi protein Mili, guided by piRNAs, cleaves expressed L1 transcripts to post-transcriptionally enforce L1 silencing in meiotic cells. The loss of both DNA methylation and the Mili piRNA pathway does not affect L1 silencing in the mitotic spermatogonia where histone H3 lysine 9 dimethylation (H3K9me2) is postulated to co-repress these elements.

Results: Here we show that the histone H3 lysine 9 dimethyltransferase G9a co-suppresses L1 elements in spermatogonia. In the absence of both a functional piRNA pathway and L1 DNA methylation, G9a is both essential and sufficient to silence L1 elements. In contrast, H3K9me2 alone is insufficient to maintain IAP silencing in spermatogonia. The loss of all three repressive mechanisms has a major impact on spermatogonial populations inclusive of spermatogonial stem cells, with the loss of all germ cells observed in a high portion of seminiferous tubules.

Conclusions: Our study identifies G9a-mediated H3K9me2 as a novel and important L1 repressive mechanism in the germ line. We also demonstrate fundamental differences in the requirements for the maintenance of L1 and IAP silencing during adult spermatogenesis, where H3K9me2 is sufficient to maintain L1 but not IAP silencing. Finally, we demonstrate that repression of retrotransposon activation in spermatogonia is important for the survival of this population and testicular homeostasis.

Keywords: G9a; H3K9me2; IAP; LINE1; Retrotransposons; piRNA and DNA methylation.

Figure 1

Expression and conditional ablation of G9a in the adult testis. (A) Immunofluorescences using anti-G9a antibody on wild type germ cells from adult testis sections are shown. Dashed lines outline the indicated cell type. (B) Overview of deletion protocol for inducible G9a ablation and analysis. (C-F) Immunofluorescences using anti-G9a (C), anti-H3K9me2 (D), anti-GLP (E) and anti-H3K9me3 (F) antibodies on spermatogonia from testis sections of the indicated genotypes are shown. (G) Scheme indicating the repressive L1 mechanisms functioning in spermatogonia of the respective genotypes.

Figure 2

Induced loss of G9a in Mili^-/- mice results in severe spermatogenic defects. (A) Hematoxylin and eosin stained adult testis sections from the indicated genotypes. The inset highlights the basal portion of the tubule containing spermatogonia and meiotic cells or spermatagonia only in the case of G9a^CKO; Mili^KO mice. (B) Representative image of G9a^CKO; Mili^KO hematoxylin and eosin stained adult testis section, the black square indicates Sertoli-only tubules. The percentage of Sertoli cell-only tubules in the respective genotypes is shown. The results are derived from four mice of the indicated genotypes and the s.e.m is shown.

Figure 3

G9a co-suppresses L1 in spermatogonia. (A) Immunofluorescence using anti-L1 ORF1 antibody on adult testis sections of the indicated genotypes are shown. The inset highlights a crop section containing spermatogonia. (B) Immunofluorescence using anti-L1 Orf1 and anti-PLZF antibodies on spermatogonia from testis sections of the indicated genotypes are shown. (C) Quantification of L1 Orf1 signal from immunofluorescence of spermatogonia cells of the indicated genotype is shown. Bars represent mean ± s.e.m. (n = 20 to 40 cells). (D) Northern blot containing testicular RNA of the indicate genotypes probed with an L1 probe is shown. Full-length L1 transcripts are indicated. 28S RNA is shown as a loading control. The two G9a^CKO;Mili^KO samples represent biological replicates. (E) qRT-PCR measurement of L1 transcripts from testicular RNA of the indicated genotypes is shown. Bars represent mean ± s.e.m. (n = 3). (F-G) Immunofluorescence using anti-L1 ORF1 (F), anti-IAP (G) and anti-γH2AX antibodies on spermatogonia from testis sections of the indicated genotypes are shown. (H) Quantification of IAP Gag protein signal from immunofluorescence of spermatogonia cells of the indicated genotype is shown. Bars represent mean ± s.e.m. (n = 20 to 40 cells). (I) Northern blot containing testicular RNA of the indicate genotypes probed with an IAP probe is shown. Full-length IAP transcripts are indicated. 28S RNA is shown as a loading control. The two G9a^CKO; Mili^KO samples represent biological replicates. (J) qRT-PCR measurement of IAP transcripts from testicular RNA of the indicated genotypes is shown. Bars represent mean ± s.e.m. (n = 3).

Figure 4

H3K9me2 resides on L1 and IAP elements in SSCs. (A-B) qPCR results from chromatin immunoprecipitation using an anti-H3K9me2 (black) and isotype control (grey) antibodies from spermatogonial stem cell lines. A schematic image describing L1 and IAP elements and the position of the primer sets used is depicted. Bars represent mean ± s.e.m. measured in triplicate from a representative experiment performed in two biological replicates.