G9a co-suppresses LINE1 elements in spermatogonia
© Di Giacomo et al.; licensee BioMed Central Ltd. 2014
Received: 27 May 2014
Accepted: 26 August 2014
Published: 11 September 2014
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.
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.
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.
KeywordsLINE1 Retrotransposons IAP G9a H3K9me2 piRNA and DNA methylation
The L1 element is the most successful mobile genetic element in mammalian genomes . Processes that ensure L1 silencing are of paramount importance to germ cell development and ultimately the quality of gametes. Spermatogonia are the mitotic germ cells of the testis comprising of the spermatogonial stem cells as well as a pool of transit-amplifying cells that support gamete formation throughout adult life. CpG promoter DNA methylation and the post-translational Piwi-interacting RNA (piRNA) pathway have proven roles in the maintenance of L1 silencing during adult spermatogenesis [2, 3]. DNA methylation of L1 promoter elements epigenetically represses L1 transcription in both somatic and germ cells [2, 4]. In parallel, piRNAs complementary to L1 sequences guide the Piwi proteins Mili or Miwi to cleave and destroy expressed L1 transcripts via an RNA interference-like mechanism [3, 5, 6]. In addition, the Piwi proteins Mili and Miwi2 direct de novo DNA methylation of L1 and IAP elements during embryonic germ cell development [5, 7–9]. In Mili -/- mice, both DNA methylation and the Piwi-piRNA pathways are lost, with L1 deregulation observed only at the onset of pachytene in meiosis . Thus, additional mechanisms must exist over and above DNA methylation and the Piwi-piRNA pathway that repress L1 in spermatogonia. The repressive H3K9me2 histone modification has been shown to be present in spermatogonia, resident across L1 elements in lepto/zygotene cells, and lost when cells reach pachytene [3, 10]. However, the importance of this potential repressive mechanism in the maintenance of L1 silencing remains unknown. Here, we sought to address the physiological significance of H3K9me2 in L1 repression and spermatogonial populations.
Results and discussion
Here we demonstrate a role for G9a and H3K9me2 in the repression of L1 elements during adult spermatogenesis. This function of G9a is redundant with CpG DNA methylation and the Mili-piRNA pathway; it is only when all three mechanisms are ablated that L1 becomes derepressed within spermatogonia. In the absence of both DNA methylation and the Mili piRNA pathway, G9a is both necessary and sufficient to maintain L1 silencing. Thus, with respect to other adult germ cell populations, spermatogonia are unique in employing three distinct mechanisms to repress L1. The SSC that maintains spermatogenesis throughout adult life resides within the population of undifferentiated spermatogonia. Therefore having three distinct L1 defensive mechanisms in place within SSCs has major protective advantages for the long-term genomic quality of the gametes. The consequence of L1 activation in spermatogonia are dire as evidenced in the G9aCKO; MiliKO mice with either the complete loss of all germ cells, or with spermatogonia being the only germ cells present within the seminiferous tubules. Thus, not only are meiotic cells acutely sensitive to L1 reactivation and the ensuing DNA damage but spermatogonia are as well. The observed genomic damage in G9aCKO; MiliKO spermatogonia could be a result of L1 derepression alone or in conjunction with the observed IAP deregulation. The fact that IAP reactivation is observed in MiliKO spermatogonia without DNA damage could indicate that other post-transcriptional mechanism (s) are in place to inhibit IAP translocation such as the APOBEC RNA editing pathway that has been shown to restrict ERVs inclusive of IAP elements [22–24]. Alternatively, the level of IAP translocation is low in MiliKO spermatogonia and does not elicit a robust DNA damage response. It is very interesting that both L1 and IAP retrotransposons that depend upon the piRNA pathway for the establishment of epigenetic silencing [5, 7–9] have fundamentally differential requirements for maintenance of their silencing in spermatogonial and meiotic cells. For both L1 and IAP the three mechanisms are in place within spermatogonia, however the derepression of IAP in MiliKO spermatogonia indicates that DNA methylation and or the piRNA pathway are predominantly required for the maintenance of IAP silencing therein. In contrast to L1, the conditional loss of Mili or specifically its endonuclease activity in meiotic cells that does not affect CpG DNA methylation patterns, has no impact on IAP silencing . As H3K9me2 is globally lost in pachytene spermatocytes , IAP repression in MiliCKO spermatocytes that are devoid of the piRNA post-transcriptional silencing pathway would indicate that CpG DNA methylation is sufficient and the key mechanism for the maintenance of IAP silencing. In summary, here we show that G9a-mediated H3K9me2 is sufficient to maintain L1 silencing in the absence of L1 CpG DNA methylation and the piRNA pathway within spermatogonia. Finally, as G9a is broadly expressed our findings may indicate a role for G9a and H3K9me2 in L1 silencing in other cell types.
The Mili - , G9a Fl and Rosa26 ERT2Cre alleles were described previously [3, 15, 25]. TMX (Sigma) was injected intraperitoneally (i.p.) at a concentration of 75 mg/kg in corn oil (Sigma) as described in the text. All mice were analyzed 15 days after the last TMX injection. All mouse breeding and experimentation was performed in the EMBL Mouse Biology Unit, Monterotondo with ethical approval from the EMBL Animal Welfare and Ethical Review Body and in accordance with current Italian legislation (Art. 9, 27. Jan 1992, no116) under license from the Italian health ministry. Requests for G9aFl mice should be addressed to Alexander Tarakhovsky (The Rockefeller University). The Mili- allele is available from European Mouse Mutant Archive (https://www.infrafrontier.eu/infrafrontier-research-infrastructure/international-collaborations-and-projects/european-mouse) on a non-collaborative basis.
Antibodies, immunofluorescence and histology
Rabbit polyclonal L1 ORF1 antisera were made through immunization of rabbits with recombinant ORF1 protein. The following antibodies were used at the indicated dilutions for IF: anti-G9a (1:50) (A. Tarakhovsky, The Rockefeller University, New York, NY, USA), anti-ORF1 L1 (1:500), mouse monoclonal anti-GLP (R&D systems, Minneapolis, MN, USA, PP-B0422-00) (1:100), anti-IAP Gag (1:500) (B. Cullen, Duke University, Durham, NC, USA), mouse monoclonal anti-γH2AX (Abcam, Cambridge, UK ab26350) (1:500), mouse monoclonal anti-H3K9me2 (Abcam, Cambridge, UK ab1220) (1:100) and rabbit polyclonal anti-Plzf (Santa Cruz, Dallas, TX, USA sc-22839) (1:100). Immunofluorescence and histology were performed as described . Quantification of L1 Orf1 and IAP signal from at least 20 to 40 cells was performed using Fiji software (http://fiji.sc/Fiji).
Quantitative PCR from total testis was performed as previously described . H2Afz was used as a loading control between samples. For MuERV-L and H2Afz detection, the primers used were as follows: MuERV-L-FW 5′- CACAGCTGCGACTGAACAAT -3′; MuERV-L-RV 5′- CTAGAACCACTCCTGGTACCAAC -3′; H2Afz-FW 5′-ACAGCGCAGCCATCCTGGAGTA-3′; H2Afz-RV 5′-TTCCCGATCAGCGATTTGTGGA-3′.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation with both H3K9me2 and isotype control antibodies were performed as previously described . Briefly, in vitro cultured SSC cells  were FACS-sorted from primary mouse embryonic fibroblast feeder layer and fixed for 10 minutes in 4% formaldehyde. Three millions cells were used as input for every CHIP experiment. Quantitative PCR on the immunoprecipitated DNA was performed using L1 primers as previously described . For IAP, primers were as follows: LTR1-FW 5′-TGGTAAACAAATAATCTGCGCATGA-3′; LTR1-RV 5′-CACTCCCTGATTGGCTGCAG-3′; LTR2-FW 5′-GTGAGAACGCGTCGAATAACAAT-3′; LTR2-RV 5′- GTGATCCGTAGTTCTGGTTCTGA-3′; Gag-FW 5′-GGACTCTTACTCTAGCTGCTAACC-3′; Gag-RV 5′-AAGACACACAAACTGAAAGGCTG-3′; Pol-FW 5′- TAATGTCCCTCGTCTTGGTGATG-3′; Pol-RV 5′-ATACATCACCGTCATTGGGAGTG-3′.
conditional knock out
histone H3 lysine 9 dimethylation
intracisternal A particle
- LINE1 or L1:
long interspersed elements 1
murine endogenous retrovirus-like
open reading frame 1
quantitative reverse-transcriptase PCR
spermatogonial stem cell
DO’C is a member of the Epigenesys network of excellence; the research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7-2007-2013) / ERC grant agreement n° ERC-310206.
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