RESUMO
In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.
Assuntos
Metilação de DNA , RNA de Interação com Piwi , Masculino , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas/metabolismo , Células Germinativas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis/genética , Mamíferos/metabolismoRESUMO
RNA-directed transposon silencing operates in the mammalian soma and germline to safeguard genomic integrity. The piRNA pathway and the HUSH complex identify active transposons through recognition of their nascent transcripts, but mechanistic understanding of how these distinct pathways evolved is lacking. TASOR is an essential component of the HUSH complex. TASOR's DUF3715 domain adopts a pseudo-PARP structure and is required for transposon silencing in a manner independent of complex assembly. TEX15, an essential piRNA pathway factor, also contains the DUF3715 domain. Here, we show that TASOR's and TEX15's DUF3715 domain share extensive structural homology. We found that the DUF3715 domain arose in early eukaryotes and that in vertebrates it is restricted to TEX15, TASOR, and TASORB orthologs. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate-specific. The branching of TEX15 and the TASOR-like DUF3715 domain likely occurred in early metazoan evolution. Remarkably, despite this vast evolutionary distance, the DUF3715 domain from divergent TEX15 sequences can functionally substitute the DUF3715 domain of TASOR and mediates transposon silencing. We have thus termed this domain of unknown function as the RNA-directed pseudo-PARP transposon silencing (RDTS) domain. In summary, we show an unexpected functional link between these critical transposon silencing pathways.
Assuntos
Proteínas de Drosophila , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Interferência de RNA , Genoma , Proteínas Argonautas/genética , RNA de Interação com Piwi , Mamíferos/genética , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genéticaRESUMO
The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Inativação Gênica , Animais , Proteínas Argonautas/genética , Feminino , Feto/citologia , Genoma , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Testículo/metabolismoRESUMO
In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.
