SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation

Abstract

GeneCore for preparation of the Methyl-seq libraries. Author contributionsA.Z. contributed to the design, execution and analysis of most experiments. T.A. helped established the IP conditions and performed the mass-spectrometry analysis under the guidance of J.R. and R.C.A. R.B. and Y.K. performed the bioinformatic analysis of the Methyl-seq data or sRNA-seq as well as RNA-seq data, respectively. T.S. prepared the sRNA-seq libraries and together with Y.K. the RNA-seq libraries of P20 testes. M.H and A.C. performed the homology alignment of the SPOC and TFIIS-M domains. L.V. performed the IF staining of HA-MIWI2 and generated the gonocytes microarray dataset. Y.R.P. performed the phylogenetic analysis under guidance of A.S. D.O'C. conceived and supervised this study. D.O'C. and A.Z wrote the final version of the manuscript.

TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing

“…Mutations in the human TEX15 are also associated with male infertility [12][13][14][15][16][17] . Furthermore, the MIWI2-TEX15 interaction was confirmed from the analysis of an independent HA-MIWI2 IP-MS published dataset 8 where the interaction is observed only in extracts prepared with Benzonase ( Supplementary Fig. 1b), a nuclease that is commonly used to solubilise chromatin-bound proteins.…”

“…3d). Methylation specifically at TE promotor elements and in young TEs is a hallmark of piRNA-directed methylation 2,8,19 and DNMT3C, which has a specialised function in germline de novo TE methylation 20,21 . Metaplot analysis revealed defective de novo methylation specifically at TE promotor elements in Tex15 −/− spermatogonia of young LINE1 families exemplified by L1Md_T and L1Md_Gf compared to the older L1Md_F; as observed in Miwi2 −/− spermatogonia ( Fig.…”

“…Indeed, we unequivocally show that TEX15 is required for de novo DNA methylation of precisely the same TEs that are regulated by the MIWI2-piRNA pathway. Interestingly, TEX15 was not found to interact with SPOCD1 in foetal gonocytes 8 , which places a function for TEX15 upstream or in parallel to SPOCD1 in the recruitment of the de novo methylation machinery. The DUF3715 domain of TEX15 is found in two other proteins, TASOR (FAM208A) and TASOR2 (FAM208B), of which TASOR is a critical component of the HUSH complex that mediates TE silencing in somatic cells [24][25][26] .…”

“…Fluorescence activated cell sorting (FACS). Gonocytes were FACS purified from control Tex15 +/− ; Miwi2 tdTom/+ and experimental Tex15 −/− ; Miwi2 tdTom/+ E16.5 testes using tdTomato expression as marker of gonocytes 8 . The isolated testes were dissected in a drop of Goni-MEM (DMEM (Life Techonologies) supplemented with penicillin-streptavidin (Life Technologies), NEAA (Life Technologies), sodium pyruvate (Life Technologies) and sodium lactate (Sigma-Aldrich)), transferred into 500 µl Trypsin-EDTA (0.25%, Gibco) and digested for 12 min at 37°C, shaking.…”

“…Effector piRNAs are loaded into the PIWI protein MIWI2 (PIWIL4) that licence its entry to the nucleus and the ribonucleoprotein particle (RNP) is proposed to guide de novo methylation by tethering to nascent TE transcripts and the recruitment of effector proteins 4,6,7 . SPOCD1 was recently identified that links MIWI2 to the de novo methylation machinery 8 but the full complement of MIWI2 effector proteins remains unknown. Here we show that TEX15 interacts with MIWI2 in foetal gonocytes and is required for piRNA-directed de novo DNA methylation of transposons.…”