C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line

Zoch A, Konieczny G, Auchynnikava T, Stallmeyer B, Rotte N, Heep M, Berrens RV, Schito M, Kabayama Y, Schöpp T, Kliesch S, Houston B, Nagirnaja L, O'Bryan MK, Aston KI, Conrad DF, Rappsilber J, Allshire RC, Cook AG, Tüttelmann F, O'Carroll D, 21.03.2024

Abstract

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.

Zoch A, Konieczny G, Auchynnikava T, Stallmeyer B, Rotte N, Heep M, Berrens RV, Schito M, Kabayama Y, Schöpp T, Kliesch S, Houston B, Nagirnaja L, O'Bryan MK, Aston KI, Conrad DF, Rappsilber J, Allshire RC, Cook AG, Tüttelmann F, O'Carroll D. C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line. Mol Cell. 2024 Mar 21;84(6):1021-1035.e11. doi: 10.1016/j.molcel.2024.01.014.

Publication: https://doi.org/10.1016/j.molcel.2024.01.014
Repository: ClinVar: SCV004098696, SCV004098697 and SCV004098698 Protein Data Bank: PDB: 8OU1 ArrayExpress: E-MTAB-11612 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE199038 ProteomeXchange: PRIDE: PXD047331

Disclaimer

The publication C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line by Zoch A, Konieczny G, Auchynnikava T, Stallmeyer B, Rotte N, Heep M, Berrens RV, Schito M, Kabayama Y, Schöpp T, Kliesch S, Houston B, Nagirnaja L, O'Bryan MK, Aston KI, Conrad DF, Rappsilber J, Allshire RC, Cook AG, Tüttelmann F, O'Carroll D is published under an open access license: https://creativecommons.org/licenses/by-nc-nd/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Curation by the MFGA team
Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.

Data set 1: Whole exome sequencing of the MERGE cohort

Exome: Whole Exome Sequencing

Species

Species
Human

Conditions

Human phenotype ontology	Participants	Comment
HP:0000027: Azoospermia		The majority (n = 1,902) had pathomechanistically idiopathic azoo- or cryptozoospermia.
HP:0030974: Cryptozoospermia		The majority (n = 1,902) had pathomechanistically idiopathic azoo- or cryptozoospermia.

Images

Figure 1: SPOCD1 is essential for human fertility and transposon silencing

(A) Table of clinical data for three identified infertile men carrying homozygous SPOCD1 variants. FSH, follicle-stimulating hormone; LH, luteinizing hormone; T, testosterone; TV, testicular volume right/left; IU, international unit; NA, not available. Reference values: FSH 1–7 IU/L, LH 2–10 IU/L, T > 12 nmol/L, TV > 12 mL. (B) Schematic representation of human SPOCD1 variants identified in the three infertile men. (C) Pedigree of individual M3457 and sequencing trace of SPOCD1 exon 15 harboring the SPOCD1c.2912T>G variation with substituted nucleotide highlighted. NOA, non-obstructive azoospermia. (D) Pedigree of individual GEMINI-88 and sequencing trace of SPOCD1 exon 16 harboring the SPOCD1c.3354_3355insA variation with inserted nucleotide highlighted from GEMINI-88. (E) Periodic acid-Schiff (PAS) and hematoxylin-stained testis sections of GEMINI-88 and control individual. SCO, Sertoli cell only. Scale bars, 50 μm. (F) Pedigree of individual M2021 and sequencing trace of part of SPOCD1 exon 8 harboring the SPOCD1c.1991_1992del variation from M2021 and brother; missing nucleotides are indicated. WT, canonical SPOCD1 allele; MUT, SPOCD1c.1991_1992del allele. (G) PAS and hematoxylin-stained testis sections of M2021. SPG, spermatogonia; SPCs, spermatocytes; RSs, round spermatids; MeiA, meiotic arrest; SCO, Sertoli cell only. Scale bars, 5 and 50 μm. (H) Testis section of M2021 and control individual stained for LINE1 ORF1p (brown 3,3′-diaminobenzidine [DAB] stain). SPG, spermatogonia. Scale bars, 50 and 5 μm.

Source: Zoch A, Konieczny G, Auchynnikava T, Stallmeyer B, Rotte N, Heep M, Berrens RV, Schito M, Kabayama Y, Schöpp T, Kliesch S, Houston B, Nagirnaja L, O'Bryan MK, Aston KI, Conrad DF, Rappsilber J, Allshire RC, Cook AG, Tüttelmann F, O'Carroll D. C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line. Mol Cell. 2024 Mar 21;84(6):1021-1035.e11. doi: 10.1016/j.molcel.2024.01.014.

Licensed under: https://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 5: Mouse strains and experimentation

Genome: Genotyping

Species

Species
Mouse

Images

Figure 2: C19ORF84 is required for male fertility and transposon silencing

(A) Western blot analysis of C19ORF84 protein abundance in E16.5 testes from C19orf84+/− and C19orf84−/− mice. (B) C19ORF84 staining of E16.5 fetal testis sections from C19orf84+/− and C19orf84−/− mice. (C) Number of embryos per plug fathered by studs with the indicated genotype mated to wild-type females. Data are mean and SEM from n = 3 C19orf84+/− (10 plugs total) and n = 4 C19orf84−/− studs (15 plugs total), ∗∗∗p < 0.001. (D) Testis weight of adult mice with the indicated genotype. Data are mean and SEM from n = 6 C19orf84+/− and n = 4 C19orf84−/− mice, ∗∗∗p < 0.001. Insert shows a representative image of testes from C19orf84+/− (left) and C19orf84−/− (right) mice. (E and F) Representative images of PAS and hematoxylin-stained epididymis (E) and testes (F) sections of two stages of the seminiferous cycle, indicating a germ cell arrest at the early pachytene stage. VII, Stage 7; PL, pre-leptotene; P, pachytene; RSs, round spermatids; eS(16), step 16 elongated spermatids; SC, Sertoli cell; Z, zygotene; m2, secondary meiocytes. (G and H) Adult testes from wild type and C19orf84−/− mice stained for LINE1 ORF1p (G) or IAP GAG (H) protein. (I) RNA-seq heatmap analysis showing fold-change of expression (relative to wild type) of the 10 most upregulated LINE1 and ERV transposons in P20 testes from C19orf84−/−, Spocd1−/−, and Miwi2−/− mice. n = 3 for all genotypes. (J and K) Adult testis sections stained for DNA damage marker γH2AX (J) or for apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (K) from wild-type and C19orf84−/− mice. Scale bars, 5 μm (B and F), 20 μm (E), 50 μm (G, H, J, and K). Germ cell nuclei are indicated with a dashed line in (B). Images shown in (B), (E)–(H), (J), and (K) are representative of data from n = 3 mice per genotype.

Source: Zoch A, Konieczny G, Auchynnikava T, Stallmeyer B, Rotte N, Heep M, Berrens RV, Schito M, Kabayama Y, Schöpp T, Kliesch S, Houston B, Nagirnaja L, O'Bryan MK, Aston KI, Conrad DF, Rappsilber J, Allshire RC, Cook AG, Tüttelmann F, O'Carroll D. C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line. Mol Cell. 2024 Mar 21;84(6):1021-1035.e11. doi: 10.1016/j.molcel.2024.01.014.

Licensed under: https://creativecommons.org/licenses/by-nc-nd/4.0/