|dc.description.abstract||Activity of transposable elements is regulated by DNA methylation. In mice, PIWI proteins MILI (MIWI-like) and MIWI2 (mouse piwi 2, also known as PIWIL4) are central to this process in the developing male germline. In that complex and multifaceted process, MILI is a cytoplasmic protein and acts on posttranscriptional level, whereas MIWI2 shows nuclear localisation and acts as a direct effector of DNA methylation via recruitment of one of de novo DNA methyltransferases and their activator (DNMT3L). How DNMT3 is recruited to the target TE loci however remains elusive.
In the recent past, O’Carroll laboratory has unravelled the interactome of MIWI2 using immunoprecipitation followed by mass spectrometry (IP-MS), leading to discovery of SPOCD1 protein (Zoch et al., 2020). SPOCD1 was found to associate with DNMT3s and DNMTL, as well as chromatin remodelling complexes. Another novel potential component of MIWI2 pathway identified in that IP-MS dataset was C19ORF84H, a newly-annotated protein of unknown function. Subsequently, C19ORF84H was also detected in the SPOCD1 interactome. C19ORF84H was demonstrated to be a nuclear and testis-specific factor. Moreover, its expression was shown to be restricted to the period of de novo methylation in foetal gonocytes, all of that marking it as a feasible factor in MIWI2 pathway.
Using CRISPR/Cas9 system, C19orf84hnull allele was generated. Males of C19orf84h-/- genotype were shown to be infertile, with pronounced atrophy of gonads and meiotic arrest. The global loss of the protein appeared to have no implications on fertility of female mice, nor did it affect embryo survival. RNA-seq data from total P20 juvenile testes linked loss of C19ORF84H with impaired control of retrotransposons, specifically of elements belonging to evolutionary young LINE-1 families, L1MdG, L1MdTf and L1MdA, and the active copies of IAP elements. Similar pattern was previously reported for Miwi2-/- and Spocd1-/- mouse lines (Carmell et al., 2007; Zoch et al., 2020). Methyl-seq data generated utilising juvenile P14 spermatogonia revealed that the observed phenotype was indeed a direct result of impaired DNA methylation of those elements.
C19ORF84H is a newly annotated-protein that does not belong to any known family of proteins. Moreover, the protein is mostly disordered with the exception being the internally located stretch (amino acids 24-81 in mice) which shows a degree of organisation. IP-MS experiments carried out in the course of this study have provided an interesting insight into the function of C19ORf84H in MIWI2 pathway, depicting its interactome as more closely related to the one of SPOCD1 than MIWI2 protein. Presence of disordered regions in C19ORF84, akin to those found in p53, STAT proteins and other critical regulators of enzymatic pathways made us speculate that alike those factors also C19ORF84H may serve a function as docking site for protein interaction, in this case via recruitment of DNMT3 and methylation of target loci. Lastly, utilising HEK293T cell line and IP-WB experiments, it was demonstrated that C19ORF84H interacts with SPOCD1 via a 10 amino acid patch that shows a degree of conservation between mammalian and reptile C19ORF84H.
In summary, my project demonstrates that C19ORF84H is crucial for spermatogenesis and participates in MIWI2-mediated silencing of retrotransposon loci. Loss of C19ORF84H impairs DNA methylation of evolutionary young LINE1 and IAP elements, but shows little effect on global methylation, similarly to what was previously observed for the loss of MIWI2 and SPOCD1 proteins. The exact mechanism by which C19ORF84H contributes to MIWI2 function is unknown, but the high degree of disorder displayed by the protein is believed to be indispensable for its function within the MIWI2 pathway. Location of SPOCD1 interacting site within C-terminal disordered region of C19ORF84H supports this claim. Overall, my project has successfully provided the first characterisation of C19ORF84H protein, focusing primarily on the potential role of the protein in MIWI2 interactome.||en