Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).

Gametes are highly specialised cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mouse, germ cells are first specified in the developing embryo as primordial germ cells (PGCs) starting around embryonic day (E) 6.251 (Fig. 1a). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming at E10.5/E11.52–11, including genome-wide loss of 5-methylcytosine (5mC)2–5,7–11 (Fig. 1a). The underlying molecular mechanisms of this process have remained enigmatic leading to our inability to recapitulate this step of germline development in vitro12–14. Using an integrative approach, we show that this complex reprogramming process involves the coordinated interplay between promoter sequence characteristics, DNA (de)methylation, Polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of Tet1 to enable the activation of a critical set of germline reprogramming responsive (GRR) genes involved in gamete generation and meiosis. Our results also unexpectedly reveal a role for Tet1 in safeguarding but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will be instructive towards recapitulating complete gametogenesis in vitro.

Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Ten-Eleven Translocation 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC)1-4. Here we demonstrate using detailed immunofluorescence analysis and ultra-sensitive LC/MS based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small molecule inhibition of Tet3 activity as well as genetic ablation impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3 driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming revealing intricate interplay between DNA demethylation, de novo methylation and Tet3 driven hydroxylation.