A role for the elongator complex in zygotic paternal genome demethylation

Abstract

The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs 1. One of the molecular events that takes place during this transition is the demethylation of the paternal genome 2,3. Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified 4. To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through siRNA-mediated knockdown in zygotes, we identified Elp3/KAT9, a component of the elongator complex 5, to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulfite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of mRNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation indicating that the SAM radical domain is involved in the demethylation process. Thus, our study not only establishes a critical role for the elongator in zygotic paternal genome demethylation, but also suggests that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.

Paternal contribution to development: Sperm genetic damage and repair in fish

“…Sperm DNA methylation pattern is close to that observed in embryonic stem cells, showing hypomethylation at developmental promoters (Hammoud et al 2009;Schagdarsurengin et al 2012). After fertilization, sperm DNA is quickly demethylated in mammals except in some specific loci (O'Doherty and McGettigan 2014;Miller et al 2010;Okada et al 2010) which are supposed to be necessary for a proper progress in early embryogenesis. However in zebrafish, whose sperm chromatin, as was explained before, is fully compacted with histones (Wu et al 2011), DNA reprogramming after fertilization follows a different mechanism, early embryos inheriting sperm DNA methylome (Jiang et al 2013).…”

Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes

“…3,[79][80][81][82][83][84][85] Consistent with yeast, mutations in Elongator genes in the mouse M. musculus, the worm C. elegans, the plant A. thaliana and human cause defects in formation of wobble uridine modifications ( Table 2). 82,[86][87][88] In vitro, Elongator complex purified from HeLa cells have histone H3 and H4 acetyltransferase activity and depletion of the human homolog hELP1, IKAP (I kappa B kinase complex associated protein) in fibroblasts, leads to hypoacetylation of histone H3, transcription impairment and cell migration defects.…”

“…86 Also observed in mice is that knockdown of the Elp1p, the Elp3p or the Elp4p homologues in oocytes impairs zygotic paternal genome demethylation. 81 In the fruit fly D. melanogaster, the Elongator complex has been implicated in several processes such as, larval-and neuro-development. 95,96 An explanation for the neurodevelopmental defects in D. melanogaster could be that Elongator complex acetylate Bruchpilot, a protein important for neuronal differentiation.…”

5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming

“…Using an RNA interference-based strategy similar to the one described by Okada et al 19 we ablated Tet1-3 by injecting short interfering RNAs (siRNAs) against Tet mRNAs into MII oocytes followed by intracytoplasmic sperm injection fertilization (Fig. 5a).…”