A role for the elongator complex in zygotic paternal genome demethylation - PubMed (original) (raw)

A role for the elongator complex in zygotic paternal genome demethylation

Yuki Okada et al. Nature. 2010.

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. One of the molecular events that takes place during this transition is the demethylation of the paternal genome. Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified. 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 short-interfering-RNA-mediated knockdown in mouse zygotes, we identified Elp3 (also called KAT9), a component of the elongator complex, to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulphite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of messenger RNA 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. Our study not only establishes a critical role for the elongator complex in zygotic paternal genome demethylation, but also indicates that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.

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Figures

Figure 1

Figure 1. Knockdown of Elp3 prevents preferential incorporation of the CxxC-EGFP reporter into the paternal pronucleus

(a) Scheme of the experimental procedure. (b, c) Time-lapse imaging of CxxC-EGFP (green; left column) and H3.3-mRFP1 (red; middle column) at various pronucleus stages of zygotic development in the absence (b) or presence (c) of siRNA that targets Elp3. ♂: male pronucleus, ♀: female pronucleus, PB: polar body. Bar = 25 μm.

Figure 2

Figure 2. Knockdown of Elp3 impairs DNA demethylation in the paternal pronucleus

(a) siRNA-mediated knockdown of Elp3 resulted in increased 5mC staining (green) in the PN5 paternal pronucleus. H3.3-mRFP1 (red) serves as a nuclear marker. H3.3-mRFP1 signal is more intense in male pronuclei than in female pronulcei due to preferential incoporation of H3.3 to the paternal genome. ♂: male pronucleus, ♀: female pronucleus, PB: polar body. Bar = 25 μm. (b) Quantification of the ratio (male/female) of 5mC intensity in Elp3 knockdown and control groups. Each dot represents a zygote. Red bars represent the average ratio of each group. The statistics of the injections are presented in the table. (c) Bisulfite sequencing of Line1-5′ and ETn indicates that knockdown of Elp3 impairs paternal DNA demethylation. Open circles and closed circles represent unmethylated and methylated CpG respectively. Each line represents an individual clone. 10 CpGs and 15 CpGs were analyzed for Line1-5′ and ETn respectively.

Figure 3

Figure 3. Knockdown of the elongator components Elp1 and Elp4 also impairs paternal DNA demethylation in zygotes

(a) siRNA-mediated knockdown of Elp1 and Elp4 resulted in increased 5mC staining (green) in the PN5 paternal pronucleus. H3.3-mRFP1 (red) serves as a nuclear marker. ♂: male pronucleus, ♀: female pronucleus, PB: polar body. Bar = 25 μm. (b) Quantification of the ratio (male/female) of 5mC intensity in Elp1, Elp4, Elp3 knockdown and control groups. Each dot represents a zygote. Red bars represent the average ratio of each group. The statistics of the injections are presented in the table.

Figure 4

Figure 4. Mutation of the cysteine-rich radical SAM domain of Elp3 impairs paternal DNA demethylation

Schematic representation of wild-type and mutant mElp3. Conserved domain (CD) of Elp3 protein sequences from NCBI are aligned with Elp3 sequences from budding yeast and mouse. Conserved amino acid residues are color coded in red. (b) Overexpression of the Cys mutant, but not the HAT mutant or wild-type, blocked paternal DNA demethylation. Representative images from PN5 stage were shown. ♂: male pronucleus, ♀: female pronucleus, PB: polar body. Bar = 25 μm. (c) Quantification of the ratio (male/female) of 5mC intensity in control and Elp3 (wild-type, Cyc mutant, or HAT mutant) mRNA injected groups. Each dot represents a zygote. Red bars represent the average ratio of each group. The statistics of the injections are presented in the table.

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