Nature. 2010 Jan 6. [Epub ahead of print]
A role for the elongator complex in zygotic paternal genome demethylation.
Okada Y, Yamagata K, Hong K, Wakayama T, Zhang Y.
 Howard Hughes Medical Institute,  Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295, USA  Present address: Career-Path Promotion Unit for Young Life Scientist, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
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.
The authors reasoned "We uncovered a critical function of the elongator complex in paternal DNA demethylation."
* First, three independent assays (reporter localization, 5mC staining, bisulphite sequencing) indicate that knockdown of Elp3 impairs paternal DNA demethylation (Figs 1 and 2).
* Second, knockdown of additional components of the elongator complex, Elp1 and Elp4, also impaired paternal DNA demethylation (Fig. 3).
* Third, a dominant-negative approach identified the radical SAM domain, but not the HAT domain, of Elp3 to be critical for the demethylation to occur (Fig. 4).
* Consistent with the involvement of the elongator complex in zygote demethylation, mRNA levels of Elp1–4 are upregulated 3–9-fold in thePN1–2 stages before the start of paternal DNA demethylation at PN3 (Supplementary Fig. 7).
The authors said "Although the exact molecular mechanism by which the elongator complex participates in the demethylation process has yet to be determined, the demonstration that a specific protein complex is involved in paternal genome demethylation in zygotes has set the stage for further studies."
The authors further indicated "Although this potential mechanism is attractive, currently we do not have evidence indicating that Elp3 directly acts upon DNA as a DNA demethylase. We note that reconstitution of the enzymatic activity may not be trivial, as the maternal genome in the same zygote is not subject to demethylation, indicating that certain features of the paternal genome might be required for the demethylation reaction to occur."
Finally, the authors said "Regardless of how the elongator complex participates in the demethylation process, our studies not only uncover a novel function for the elongator complex, but also set the stage for understanding the functional significance of paternal genome demethylation."
cDNA that encodes the CxxC domain (amino acids 1144–1250) of mouse Mll-1 (NCBI accession NP_005924) was cloned by RT–PCR. cDNA for H3.3 was provided by Y. Nakatani 28. These cDNAs were subcloned into a pcDNA3.1-poly(A)83 vector with a C-terminal EGFP or mRFP1. pcDNA3.1-EGFP-MBD-poly(A)83 and pcDNA3.1-H2B-mRFP1-poly(A)83 were previously described 19. These plasmids were used for in vitro transcription using the RiboMAX Large Scale RNA production System T7 (Promega). Synthesized mRNAs were purified with Illustra MicroSpin G-25 columns (GE Healthcare) before being used for injection. The mouse Elp3 cDNA was amplified by RT–PCR and was subcloned into a pCDNA3.1-poly(A)83 vector with a Flag tag at the N terminus. Both the cysteine and the HAT mutants of Elp3 were generated by PCR-based mutagenesis and confirmed by sequencing. The primers used for generation of these mutants were as follows: Cys-F, 5′-ACAGGGAATATATCTATATACTCCCCCGGAGGACCTG-3′; Cys-R, 5′-CAGGTCCTCCGGGGGAGTATATAGATATATTCCCTGT-3′; HAT-F, 5′-AATTTCAGCATCAGTTCGCCTTCATGCTGCTGATGG-3′; HAT-R, 5′-CCATCAGCAGCATGAAGGCGAACTGATGCTGAAATT-3′. The underlined nucleotides are substituted in the mutants.
Mice and oocyte/zygote preparation
All animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee (IACUC protocol 07-006) and the Animal Experiment Handbook at the Kobe Center for Developmental Biology (RIKEN). Four-to-twelve-week-old BDF1 mice (C57BL6 × DBA2, Charles River or Japan SLC) were used for all the experiments. MII oocytes, collected from female mice treated with PMSG (Harbour-UCLA) and hCG (Sigma Aldrich), were cultured in M16 medium (EmbryoMax, Millipore) or CZB medium at 37 °C with 5% CO2 before being used in experiments. Gadd45b-deficient zygotes were obtained by mating of Gadd45b knockout mice pairs 14.
5mC staining and time-lapse imaging
Zygotes were fixed with 4% paraformaldehyde for at least 1.5 h at 4 °C. After washing with PBS, the zygotes were permeabilized with 0.4% Triton X-100 for 30 min at room temperature. Cells were then washed with PBS containing 0.05% Tween20 (PBST) and treated with 4 N HCl for 30 min at room temperature before being neutralized with 0.1 M Tris-HCl (pH 8.5) for 10 min, and then washed with PBST containing 0.5 M NaCl. In the following procedure, all solutions and buffers contain 0.5 M NaCl. After blocking with 1% BSA in PBST, cells were incubated with anti-5mC antibody (1:100 dilution, Eurogentic) for 0.5–1 h at 37 °C, and the positive signal was detected by FITC-conjugated donkey anti-mouse IgG (Jackson Immunoresearch). Fluorescent images were taken using a confocal microscope with a spinning disk ( CSU-10, Yokogawa). The same confocal microscope system, combined with an on-stage incubation chamber, was used for time-lapse imaging. For both live and fixed zygotes, images were acquired as multiple 2 μm Z-axis intervals, and stacked images were reconstituted using Axiovision (Zeiss) or MetaMorph (Universal Imaging Co). The intensity of 5mC in each pronucleus was calculated by MetaMorph as shown in Supplementary Fig. 6.
mRNA and siRNA injection, RT-qPCR and bisulphite sequencing
About 3–5 pl of siRNAs (2 μM) purchased from Ambion (Supplementary Table 1) were co-injected with H3.3–mRFP1 (25 μg ml-1) and CxxC–EGFP mRNAs (25 μg ml-1) simultaneously. After 8 h of cultivation, cells were subjected to ICSI (Fig. 2a). For determination of knockdown efficiency, RNA isolated from 10–20 zygotes at PN4–5 stage was used for reverse transcription using SuperScript III Cell Direct cDNA synthesis kit (Invitrogen) followed by quantitative PCR (qPCR) using SYBR GreenER (Invitrogen). To determine the expression dynamics of Elp1–4 during pronuclear stages (Supplementary Fig. 6), MII oocytes were subjected to in vitro fertilization. Groups of zygotes (100–120) were collected at 4 h after insemination (PN1–2), and then every 2 h (PN3, 4, 5, respectively) followed by acidic Tyrode’s treatment to remove cumulus cells. The extracted RNAs were subject to reverse transcription. Results were normalized with 18S rRNA as a standard. Primer sequences for qPCR are listed in Supplementary Table 2.
For bisulphite sequencing, either Elp3 siRNA or control siRNA was co-injected with H3.3–mRFP1 mRNA to MII oocytes followed by ICSI after 6–8 h of siRNA/mRNA injection. Male pronuclei, which were distinguished from female pronuclei based on their size, distance from polar bodies, and more intense H3.3–mRFP1 fluorescence, were harvested from zygotes of PN3–4 stages by breaking the zona and cytoplasm using Piezo drive (Prime Tech) and aspirating using a micromanipulator. Forty-three male pronuclei from control siRNA-injected zygotes and 47 male pronuclei from siElp3-injected zygotes were collected and subjected to bisulphite conversion using the EZ DNA Methylation-Direct Kit (Zymo Research). Nested PCR was performed using Platinum Taq DNA polymerase (Invitrogen) or ExTaq HS (TaKaRa). The sequences of the PCR primers and PCR conditions are listed in Supplementary Table 3 (refs 29, 30).
Cell culture and transfection
Immortalized p53 knockout and p53/Dnmt1 double knockout MEFs were previously described31. The knockout MEFs, double knockout MEFs, and NIH3T3 cells were maintained in DMEM supplemented with 10% FBS. pcDNA3-EGFP-pA83 plasmids containing the MBD domain and CxxC motif were transfected using Fugene6 (Roche). NIH3T3 cells that stably express CxxC–EGFP were selected under 1 mg ml-1 G418. 5-Aza-2′deoxycytidine (Sigma Aldrich) was applied at the concentration of 5 μM for 72 h.
To determine knockdown efficiency, RNA isolated from 10–20 zygotes at PN4–5 stage was used for RT–qPCR using the SuperScript III cDNA synthesis kit and SYBR GreenER (Invitrogen).
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