Role of DNA demethylation in pre-implantation embryo development

Another nice paper from Yi-Lab, published two months ago. Clean and get to the point! Just like the previous one.

Nature. 2010 Aug 26;466(7310):1129-33.

Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.

Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y.

DNA methylation is one of the best-characterized epigenetic modifications. Although the enzymes that catalyse DNA methylation have been characterized, enzymes responsible for demethylation have been elusive. A recent study indicates that the human TET1 protein could catalyse the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process. Here we extend this study by demonstrating that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction. Tet1 has an important role in mouse embryonic stem (ES) cell maintenance through maintaining the expression of Nanog in ES cells. Downregulation of Nanog via Tet1 knockdown correlates with methylation of the Nanog promoter, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification.

PMID: 20639862
DOI: 10.1038/nature09303

The approaches used in the paper that I really like.

• An in vitro activity assay was performed using a modified procedure described previously 20. Briefly, 5 μg of purified proteins were incubated with 0.5 μg of double-stranded oligonucleotide substrates in 50 mM HEPES, pH 8, 75 μM Fe(NH4)2(SO4)2, 2 mM ascorbate, and 1 mM α-KG for 3 h at 37 °C. Oligonucleotide substrates were purified using Qiaquick Nucleotide Removal kit (Qiagen) and then digested with MspI. The 5′ end of the digested DNA was treated with calf alkaline phosphatase, and labelled with [γ-32P]ATP and T4 polynucleotide kinase. Labelled fragments were ethanol-precipitated, and digested with 10 μg of DNase I and 10 μg of phosphodiesterase I in the presence of 15 mM MgCl2, 2 mM CaCl2 at 37 °C. One microlitre of digestion products was spotted on a PEI-cellulose TLC plate (Merck) and separated in isobutyric acid:water:ammonium hydroxide (66:20:2) running buffer. After drying, the TLC plate was exposed to X-ray film.
• To knockdown Tet proteins, lentiviral transduction was performed in mouse ES cells as described previously 19. Short-hairpin RNA (shRNA) sequences (Supplementary Table 2) were cloned into the pTY vector under the U6 promoter. To rescue Tet1 knockdown with Nanog, cDNA of Nanog was placed downstream of the puromycin-resistant gene and foot-and-mouth disease virus 2A segment (Supplementary Fig. 12), which enables multicistronic expression of transgenes in ES cells using a single promoter 21.
• For pre-implantation embryo staining, embryos at various developmental stages were incubated in blocking solution containing anti-Tet1, anti-Cdx2, or anti-Oct4 antibody for 1 h at room temperature. After washing with PBS, cells were incubated in FITC or Rhodamine-conjugated secondary antibody for 1 h at room temperature. Embryos were counterstained with DAPI. Fluorescent images were captured using a confocal microscope with a spinning disk (CSU-10, Yokogawa) and an EM-CCD camera (ImagEM, Hamamatsu). All images were acquired as 2 μm Z-axis intervals and reconstituted using Axiovision (Zeiss).
• Five- to six-week-old female BDF1 (C57BL6 X DBA2) hybrid mice were superovulated by injecting 7.5 International Units (I.U.) of PMSG (Harbour, UCLA) and 7.5 I.U. of hCG (Sigma Aldrich). For knockdown at the two-cell stage, siRNAs (KD1 and KD2, 2 μM each) that target Tet1 or siControl were co-injected into one of the blastomeres with histone H2B fused to monomeric red fluorescent protein (mRFP, 50 ng μl−1). The injected embryos were cultured in KSOM media (EmbryoMax, Millipore) to blastocyst stage.
• Dot-blot assay. Different amounts of standard DNA (949 bp, Zimo Research, catalogue no. D5405) where C is either C, 5mC, or 5hmC, or genomic DNA were denatured with 0.1 N NaOH and spotted on nitrocellulose membranes (BioRad; catalogue no. 162-0112). The membrane was baked at 80 °C and then blocked in 5% skimmed milk in TBS containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were then incubated with 1:10,000 dilution of 5hmC (Active Motif) or 1:1,000 dilution of 5mC (Eurogentec) overnight at 4 °C. After three rounds of washes with TBST, membranes were incubated with 1:2,000 dilution of HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody, respectively. The membranes were then washed with TBST and treated with ECL.

NICE!

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