METTL3 knockout in mouse stem cells activates transposable elements and 2-cell-like genes

Researchers reported in Cell Regeneration that knockout of Mettl3 in mouse embryonic stem cells (mESCs) leads to widespread upregulation of transposable elements (TEs) and 2-cell (2C)-like genes. The effect depends on m6A enzyme activity and does not depend on culture conditions biomedcentral.comCell Regenerationbiomedcentral.comOpen source ↗.

In embryo chimera experiments with a transient METTL3 degradation system (dTAG), METTL3-deficient mESCs contributed to trophectoderm lineages at the blastocyst stage. The authors interpret this as expanded developmental potential and link the loss of m6A to a 2C-like state with features of extended potency.

Knockout design and culture conditions

METTL3 is the catalytic core of the complex that deposits m6A on messenger RNA. The team used CRISPR-Cas9 to excise exons 4 through 7 of the endogenous Mettl3 locus, which removed the DPPW catalytic motif in exon 6. The deletion caused a frameshift, and immunoblotting detected no METTL3 protein. Mass spectrometry showed the m6A-to-adenosine ratio in purified mRNA fell to virtually undetectable levels.

The authors compared wild-type and knockout cells in three media: serum with leukemia inhibitory factor, serum-free N2B27 with two small-molecule inhibitors and the same factor, and serum with both inhibitors. In serum without inhibitors, knockout cells spread as spindle-shaped monolayers and did not form colonies. With inhibitors of glycogen synthase kinase 3 and MEK in serum-free medium, they formed flattened colony-like structures. The loss of mRNA m6A was the same in all media biomedcentral.comCell Regenerationbiomedcentral.comOpen source ↗.

Transposable elements and catalytic activity

Quantitative PCR and bulk RNA sequencing showed that TEs were derepressed in METTL3-deficient cells in both serum and chemically defined media. Transcripts of LINE-1 families and intracisternal A-particles were higher than in wild-type controls.

In rescue experiments, full-length wild-type METTL3 returned retrotransposon transcript levels to the wild-type baseline. A catalytically inactive mutant, with DPPW changed to APPA at amino acids 395 through 398, did not restore repression. Neither did a truncated variant spanning amino acids 242 through 448, which lacks the zinc finger domain and the catalytic core. The authors conclude that TE repression in mESCs depends on the catalytic activity of METTL3.

Earlier studies disagreed on how METTL3 loss affects mobile elements, including the direction of change for LINE elements under different culture formulations. To separate new transcript synthesis from altered decay of unmethylated RNA, the authors used a DRB chase combined with transient transcriptome sequencing (DRB-TT-seq) biomedcentral.comCell Regenerationbiomedcentral.comOpen source ↗.