SRA

Naive pluripotent epiblast cells of the preimplantation murine embryo and their in vitro counterpart, embryonic stem (ES) cells, have the capacity to give rise to all cells of the adult. Such developmental plasticity is associated with global genome hypomethylation. It is unclear whether genome methylation is dynamically regulated only via differential expression of DNA methyltransferases (DNMTs) and Ten-eleven Translocation (TET) enzymes, which oxidase methylated DNA. Here we show that LIF/Stat3 signalling induces genomic hypomethylation via metabolic reconfiguration. In Stat3-/- ES cells we observed decreased alpha-ketoglutarate (?KG) production from reductive Glutamine metabolism, leading to decreased TET activity, increased Dnmt3a/b expression and to a global increase in DNA methylation. Notably, genome methylation is dynamically controlled by simply modulating aKG availability, mitochondrial activity or Stat3 activation in mitochondria, indicating an effective crosstalk between metabolism and the epigenome. Stat3-/- ES cells also show increased methylation at Imprinting Control Regions accompanied with differential expression of >50% of imprinted genes. Single-cell transcriptome analysis of Stat3-/- embryos confirmed dysregulated expression of Dnmt3a/b, Tet2, and imprinted genes in vivo. Our results reveal that the LIF/Stat3 signal bridges the metabolic and epigenetic profiles of naive pluripotent cells, ultimately controlling genome methylation and imprinted gene expression. Several imprinted genes regulate cell proliferation and are often misregulated in tumours. Moreover, a wide range of cancers display Stat3-overactivation, raising the possibility that the molecular module we described here is exploited under pathological conditions. Overall design: RRBS profiles of wild-type (S3+/+) and knock-out (S3-/-) mES cells were generated by high-throughput sequencing, using Illumina NextSeq 500. Cells were either cultured in 2iLIF; 2 experimental replicates for each condition. Total RNA profiles of wild-type (S3+/+), knock-out (S3-/-) and mitochondrial Stat3 (MitoS3.A, MitoS3.B) mESCs were generated by high-throughput sequencing, using Illumina HiSeq4000. Cells were either cultured in 2i, 2iLIF or without 2iLIF for 48 hours; 2 experimental replicates for each condition. RRBS profiles of wild-type (WT) and Dmmt3a/b knock-out (Dnmt3a/b dKO) mES cells were generated by high-throughput sequencing, using Illumina NextSeq 500. Wild-type E14 cells were stably cultured either in 2iLIF or 2i; Dnmt3a/b dKO cells were stably cultured in 2i; 2 different KO clones were used (Dnmt3a/b dKO.A, Dnmt3a/b dKO.B); 2 experimental replicates for each condition.