Tet2 regulates Sin3a recruitment at active enhancers in embryonic stem cells

In this study: (1) we distinguished Tet2 target genes that are regulated by its catalytic vs. noncatalytic functions in ESCs by transcriptomic profiling of Tet2 wildtype (WT), Tet2 catalytic mutant (Mut), and Tet2 knockout (KO) mouse ESCs by RNA-seq. (2) We mapped genome-wide DNA methylation of Tet2-WT, Tet2-Mut, and Tet2-KO ESCs by WGBS, to establish a critical role for Tet2 in demethylating promoters and enhancers of its catalytic target genes. (3) We determined how the genome-wide occupancy of the epigenetic modifiers Sin3a and Sap30 and the enrichment of H3K27ac, are affected in Tet2-Mut and Tet2-KO ESCs versus Tet2-WT by CUT&Tag and identified that Tet2 deficiency diminishes Sin3a occupancy at promoters and enhancers. (4) We mapped Sin3a levels genome-wide in Tet1/2 double catalytic mutant (DMUT) and Tet1/2 double knockout (DKO) ESCs, and found that deficiency of both Tet1 and Tet2 resulted in decreased levels of Sin3a in a subset of active enhancers. We studied the role of Tet2 noncatalytic functions in the regulation of ESC gene expression by performing transcriptomic analysis of Tet2-WT, Tet2-Mut, and Tet2-KO ESCs by RNA-seq to identify differentially expressed genes. RNA-Seq: Three independent ESC for Tet2-WT and two clones for each Tet2-Mut and Tet2-KO were cultured on feeders, pre-plated to remove feeders, and seeded on gelatin overnight. Total RNA was extracted (Omega E.Z.N.A Total RNA kit). Library preparation and RNA-seq were performed at Novogene (https://en.novogene.com/) on an Illumina Novoseq 6000 platform. About 20-30 million reads were generated per sample. Details of RNA-seq and data analysis are described in the methods sections of the manuscript.  WGBS: One clone of each genotype of ESCs was seeded on feeders and plated on gelatin overnight. High-quality DNA was extracted (Quick-DNA miniprep kit (Zymo, D3024) and sent for WGBS analysis at BGI genomics company (https://en.genomics.cn/). Lamda DNA spike-in was added to confirm the bisulfite conversion efficiency. Details of WGBS and data analysis are described in the methods sections of the manuscript. CUT&Tag: For Sin3a, Sap30 and H3K27ac genome-wide occupancy experiments, one ESC line of each genotype (Tet2-WT, Tet2-Mut, and Tet2-KO) was cultured on feeders, pre-plated to remove feeders, and seeded on gelatin overnight. For Tet1/2 DMUT and DKO ESCs, two independent clones of each genotype were used. ESCs were crosslinked, bound to Con-A beads, incubated with primary antibody and treated with pre-loaded pA-Tn5 adapter complex. Details of CUT&Tag procedure and data analysis are described in detail in the methods section of the manuscript. Libraries were prepared using NEBNext HiFi 2x PCR Master and subjected to 75-bp paired-end sequencing using Illumina NextSeq 500 platform at the Einstein Epigenomics core following their protocols.