Tet1 regulates embryonic stem cell differentiation programs and development independent of its catalytic activity.

In this study: (1) we characterized the Tet1 non-catalytic functions in the regulation of ESC gene expression programs by performing transcriptomic analysis of Tet1 wild type (WT), Tet1 catalytic mutant (Mut) and Tet1 knockout (KO) mouse ESCs by RNA-seq to identify differentially expressed genes. (2) We mapped the genome-wide occupancy of endogenously FLAG-tagged Tet1-WT and Tet1-Mut in ESCs by CUT&Tag using a specific antibody against FLAG. (3) We determined how the genome-wide occupancy of the epigenetic modifiers: Ezh2, Sin3a, Chd4 and the enrichment of histone marks: H3K27me3, H3K4me3 and H3K27ac, are affected in Tet1-KO ESCs versus Tet1-WT and Tet1-Mut by CUT&Tag or CUT&RUN. (4) We analyzed methylation levels and distribution in Tet1-WT, Tet1-Mut and Tet1-KO ESCs by WGBS, to confirm Tet1 non-catalytic targets with no differential methylation. (5) We explored whether Tet1 non-catalytic functions play a role in chromatin accessibility by performing ATAC-seq in Tet1-WT, Tet1-Mut and Tet1-KO ESCs. Overall design: We examined the role of Tet1 non-catalytic functions in regulation of ESC gene expression programs by performing transcriptomic analysis of Tet1-WT, Tet1-Mut, Tet1-KO ESCs by RNA-seq to identify differentially expressed genes. RNA-Seq: Three independent ESC of each genotype (Tet1-WT (Tet1+/+), Tet1-Mut (Tet1m/m), Tet1-KO (Tet1–/–)) 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), barcoded and used to prepare libraries. ERCC spike in controls were included. The libraries were subjected to 100 bp paired-end sequencing using Illumina NextSeq 500 platform at the Einstein Epigenomics core following their protocols. We generated ~37 million reads per sample. Details of RNA-seq and data analysis are described in the methods sections of the manuscript. CUT&Tag/CUT&RUN-seq: Genome-wide occupancy experiments were performed on one ESC line of each genotype, which was cultured on feeders, pre-plated to remove feeders and seeded on gelatin overnight. CUT&Tag sequencing analysis of embryonic stem cells expressing FLAG-tagged WT-Tet1 and FLAG-tagged Tet1-catalytic mutant from the endogenous Tet1 locus. FLAG-Tet1-WT and FLAG-Tet1-Mut binding sites were defined by enrichment over non-specific enriched regions identified in IgG and parental untagged Tet1-WT cells. Ezh2/H3K27me3/H3K4me3/Sin3a/HEK27ac CUT&Tag and Chd4 CUT&RUN sequencing analysis of Tet1-WT, Tet1-Mut, Tet1-KO ESCs: Specific binding sites were defined by enrichment over IgG control. CUT&Tag and CUT&RUN libraries were subjected to 75 bp and 35 bp paired-end sequencing, respectively, using Illumina NextSeq 500 platform at the Einstein Epigenomics core following their protocols. Details of CUT&Tag/CUT&RUN and data analysis are described in the methods sections of the manuscript. WGBS-seq: WGBS was performed on two independent ESC lines of each genotype (Tet1-WT, Tet1-Mut, Tet1-KO) which were pre-plated and seeded on gelatin overnight. High quality DNA (extracted by Quick-DNA Miniprep Kit) was bisulfite converted and sequenced by BGI 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. ATAC-seq: Three independent ESC lines of each genotype (Tet1-WT, Tet1-Mut, Tet1-KO) were used to identify differences in chromatin accessibility by ATAC-seq. Transposed DNA fragments were used to prepare 200-1000 bp libraries. Libraries were sequenced at the Einstein Epigenomics core with a 75 bp paired-end protocol on an Illumina NextSeq 500 platform. Details of WGBS and data analysis are described in the methods sections of the manuscript.