Catalytic dependent and independent roles of Tet3 in activation of ectodermal and silencing of mesodermal programs during neuroectoderm specification
In this study: (1) we distinguished Tet3 target genes that are regulated by its catalytic vs. noncatalytic functions in neuroectoderm (NE) cells by transcriptomic profiling of Tet3 wildtype (WT), Tet3 catalytic mutant (Mut), and Tet3 knockout (KO) NE cells by RNA-seq. (2) We mapped genome-wide DNA methylation of Tet3-WT, Tet3-Mut, and Tet3-KO NE cells by WGBS. (3) We determined Tet3 genome-wide occupancy in Tet3-WT NE cells by CUT&Tag. (4) We mapped 5hmC rich regions in Tet3-WT, Tet3-Mut, and Tet3-KO NE cells by hmeDIP. (5) We overexpressed  (OE) the Tet3 target gene Dnmt1 (D) or an empty vector (EV) in Tet3-WT, Tet3-Mut, and Tet3-KO NE cells and assessed gene expression by RNA-seq. (6) We overexpressed (OE) Tet3 (T) or an empty vector (EV) in Tet3-WT, Tet3-Mut, and Tet3-KO NE cells and examined gene expression changes in these cells by RNA-seq. Overall design: We investigated the catalytic and noncatalytic roles of Tet3 in gene regulation during ESC differentiation toward neuroectoderm (NE). Analysis and integration of RNAseq, WGBS, hmeDIP, and CUT&Tag data revealed novel functions of Tet3 in NE specification. For all omics experiments, Tet3-WT, Tet3-Mut, and Tet3-KO ESCs were differentiated to NE by LIF withdrawal and 1uM retinoic acid (RA) treatment for three days. Cells were collected for each experiment at day 3 (72 hours of differentiation). All RNAseq experiments: RNA was extracted from Tet3-WT, Tet3-Mut, and Tet3-KO NE cell lines (Qiagen RNeasy mini kit) at 72 hours of differentiation. Library preparation and RNAseq were performed by Novogene (https://en.novogene.com/) on an Illumina Novoseq 6000 platform and 20-30 million reads were generated per sample. WGBS: DNA was extracted from one clone of Tet3-WT, Tet3-Mut, and Tet3-KO NE cells (Quick-DNA miniprep kit - Zymo, D3024) and subjected to WGBS analysis at BGI genomics company (https://en.genomics.cn/). Lamda DNA spike-in confirmed the efficiency of bisulfite conversion (>99.0%). CUT&Tag: One Tet3-WT ESC cell line was differentiated to NE (3 days) and used to determine Tet3 genome-wide occupancy in NE cells. Cells were crosslinked, bound to Con-A beads, incubated with primary antibody and treated with pre-loaded pA-Tn5 adapter complex. Libraries were prepared with NEBNext HiFi 2x PCR Master mix and sent for 75-bp paired-end sequencing on an Illumina NextSeq 500 platform at the Einstein Epigenomics core following their protocols. hmeDIP: DNA was extracted from one clone of Tet3-WT, Tet3-Mut, and Tet3-KO NE cells  (Quick-DNA miniprep kit - Zymo, D3024). Pull down of 5hmC enriched regions was perfromed using the Active Motif hmeDIP kit (#55010) and subjected to 75bp paired-end sequencing using Illumina NextSeq 500 platform at the Einstein Epigenomics Core. Details of RNAseq, WGBS, hmeDIP and CUT&Tag procedure and data analyses are described in detail in the methods section of the manuscript.