Journal: Nucleic Acids Research

Article Title: Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation

doi: 10.1093/nar/gkad231

Figure Lengend Snippet: Activation of Wnt/β-catenin signalling by de-repression of the Tcf7l1 gene-regulatory network in Tet1 −/− cells. ( A ) Top 10 transcription factors (TFs) predicted to regulate up-regulated DEGs in Tet1 KO cells at day 3. ( B ) Time-course expression heatmap of genes involved in Wnt positive and negative regulation and Nodal signalling, shown as the Z -score of RNA-seq TPM values. ( C ) Expression of direct targets of Wnt ( Axin2 ) and Nodal signalling ( Nodal ) in the Epiblast cluster, shown as normalized counts. ( D ) Schematic of directed differentiation towards antNPCs with signalling inhibitors added from day 2. ( E ) UMAP of integrated scRNA-seq samples collected at day 5. WT and Tet1 KO cells are either untreated or treated with 5 μM of the Wnt inhibitor XAV939 (XAV) or 2.5 μM of the Nodal signalling inhibitor SB431542 (SB). For clarity the UMAP is split per genotype and treatment. ( F ) UMAP clusters coloured by lineage identity. ( G ) Proportion of cell types annotated by lineage cluster per sample, using the colour scheme per lineage cluster shown in (F). ( H ) Selected top 10 differential regulons in Tet1 KO vs WT cells in Epiblast cluster ranked based on number of overlapping bulk RNAseq collated DEGs. ( I ) Expression level and regulon activity (number of genes in the regulon is indicated within the parenthesis) of Tcf7l1 in Epiblast cluster cells. ( J ) ChIP qPCR analysis of TET1 binding at Tcf7l1 transcription start site (TSS) at day 0 and day 2 of differentiation. Data are shown as mean ± SEM of n = 4 biological replicates, from two independent differentiations using two different ESC lines per genotype. ( K ) Gene expression of primitive streak markers T and Foxa2 at day 3 and day 5 of neurobasal differentiation (without inhibitor treatment) from Tet1 KO versus WT ESCs and Tcf7l1 KO vs WT R1 ESCs with a similar genetic background, measured using qPCR. Data are shown as mean ± SEM of n = 3 biological replicates from independent differentiations using two ESC lines of Tet1 KO and WT cells, and one ESC line of Tcf7l1 KO and R1 cells. ( L ) Heatmap of qPCR gene expression data of ectoderm ( Sox1 , Lhx5 , Pax6 ), endoderm ( Sox17 , Spink1 , Cpm ) and mesoderm ( Twist1 , Myl7 ) lineage markers in Tet1 KO and Tcf7l1 KO ESCs vs their respective controls. Data are shown as Z -score of log 10 transformed fold induction. ( M ) Western blot for TCF7L1 in serum + LIF-cultured ESCs treated for 48 h with 2 μg/ml doxycycline (DOX) to over express Tcf7l1 in Tet1 KO and WT stably transfected over-expression clonal lines. ( N ) Expression of primitive streak markers T and Foxa2 at day 3 of differentiation (without inhibitor treatment) from Tet1 KO and WT ESC lines overexpressing Tcf7l1 . 2 μg/ml of DOX was added at day 1 or day 2. Data are shown as mean ± SEM of n = 6 biological replicates from two independent differentiations using three different ESC lines per genotype. ( O ) Wnt activity based on a transient transfection TOP/FOP-flash reporter assay measured at day 3 of differentiation in Tet1 KO and WT ESCs, treated with 2 μg/ml DOX from day 1 to over-express Tcf7l1 . TOP-Flash contains a minimal fos-promoter coupled to Tcf-binding sites upstream of a luciferase reporter; FOP-Flash contains mutated Tcf-binding sites. Data are shown as mean ± SEM of n = 4 biological replicates using three different ESC lines per genotype.

Article Snippet: 2iL media consist of basal N2B27 medium as a 1:1 mixture of DMEM/F12 (Invitrogen, 11320-074) and Neurobasal medium (Invitrogen, 21103-049), 0.5× N2 (Invitrogen, 17502-048), 0.5× B27 (Invitrogen 17504-044), 2 mM l -glutamine, 0.1 mM 2-mercaptoethanol and 50:50 U:μg/ml penicillin/streptomycin, that is supplemented with 1 μM MEK inhibitor PD0325901, 3 μM GSK3 β inhibitor CHIR99021 and 0.01 ng/ml LIF (Peprotech, 250-02).

Techniques: Activation Assay, Expressing, RNA Sequencing, Activity Assay, ChIP-qPCR, Binding Assay, Gene Expression, Transformation Assay, Western Blot, Cell Culture, Stable Transfection, Transfection, Over Expression, Reporter Assay, Luciferase