Figure 5. Panel A shows Venn diagram... | Rockefeller University Press

Figure 5.

Mettl8 promotes m3C modification of Tcf7 mRNA and its genome-specific loops of Tox in CD8+T cells. (A) Venn plot illustrates the overlap of downregulated genes from RNA-seq, m3C-seq, and Mettl8-binding genes from RIP-seq. (B) Mettl8 occupancy at the Tcf7 gene loci is revealed through m3C-seq (WT and Mettl8−/−) of EG7-OVA tumor-infiltrating OT-I cells and RIP-seq (Mettl8-tdTomato-Flag) of B16F10 tumor-infiltrating CD44+ CD8+ T cells. The binding peaks on Tcf7 loci are depicted. The m3C tracks are all plotted on a consistent scale. (C) The RNA decay assay demonstrates the remaining Tcf7 mRNA of CD8+ T cells from the spleens of WT and Mettl8−/− mice detected by qRT-PCR, normalized to t = 0. (D) Heatmaps display changes in total Tcf1-targeting genes between WT and Mettl8−/− EG7-OVA tumor-infiltrating OT-I cells and Mettl8-targeting genes in B16F10 tumor-infiltrating CD44+ CD8+ T cells of Mettl8-tdTomato-Flag mice as detected by CUT&Tag. (E) Diamond graphs exhibit chromatin interactions in WT and Mettl8−/− tumor-infiltrating OT-I cells at the Tox gene loci (top), with CUT&Tag and ATAC-seq tracks, and gene structures on the bottom. An enlarged view highlights the signal profiles across the Tox gene region. (F) co-IP of Tcf1 by anti-Flag magnetic beads in CD3+ T cells from the spleens of Mettl8-tdTomato-Flag (RPT) and WT mice. IB, immunoblot. (G) co-IP of Tcf1 by Flag-tagged Mettl8 protein with anti-Flag magnetic beads after co-transfection into HEK293T cells. (H) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (I) Schematic diagram of the tumor model: Mettl8fl/flCd4cre mice were subcutaneously injected with 2 × 105 B16F10 cells and harvested after 13 days. (J) Representative flow cytometry plots and cumulative data show the frequency of Tcf1+ Tox+ cells gated on tumor-infiltrating CD8+ CD44+ T cells (right). n = 6 per group. (K) Schematic diagram of the OT-I–transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 105 EG7-OVA cells, followed by 2 × 106 WT or Mettl8−/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. Representative flow cytometry plots and cumulative data show the frequency of Tox+ cells gated on Tcf1+ OT-I cells. n = 6 per group. (L) The MFI of Tox gated on Tcf1+ OT-I cells of the mice in K. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F5. Refer to the image caption for details.

Panel A shows Venn diagram comparing downregulated genes across sequencing datasets. Panel B shows genome browser tracks showing M e t t l 8 binding at T c f 7 locus. Panel C shows line graph of T c f 7 m R N A decay over time. Panel D shows heatmap of T c f 1 target genes and M e t t l 8 targets. Panel E shows chromatin interaction diamond plots at T o x gene locus. Panel F shows co immunoprecipitation blot showing interaction between M e t t l 8 and T c f 1. Panel G shows co immunoprecipitation validation of M e t t l 8 and T c f 1 interaction. Panel H shows UMAP plots displaying single cell gene expression patterns. Panel I shows schematic diagram of B 16 F 10 tumor experiment timeline. Panel J shows flow cytometry plots and quantification of T c f 1 positive T o x cells. Panel K shows flow cytometry plots and quantification of T o x positive O T I cells. Panel L shows the histogram and quantification of T o x expression intensity.

Mettl8 promotes m ³ C modification of Tcf7 mRNA and its genome-specific loops of Tox in CD8 ⁺ T cells. (A) Venn plot illustrates the overlap of downregulated genes from RNA-seq, m³C-seq, and Mettl8-binding genes from RIP-seq. (B) Mettl8 occupancy at the Tcf7 gene loci is revealed through m³C-seq (WT and Mettl8^−/−) of EG7-OVA tumor-infiltrating OT-I cells and RIP-seq (Mettl8-tdTomato-Flag) of B16F10 tumor-infiltrating CD44⁺ CD8⁺ T cells. The binding peaks on Tcf7 loci are depicted. The m³C tracks are all plotted on a consistent scale. (C) The RNA decay assay demonstrates the remaining Tcf7 mRNA of CD8⁺ T cells from the spleens of WT and Mettl8^−/− mice detected by qRT-PCR, normalized to t = 0. (D) Heatmaps display changes in total Tcf1-targeting genes between WT and Mettl8^−/− EG7-OVA tumor-infiltrating OT-I cells and Mettl8-targeting genes in B16F10 tumor-infiltrating CD44⁺ CD8⁺ T cells of Mettl8-tdTomato-Flag mice as detected by CUT&Tag. (E) Diamond graphs exhibit chromatin interactions in WT and Mettl8^−/− tumor-infiltrating OT-I cells at the Tox gene loci (top), with CUT&Tag and ATAC-seq tracks, and gene structures on the bottom. An enlarged view highlights the signal profiles across the Tox gene region. (F) co-IP of Tcf1 by anti-Flag magnetic beads in CD3⁺ T cells from the spleens of Mettl8-tdTomato-Flag (RPT) and WT mice. IB, immunoblot. (G) co-IP of Tcf1 by Flag-tagged Mettl8 protein with anti-Flag magnetic beads after co-transfection into HEK293T cells. (H) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (I) Schematic diagram of the tumor model: Mettl8^fl/flCd4^cre mice were subcutaneously injected with 2 × 10⁵ B16F10 cells and harvested after 13 days. (J) Representative flow cytometry plots and cumulative data show the frequency of Tcf1⁺ Tox⁺ cells gated on tumor-infiltrating CD8⁺ CD44⁺ T cells (right). n = 6 per group. (K) Schematic diagram of the OT-I–transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10⁵ EG7-OVA cells, followed by 2 × 10⁶ WT or Mettl8^−/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. Representative flow cytometry plots and cumulative data show the frequency of Tox⁺ cells gated on Tcf1⁺ OT-I cells. n = 6 per group. (L) The MFI of Tox gated on Tcf1⁺ OT-I cells of the mice in K. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F5.