Extended Data Fig. 9: Genome-scale CRISPR screening identifies mitochondrial metabolism as an important downstream pathway of REGNASE-1 and BATF.
From: Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy
a, Chromatin accessibility heat maps normalized by row (z-score) for 7,480 genes with significantly increased chromatin accessibility (by |fold change (log2-transformed ratio)| > 0.5; P < 0.05) in Regnase-1-sgRNA-transduced OT-I cells as compared to control-sgRNA-transduced cells. Specifically, OT-I cells transduced with control sgRNA (mCherry+) (n = 4), Regnase-1 sgRNA (ametrine+) (n = 4), Batf sgRNA (GFP+) (n = 2) or Batf and Regnase-1 sgRNAs (GFP+ and ametrine+) (n = 4) were transferred into tumour-bearing hosts individually. OT-I cells were isolated from TILs at day 7 for ATAC-seq analysis. We annotated the differential accessibility regions in ATAC-seq for the nearest genes, and identified 7,480 genes with significantly increased chromatin accessibility in REGNASE-1-null cells as compared to wild-type cells. BATF co-deletion reversed the upregulated chromatin accessibility for a large proportion of these genes (5,052 in total). Also, 2,527 of these 5,052 genes showed significantly downregulated chromatin accessibility in BATF-null cells as compared to wild-type cells. b, Functional enrichment plots of the top-10 significantly (FDR < 0.05) enriched pathways in top-ranking depleted genes (n = 4 sgRNAs for each gene) identified in the genome-scale CRISPR screening (by less than −3.5 log2(TIL/input ratio); adjusted P < 0.05). c, GSEA enrichment plots of TIL Regnase-1-sgRNA-transduced OT-I cells using the OXPHOS Hallmark gene set. Specifically, control-sgRNA- and Regnase-1-sgRNA-transduced OT-I cells were mixed and transferred into tumour-bearing mice, and tumour-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by RNA-seq. d, Representative images (top) and quantification of mitochondrial volume (stained with TOM20, white) per cell (bottom) in control-sgRNA- (mCherry+; red) and Regnase-1-sgRNA-transduced OT-I cells (ametrine+; green) in tumours at 7 days after adoptive transfer (n = 4 mice). e, Oxygen consumption rate (OCR) bioenergetic profiling of control-sgRNA- and Regnase-1-sgRNA-transduced OT-I cells cultured in vitro for basal (left) and maximal (right) OCR (n = 9 samples per group). f, List of the top-2 significantly (FDR < 0.05) upregulated and top-8 significantly downregulated pathways in TIL Batf-and-Regnase-1-sgRNAs- versus Regnase-1-sgRNA-transduced OT-I cells (n = 3 samples per group) isolated from TILs, as revealed by performing GSEA using the Hallmark gene sets. Specifically, Regnase-1-sgRNA- and Batf-and-Regnase-1-sgRNA-transduced OT-I cells were mixed and transferred into tumour-bearing mice, and tumour-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by microarray. g, GSEA enrichment plots of TIL Batf-and-Regnase-1-sgRNAs- versus Regnase-1-sgRNA-transduced OT-I cells (n = 3 samples per group) using the OXPHOS gene set. h, OT-I cells transduced with control sgRNA (mCherry+; spike) were mixed at a 1:1 ratio with cells transduced with control sgRNA (ametrine+), Regnase-1 sgRNA (ametrine+), Batf sgRNA (GFP+) or Batf and Regnase-1 sgRNA (GFP+ and ametrine+), and transferred into tumour-bearing hosts individually (n = 4 mice per group). Mice were analysed at 5 days after adoptive transfer for quantification of the relative MFI of TMRM (left) and Mitotracker (right), normalized to spike in tumour-infiltrating OT-I cells. i, Chromatin accessibility heat maps normalized by row (z-score) for mitochondrial genes with significantly increased chromatin accessibility (by |fold change (log2-transformed ratio)| > 0.5; P < 0.05) in Regnase-1-sgRNA-transduced OT-I cells compared to control-sgRNA-transduced cells, determined by ATAC-seq as described in a. We annotated the differential accessibility regions in ATAC-seq for the nearest genes, and superimposed these genes with 1,158 mitochondrial genes defined in the MitoCarta 2.0 database. A total of 341 mitochondrial genes showed significantly upregulated chromatin accessibility in the absence of REGNASE-1, 214 of which were blocked by BATF co-deletion in BATF-null REGNASE-1-null cells. Moreover, 96 of these 214 genes showed significantly downregulated chromatin accessibility in BATF-null cells as compared to wild-type cells. Mean ± s.e.m. (d, e, h). *P < 0.05, **P < 0.01, ***P < 0.001. Two-sided Fisher’s exact test (a, i), right-tailed Fisher’s exact test (b), Kolmogorov–Smirnov test followed by Benjamini–Hochberg correction (c, f, g), two-tailed unpaired Student’s t-test (d, e) or one-way ANOVA (h). Data are representative of two (d, e) independent experiments, or pooled from two (h) independent experiments.
