Figure 4. Identification of direct target genes of the four survival TFs.

(A) A schematic diagram displaying integrative analysis of DNA-footprinting using ATAC-seq data and RNA-seq to identify each TF’s direct target genes. Chromatin-bound TF protects DNA elements from Tn5 transposase cleavage, creating single-nucleotide-resolution DNA “footprints” during ATAC-seq. Mapping these footprints would identify DNA regions directly bound by the TF, and the genes linked to TF footprinted regions. To identify genes that are directly regulated by each survival TF, RNA-seq was performed on RGCs with or without prior CRSIPR ablation of this TF at day 3 following injury. Genes that are footprinted by this TF and differentially expressed (up- or down-regulated, absolute logFC >0 and FDR < 0.1) upon ablation of this TF are considered as its direct target genes. N = 4–5 biological repeats in each condition, except for the group with ATF4 ablation (n = 2 biological repeats after outlier removal).

(B) A Chow -Rusky Venn diagram and an Upset plot showing the overlap of each TF’s direct targets. Similar to regular Venn diagram graphics, the Chow-Rusky Venn diagram is divided into 2ⁿ connected regions (n = 4 TF combinations). The regions forming loops indicate overlapped targets of colored TFs (e.g. 140 targets common to 4 TFs), while the other open connected regions indicate targets exclusive to certain sets of TFs (e.g. 820 targets unique to ATF3 that is red colored, but not to the other 3 TFs). In the Upset plot, the set size indicates the number of direct targets for each TF. The intersection size indicates the number of overlapped targets unique to dotted TF(s).

(C) A heatmap of correlation matrix showing similarity and dis-similarity in the expression levels of each TF’s direct target genes. Normalized RNA-seq counts of each TF’s direct target genes were correlated and clustered by the distances among samples from control RGCs (nontargeting sgRNA) or treated RGCs receiving individual CRISPR TF ablations. Colors in the heatmap indicate Pearson’s correlations between two samples. ATF3 and CHOP are more similar in the expression levels of their target genes, while ATF4 and C/EBPγ are more similar.

(D) Shared or unique target gene sets of each TF. Positive targets are defined as genes that are down-regulated (log2FC < 0, FDR < 0.1) when this TF is ablated. Negative targets are defined as genes that are up-regulated (log2FC > 0, FDR < 0.1) when this TF is ablated. Based on gene expression similarity in (C), each TF’s positive or negative targets were grouped together and clustered into three gene sets: a) genes that are uniquely controlled by ATF3 and CHOP, b) genes that are uniquely controlled by ATF4 and C/EBPγ, and c) genes that are shared by ATF3/CHOP and ATF4/C/EBPγ groups. Gene ontology on these three gene sets were performed and related GO terms with enrichment FDR value < 0.05 were presented.