Fig. 4: SOX4 binding silences hepatocyte genes while priming biliary genes by altering binding patterns.

a GSEA was performed with genes annotated to 4dpi ATAC-Seq peaks to compare empty vector and Sox4 hepatocytes using the hepatocyte-enriched and Rep_early-enriched signatures (see Supplementary Data 1, Fig. 2j, Methods). Heatmap visualization of the 5,272 genes near newly closed peaks (b) and 5,823 genes near newly opened peaks c using the RNA-Seq data of the DDC-induced reprogramming experiment (Fig. 2i, j) (n = 3 animals). Genes were categorized to four (b) or three (c) clusters by k-means clustering. Representative hepatocyte (39/50) and biliary/reprogramming genes (33/49 from a manually curated gene list; Supplementary Table 1) are shown on the right. Newly closed (d) and opened peaks (f) in Sox4 expressing hepatocytes at 4 dpi compared with empty vector hepatocytes (Fig. 3a) were annotated with the nearest genes, and this gene list was used as the input for GO enrichment analysis (n = 3 animals). GO analysis of RNA-Seq was also performed using genes downregulated (e) and upregulated (g) in Rep_intermediate cells vs Hep during DDC-induced reprogramming (n = 3 animals). The same analysis comparing different stages, namely Rep_early vs Hep and Rep_late vs Hep are also shown in Supplementary Fig 12a, b. GO terms shared between the newly-closed and newly opened region-associated gene set and DDC-induced reprogramming context at any reprogramming stages are highlighted in bold blue (d, e) and red (f, g) texts, respectively. h GSEA was performed with genes annotated to the SOX4 CUT&RUN peaks identified either at 18 hpi and 4 dpi using the hepatocyte-enriched and Rep_early-enriched signatures as described earlier (Supplementary Data 1, Fig. 2j, Methods). i Global footprint analysis depicted as a volcano plot. The analysis used all motifs assigned as “bound” by TOBIAS in either empty vector or Sox4 hepatocytes (n = 3 animals). Differential footprints are defined as those with Log2(fold-change of footprint score) >0.15 and log10(p value) < −100. Statistical testing was performed using the TOBIAS tool with the default setting. a, h Adjusted P values and NES values were calculated using the R fgsea package.