Physiological reprogramming in vivo mediated by Sox4 pioneer factor activity

Abstract

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Fig. 1.. Exogenous expression of Sox4 is sufficient to induce biliary phenotypes in adult hepatocytes.

(a) Experimental design. AAV8 packaged with HA-tagged Sox4, Sox9, both (Sox4/9), or neither (EV), under the control of the TBG promoter, were injected into Rosa-LSL-Cas9-EGFP mice. After being maintained on a normal diet for 4–7d, liver tissue and percoll-enriched hepatocytes were harvested and used for immunofluorescence, flow cytometry and qRT-PCR, respectively. (b) Reprogramming efficiency assessed by flow cytometry at 7 dpi using Cd24 (early-to-intermediate) and Epcam (intermediate-to-late) markers (n = 3–5 per group). Representative flow plots are shown in Supplementary Fig. 4. (c) Representative immunofluorescence of a panel of reprogramming markers in liver tissue harvested from mice injected with AAV-empty vector (EV) or AAV-TBG-HA-Sox4 (Sox4) at either 4 dpi or 7 dpi timepoints. Arrows indicate EGFP+ hepatocyte-derived cells expressing the indicated reprogramming marker. Scale bar = 50 μm.

Fig. 2.. The chromatin landscape following ectopic expression of Sox4 recapitulates that of partially reprogrammed cells.

(a) Differential peak analysis identified 20,329 opened regions and 14,564 closed regions resulting from Sox4 expression in hepatocytes compared with empty vector (EV). DiffBind and DESeq2 packages were used with an FDR cutoff set to 0.01. (b) Results of DNA footprint analysis for the Sox4 binding motif comparing empty vector and Sox4 hepatocytes at newly opened and unchanged regions as defined in (a). TOBIAS software was used with the default setting, and motifs assigned “bound” either for empty vector or Sox4 hepatocytes were used for visualization of the averaged signals. (c) PCA mapping for ATAC-Seq of empty vector (EV) and Sox4 hepatocytes (n = 3 per group). ATAC-Seq was performed using percoll-enriched hepatocytes at 4 dpi of AAV-empty vector and AAV-HA-Sox4 injection. The data are shown in comparison with previously published data^,. The data obtained in this study were downsampled by approximately 1/100-fold to adjust read depth to be comparable to the previous data sets. Note that “Rep_int (intermed)” in this panel does not indicate Cd24+ cells but rather indicates Sox9+ cells sorted from Sox9-RFP reporter mice treated with DDC. (d) Quantification of Sox4 total binding was estimated using E-coli spike-in genomic DNA. Scale factors were calculated as (Total read counts aligned to mouse genome/Total read counts aligned to E-coli genome). Data are shown as fold-change compared to the mean value at 18 hpi data (n = 3). (e) Averaged aggregate plots for spike-in-scaled Sox4 CUT&RUN (C&R)-Seq data at newly closed and newly opened regions at the designated time points. (f) Sox4 CUT&RUN (C&R) signals (right two columns) are visualized as heatmaps for the newly opened and closed regions defined by ATAC-Seq (left two columns). The corresponding averaged aggregate plots for Sox4 CUT&RUN are shown in Extended Data Fig. 4f. Note that Sox4 data are normalized genome-wide in each sample (18 hpi and 4 dpi) and do not support quantitative comparison between the two timepoints. The numbers in brackets indicate number of regions.

Fig. 3.. 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 as described earlier (Table S1, Extended Data Fig. 3i, Methods). (b) Heatmap visualization of the 5,272 genes near newly closed peaks using the RNA-Seq data of the DDC-induced reprogramming experiment (Extended Data Fig. 3h). Genes were categorized to four clusters by k-means clustering. Representative hepatocyte genes (39/50 from a manually curated gene list; Table S2) are shown on the right. (c) Heatmap visualization of the 5,823 genes near newly opened peaks using the RNA-Seq data. The genes were categorized to three clusters by k-means clustering. Representative biliary/reprogramming genes (33/49 from a manually curated gene list; Table S2) are shown on the right. (d) 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 (Table S1, Extended Data Fig. 3i, Methods). “NA” was assigned when p.adj < 1e-50. (e) 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. Differential footprints are defined as those with Log2(fold-change of footprint score) > 0.15 and log10(pvalue) < −100.

Fig. 4.. Sox4 suppresses hepatocyte identity by inactivating hepatocyte enhancers.

(a) Newly closed regions with or without overlaps with 18hpi-Sox4 peaks were annotated with their nearest genes, and expression of each gene in empty vector or Sox4-expressing hepatocytes was compared with the DDC-induced reprogrammed cells. ****p < 0.0001. p-value was calculated by Wilcoxon rank sum test. (b) Heatmap representation of MNase-seq at low and high levels for the newly closed regions with or without overlaps with Sox4 peaks at 18 hpi along with active liver promoters and active liver enhancers. The corresponding averaged aggregate plots are shown in (c). (c) Averaged aggregate plots of hepatocyte MNase-Seq in newly closed regions with or without overlaps with Sox4 peaks (18 hpi) along with active liver promoters and active liver enhancers. (d) Heatmap representation of ChIP-seq for core histone H2B and H3 for the same regions as in (b). The corresponding averaged aggregate plots are shown in (e). (e) Averaged aggregate plots of H2B and H3 ChIP-Seq for the same regions as in (b). (f) ATAC-Seq and CUT&RUN-Seq of Sox4, H3K27ac, H3K4me1 and H3K27me3 signals visualized as heatmaps at active liver enhancer regions. The corresponding averaged aggregate plots are shown in Extended Data Fig. 6d,e. (g) Genome browser view of the Cyp2e1 gene is shown as an example of Sox4-induced closing of a hepatocyte gene-associated region. ChIP-Seq data were obtained from GEO datasets (GSE137066 for Hnf4a; GSE53736 for Rxra) reflecting normal liver cells.

Fig. 5.. Sox4 evicts hepatocyte transcription factors by hijacking their binding motifs.

(a) HOMER motif analysis with known motifs for 18hpi- and 4dpi- enriched Sox4 CUT&RUN peaks. 18hpi-enriched Sox4 peaks (n = 2,327) and 4dpi-enriched Sox4 peaks (n = 3,136) were identified using DiffBind and DESeq2 packages. The Hnf4a motif, highlighted in red, ranked first among 18 hpi-enriched Sox4 binding sites. (b) Consensus binding sequences of several hepatocyte transcription factors exhibiting partial overlap with that of Sox4 (overlapped sequences are underlined). (c) Averaged aggregate plots of DNA footprinting of Hnf4a and Rxra at the 18hpi-enriched Sox4 binding sites, 4dpi-enriched Sox4 binding sites and common Sox4 binding sites (upper panel). The data are shown as the comparison between empty vector and Sox4 hepatocytes. Motifs assigned “bound” by TOBIAS for either the empty vector or Sox4 hepatocytes were used for the analysis. The lower violin plots show the comparison of the footprint scores, indicative of the binding probability of the transcription factors, at each region. Bars indicate median values. p-values were calculated by Wilcoxon rank sum test.

Fig. 6.. Sox4 opens putative biliary cell enhancers in hepatocytes.

(a) EMSA assay to evaluate the binding ability of purified recombinant FLAG-Sox4 to naked LIN28B DNA and in vitro assembled nucleosomal LIN28B DNA. (b) Sox4 CUT&RUN-Seq data (right column) are shown for the more opened regions (MOR) and de novo opened regions (DOR), which correspond to newly opened regions with weak ATAC peaks in empty vector and those without ATAC peaks in empty vector (left two columns). Numbers in brackets indicate number of regions. (c) Heatmaps of previously published MNase-Seq data obtained for adult hepatocytes at low and high levels of MNase . The regions are centered with all the intersectable ATAC peaks of either empty vector or Sox4-expressing hepatocytes. Numbers in brackets indicate number of regions. (d) Averaged aggregate plots corresponding to the heatmaps shown in (c). (e) Heatmaps of previously published H2B and H3 ChIP-Seq obtained for adult hepatocytes . Regions are centered with all intersectable ATAC peaks of either empty vector or Sox4 hepatocytes. (f) Averaged aggregate plots corresponding to the heatmaps shown in (d). (g) Genome browser views of two examples for Sox4-induced opening at biliary epithelial cell genes: Itga3 (intermediate-to-late reprogramming marker) and Cd24a (early-to-intermediate reprogramming marker). Negative values for Sox4 tracks are not shown.