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Enhancer selection dictates gene expression responses in remote organs during tissue regeneration

Abstract

Acute trauma stimulates local repair mechanisms but can also impact structures distant from the injury, for example through the activity of circulating factors. To study the responses of remote tissues during tissue regeneration, we profiled transcriptomes of zebrafish brains after experimental cardiac damage. We found that the transcription factor gene cebpd was upregulated remotely in brain ependymal cells as well as kidney tubular cells, in addition to its local induction in epicardial cells. cebpd mutations altered both local and distant cardiac injury responses, altering the cycling of epicardial cells as well as exchange between distant fluid compartments. Genome-wide profiling and transgenesis identified a hormone-responsive enhancer near cebpd that exists in a permissive state, enabling rapid gene expression in heart, brain and kidney after cardiac injury. Deletion of this sequence selectively abolished cebpd induction in remote tissues and disrupted fluid regulation after injury, without affecting its local cardiac expression response. Our findings suggest a model to broaden gene function during regeneration in which enhancer regulatory elements define short- and long-range expression responses to injury.

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Fig. 1: cebpd is induced in local and remote tissues during heart regeneration.
Fig. 2: cebpd expression features with different stimuli and in various tissues.
Fig. 3: Local and remote Cebpd requirements during heart regeneration.
Fig. 4: Profiling and transgenesis identify an enhancer downstream of cebpd that responds to injury.
Fig. 5: Corticosteroid signals regulate distant cebpd expression during heart regeneration.
Fig. 6: CEN is required for distant cebpd activation and fluid regulation after cardiac injury.

Data availability

RNA-seq and ATAC-seq data are deposited in the NCBI GEO database under accession nos. GSE158079 and GSE193630. The brain, kidney, heart, muscle and kidney H3K27ac, H3K4me3, WGBS and the deep sequencing of brain Hi-C data are downloaded from GEO: GSE13405534. Other heart-related ChIP-seq data were downloaded from GSE818624, GSE7589431 and GSE9692840. Unique reagents generated in this study, and all data supporting the findings of this study, are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Duke Zebrafish Core for animal care, Duke Center for Genomic and Computational Biology for advice, A. Dickson and K. Oonk for assistance with ISH, J. Kang, Y. Diao, J. A. Goldman, M. Pronobis, V. Cigliola, R. Yan, K. Ando, L. Slota-Burtt and R. Karra for comments on the manuscript and Y. Diao, J. Rawls, B. Black and N. Bursac for discussions. We acknowledge research support from NIH (R01 HL155607 to J.C.; R35 GM 124820 to F.Y.; R35 HL150713 and R01 HL136182 to K.D.P.) and from AHA and Fondation Leducq to K.D.P.

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Authors and Affiliations

Authors

Contributions

Conceptualization was provided by F.S. and K.D.P., wet lab investigations by F.S., A.R.S., J.C. and A.S., bioinformatic analysis by J.O., L.S., Y.L., H.Y., F.Y. and G.E.C., writing of the manuscript by F.S. and K.D.P., funding acquisition by K.D.P., G.E.C. and F.Y., and supervision by K.D.P., G.E.C. and F.Y.

Corresponding author

Correspondence to Kenneth D. Poss.

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The authors declare no competing interests.

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Nature Cell Biology thanks Josep Saura and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Bioinformatic analysis of brain RNA-seq during cardiac regeneration.

a, Gene Ontology analysis demonstrating biological pathways with gene enrichment. p < 0.05. Counts indicate the number of genes with significantly changed expression in each biological pathway. b, HOMER analysis demonstrating enriched transcription factor binding motifs at the promoter regions (upstream 2 kb to downstream 500 bp) of genes with increased brain RNA levels during heart regeneration.

Extended Data Fig. 2 Expression of cebpd is not induced in several tissues in adult zebrafish during heart regeneration.

ISH on sections of nasal epithelium, spleen, liver, skeletal muscle, and intestine, indicating that cebpd expression is not noticeably induced in these tissues during heart regeneration. Tissues were harvested 7 days after tamoxifen administration, which induced CM ablation in CreER + animals. n = 10 animals for all groups. Scale bars: 100 µm.

Extended Data Fig. 3 Cardiac muscle regeneration and fluid regulation in cebpd mutant animals.

a, Representative sections of cardiac ventricles 30 days after resection (dpa) in wild-type and cebpd-/- animals, stained with AFOG. Scale bar: 100 µm. n reported in (b). b, Semiquantitative assessment of cardiac injuries based on muscle and scar morphology, indicating no significant difference between cebpd-/- animals and their wild-type siblings. 1: Robust regeneration. 2: Partial regeneration 3: Blocked regeneration. A single trial was performed, with n = 11 animals for each group. Data were analysed using a Fisher’s exact test. c, Sections of 7 days post resection (dpa) ventricles staining with markers for CM nuclei (Mef2; green) and cell cycle entry (EdU; red). Dashed lines outline the approximate resection injury site. Scale bars: 100 µm. d, Quantification of the CM cycling index at 7 dpa indicating no significant differences in cebpd knockout animals compared to their wild-type siblings. A single trial was performed with n = 8 cebpd+/+animals, 9 cebpd+/− animals, and 12 cebpd−/− animals. Mean ± s.d.. Data were analysed using an unpaired two-tailed Student’s t-test. e, Fluorescence images of tailfin vasculature indicating that significantly less fluorescent dextran was able to reach the circulatory system of cebpd-/- fish after cardiac injuries. Data are quantified in Fig. 3. n = 16 CreER- and 21 CreER + animals for cebpd+/+, and n = 22 CreER- and 18 CreER + animals for cebpd-/-. Scale bars: 500 µm.

Source data

Extended Data Fig. 4 Transcriptome profiling of cebpd mutant brain and kidney during heart regeneration.

a,b, Volcano plot showing differential gene expression in the cebpd-/- brain (a) and kidney (b) during heart regeneration. 521 genes (158 genes increased and 363 genes decreased) and 1662 genes (787 increased and 875 decreased) were differentially regulated in the brain and kidney of cebpd-/- animals, respectively. Full lists of differentially expressed genes are shown in Supplementary Tables 2 and 3. Pink dots: highlighted genes. Blue dots: genes with decreased RNA levels (p < 0.05, FC < −1.2). Grey dots: genes with no significant changes. Orange dots: genes with increased RNA levels (p < 0.05, FC > 1.2). c, RNA-seq browser track of aqp7 showing decreased transcript levels of aqp7 (Log2FC = −1.062, p = 0.0012) in cebpd-/- kidney during heart regeneration. d, RNA-seq browser track of aqp10a showing decreased transcript levels of aqp10a (Log2FC = −1.045, p = 0.0020) in cebpd-/- kidney during heart regeneration. e, RNA-seq browser track of slc5a8l showing decreased transcript levels of slc5a8l (Log2FC = −0.515, p = 0.0034) in cebpd-/- kidney during heart regeneration. f,g, Gene Ontology analysis of differentially expressed genes in cebpd-/- brain during heart regeneration demonstrating top biological pathways (f) and molecular functions (g) with gene enrichment. Counts indicate the number of genes with significantly changed expression in Gene Ontology term. h,i, Gene Ontology analysis of differentially expressed genes in cebpd-/- kidney during heart regeneration demonstrating top biological pathways (h) and molecular functions (i) with gene enrichment. Counts indicate the number of genes with significantly changed expression in Gene Ontology term.

Extended Data Fig. 5 Two BAC sequences direct distinct gene expression patterns in zebrafish.

a, Two BAC transgenic lines show distinct larval EGFP fluorescence patterns. Boxed area is magnified on bottom left, and a magnified dorsal view is shown on bottom right. 108cebpd12:EGFP is prominent in skeleton and strong in jaw, indicated by yellow arrows. 35cebpd84:EGFP is weak in skin with low expression in jaw, indicated by yellow arrows. Scale bars: 500 µm. This expression is consistent in all animals used in this study. b, Two BAC transgenic lines show distinct adult EGFP fluorescence patterns. 108cebpd12:EGFP is strong in caudal fin rays and nasal cavity, indicated by yellow arrows. 35cebpd84:EGFP is weak in skin, indicated by yellow arrows. Scale bars: 500 µm. This expression is consistent in all animals used in this study.

Extended Data Fig. 6 Transcriptome and epigenetic analysis of whole kidney marrow.

a, Schematic of CM ablation and whole kidney marrow (WKM) collection for RNA-seq and ATAC-seq. b, Volcano plot showing differential gene expression in WKM after cardiac injury. Blue dots: genes with decreased RNA levels (p < 0.05, FC < −1.2). Grey dots: genes without significant changes. Orange dots: genes with increased RNA levels (p < 0.05, FC > 1.2). c, Gene ontology analysis of top biological pathways with gene enrichment. p < 0.05. Counts indicate the number of genes with significantly changed expression in each biological pathway. d, Heatmap of chromatin regions with changes in accessibility in the brain and kidney after CM ablation. p < 0.05. e, Heatmaps of RNA-seq and ATAC-seq data representing putative enhancer elements linked to genes with significant transcriptional changes in WKM after a cardiac injury. f, BiFET analysis indicating enriched transcription factor binding to open regions in WKM chromatin regions cardiac injury. Red: p < 0.05. Blue: p ≥ 0.05.

Extended Data Fig. 7 Epigenetic features of CEN and its sequence conservation across species.

a, A/B compartment analysis indicates a chromatin B to A compartment switch at cebpd and CEN loci. Dashed lines indicate cebpd and CEN region. b, Browser tracks indicating assays from this study and others. Brain ATAC-seq and Brain RNA-seq, uninjured and 7 days after CM ablation (7 dpi; this study); CM H3.3 occupancy, uninjured and 7 dpi (GSE81862); H3K27Ac occupancy, uninjured and 7 dpi (GSE75894); H3K27me3 and H3K4me3 occupancy in ventricular Gata4+ CMs, uninjured and 5 days post resection (GSE96928). Dashed lines indicate CEN. c, mVista plot of genomic regions around cebpd indicating high conservation of zebrafish CEN with cyprinid fish and low conservation with amphibians and mammals. Calculation window = 100 bp, conservation identity = 70%.

Extended Data Fig. 8 Expression of corticosteroid receptors in the brain and kidney.

a,b, RNA-seq browser track of nr3c1 (a) and nr3c2 (b) indicating that expression of nr3c1 in both brain and whole kidney marrow (WKM) are not significantly changed during heart regeneration, indicated as ‘injured’ in tracks. c, ISH on sections of brain and kidney from uninjured animals demonstrating nr3c1 and nr3c2 expression in multiple regions including ependymal cells and renal tubules. n = 5 animals for each group. Scale bar: 100 µm.

Extended Data Fig. 9 CEN is required for fluid homeostasis during heart regeneration.

Fluorescence images of tailfin vasculature indicating that less fluorescent dextran transferred to the circulatory system of CEN−/− fish after cardiac injuries. A single trial with n = 9 animals for both groups of CEN+/+, and n = 11 CreER- and n = 10 CreER + animals for CEN−/− animals, was performed. Data are quantified in Fig. 6. Scale bars: 500 µm.

Extended Data Fig. 10 Model describing CEN element functions in local and remote tissues.

(Top) Cardiac injury induces cebpd expression locally, regulating epicardial activation during heart regeneration. CEN is sufficient but not required for directing local cebpd induction, likely due to existence of redundant enhancers. (Bottom) Cardiac injury leads to transcriptional activation by bound corticosteroid receptors in remote tissues. CEN exists in an open, permissive structure, topologically close to its promoter and poised for corticosteroid receptor binding. Ligand-bound corticosteroid receptors trigger CEN-directed cebpd expression in distant tissues, contributing to fluid homeostasis in animals undergoing injury-induced regeneration.

Supplementary information

Reporting Summary.

Peer Review File.

Supplementary Tables

Supplementary Table 1 Differentially expressed transcripts from brain RNA-seq after cardiomyocyte ablation (FC > 1.2, P < 0.05). Two-sided Wald significance test performed with DESeq2. Supplementary Table 2 Differentially expressed transcripts from RNA-seq of wild-type and cebpd−/− brains after cardiomyocyte ablation (FC > 1.2, P < 0.05). Two-sided Wald significance test performed with DESeq2. Supplementary Table 3 Differentially expressed transcripts from RNA-seq of wild-type and cebpd−/− kidneys after cardiomyocyte ablation (FC > 1.2, P < 0.05). Two-sided Wald significance test performed with DESeq2. Supplementary Table 4 Differentially accessible chromatin regions from brain ATAC-seq after cardiomyocyte ablation (P < 0.05). Two-sided Wald significance test performed with DESeq2. Supplementary Table 5 Differentially expressed transcripts from whole kidney marrow RNA-seq after cardiomyocyte ablation (FC > 1.2, P < 0.05). Two-sided Wald significance test performed with DESeq2. Supplementary Table 6 Differentially accessible chromatin regions from whole kidney marrow ATAC-seq after cardiomyocyte ablation (P < 0.05). Supplementary Table 7 BiFET analysis of transcription factor binding sites in open regions of whole kidney marrow chromatin samples. MACS2-called peaks were analysed based on the hypergeometric distribution and P values were adjusted by the Benjamin–Hochberg procedure. Supplementary Table 8 Differential ATAC-seq chromatin regions paired with differentially expressed genes in the whole kidney marrow after cardiomyocyte ablation. Supplementary Table 9 BiFET analysis of transcription factor binding sites in open regions of brain chromatin samples. MACS2-called peaks were analysed based on the hypergeometric distribution and P values were adjusted by the Benjamin–Hochberg procedure. Supplementary Table 10 Differential ATAC-seq chromatin regions paired with differentially expressed genes in the brain after cardiomyocyte ablation. Supplementary Table 11 Footprint analysis of enriched transcription factor occupancy in brain during heart regeneration. Two-sided Fisher’s exact test. Supplementary Table 12 Summary of reagents and strains. a, List of experimental models. b, List of antibodies. c, List of chemicals. d, List of recombinant DNA. e, List of primers for generating ISH probes. f, List of primers for qPCR. g, List of software and algorithms.

Source data

Source Data Fig. 2

Statistical source data for Fig. 2.

Source Data Fig. 3

Statistical source data for Fig. 3.

Source Data Fig. 5

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Statistical source data for Extended Data Fig. 3.

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Sun, F., Ou, J., Shoffner, A.R. et al. Enhancer selection dictates gene expression responses in remote organs during tissue regeneration. Nat Cell Biol 24, 685–696 (2022). https://doi.org/10.1038/s41556-022-00906-y

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