Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration

Abstract

During liver regeneration, most new hepatocytes arise via self-duplication; yet, the underlying mechanisms that drive hepatocyte proliferation following injury remain poorly defined. By combining high-resolution transcriptome and polysome profiling of hepatocytes purified from quiescent and toxin-injured mouse livers, we uncover pervasive alterations in messenger RNA translation of metabolic and RNA-processing factors, which modulate the protein levels of a set of splicing regulators. Specifically, downregulation of the splicing regulator ESRP2 activates a neonatal alternative splicing program that rewires the Hippo signaling pathway in regenerating hepatocytes. We show that production of neonatal splice isoforms attenuates Hippo signaling, enables greater transcriptional activation of downstream target genes, and facilitates liver regeneration. We further demonstrate that ESRP2 deletion in mice causes excessive hepatocyte proliferation upon injury, whereas forced expression of ESRP2 inhibits proliferation by suppressing the expression of neonatal Hippo pathway isoforms. Thus, our findings reveal an alternative splicing axis that supports regeneration following chronic liver injury.

Fig. 1: Mature hepatocytes activate neonatal gene expression patterns during liver regeneration.
Fig. 2: Regenerating hepatocytes exhibit pervasive shifts in ribosome occupancies at transcripts encoding RNA processing and metabolic factors.
Fig. 3: Altered translation of alternative splicing factors and activation of a neonatal splicing program in regenerating hepatocytes.
Fig. 4: Regeneration-regulated alternative exons are enriched in unstructured protein domains and phosphorylation sites.
Fig. 5: ESRP2 downregulation supports hepatocyte proliferation during liver regeneration.
Fig. 6: ESRP2 downregulation reprograms alternative splicing to generate neonatal isoforms of Hippo pathway proteins.
Fig. 7: Exclusive production of neonatal protein isoforms attenuates Hippo signaling, activates gene expression, and promotes hepatocyte proliferation.

Data availability

All raw RNA-seq and polysome profiling data files are available for download from the Gene Expression Omnibus under accessions GSE105944 and GSE106140. Data underlying the analyses in Figs. 1f–h, 2d, 3c,e, and 4a, and Supplementary Fig. 3j are available in Supplementary Dataset 2. Source data for Figs. 3a, 5d–h, and 7d, e, and Supplementary Figs. 4 and 7 are available in Supplementary Dataset 3. All other data are available from the authors upon reasonable request.

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Acknowledgements

We thank the members of the Kalsotra and Anakk laboratories for their valuable discussions and comments on the manuscript. This research was supported through NIH (R01HL126845) and March of Dimes (5-FY14-112) grants to A.K. and NIH (R01AI081710) to S.A. W.A. was supported by the NIH predoctoral NRSA fellowship (F30DK108567). J.S. was partly supported by the NIH Chemistry–Biology Interface Training Grant (5T32-GM070421) and the American Heart Association predoctoral fellowship (17PRE33670030). A.B. was partly supported by the Herbert E. Carter fellowship in Biochemistry, UIUC. Three cores at UIUC supported this project: Transgenic Mouse Facility Core, High-Throughput Sequencing and Genotyping Core, and Histology and Microscopy Core.

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S.B., W.A., J.S., and A.K. conceived the project and designed the experiments. S.B., W.A., J.S., A.B., J.C., and E.H.R. performed experiments. R.P.C. and S.A. provided reagents. S.B., W.A., J.S., and A.K. interpreted results and wrote the manuscript. All authors discussed the results and edited the manuscript.

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Correspondence to Auinash Kalsotra.

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Supplementary Figure 1 Validation of DDC-induced liver injury/regeneration in mice and reproducibility between hepatocyte-specific RNA-seq experiments.

a,b, Alanine aminotransferase (ALT) (a) and aspartate aminotransferase (AST) (b) levels in the serum of chow-fed mice (n = 6 animals) or 4-week DDC-fed mice (n = 3 animals). For a and b, parametric t test, unpaired with Welch’s correction, two-sided. The center value represents the mean, and error bars show s.d. c, Representative histology and immunofluorescence of chow- and DDC-fed mouse liver sections. First column: black arrows point to porphyrrin plugs, hepatic necrosis, and inflammatory cells. PV, portal vein. Second column: white arrows point to ductular hyperplasia (KRT19, red). Third column: white arrows point to proliferating hepatocyte nuclei co-stained for Hnf4-a (green) and pHistone3 (red). These experiments repeated with n = 3 animals in each condition. d, TPM (n = 2 biologically independent animals/condition) of cell-type-specific markers from hepatocytes and whole liver RNA-seq for chow- and DDC-fed conditions. e, Relative mRNA expression (qPCR) of hepatocyte- and non-parenchymal cell (NPC)-specific gene markers in hepatocytes isolated from embryonic day 18 (E18) mouse livers. fh, Scatterplots of TPM values derived from paired-end RNA-seq biological replicate experiments. The number of genes with TPM >1 and Pearson correlation coefficients are indicated.

Supplementary Figure 2 Overlap of mRNA abundance and gene ontologies between development and regeneration clusters.

a, Histogram of the percentile score distribution of E18-specific (left; bottom 50% adult, top 50% E18) and adult chow-specific (right; top 50% adult, bottom 50% E18) genes. b, Gene Ontology (GO) terms of upregulated and downregulated genes in development and regeneration. ce, Enrichment map of GO categories for each cluster are shown below (P < 0.05). The node size is proportionate to the number of genes related to each GO category, and the thickness of edges is proportionate to the number of shared genes between categories. For be, the sample size used was the same as in Fig. 1, n = 2 biologically independent animals/condition. Gene ontology enrichment analysis was performed using DAVID, with the expressed gene set as background and adjusting P values to account for multiple testing.

Supplementary Figure 3 Experimental approach and reproducibility of hepatocyte-specific in vivo polysome profiling from mouse livers.

a, Schematic description of the experimental procedure. Mouse livers were perfused with cycloheximide to stall ribosomes prior to collagenase digestion. The cytoplasmic lysate from purified hepatocytes was partitioned in a 15–45% sucrose gradient. A representative polysome profile is shown from adult mouse hepatocytes. RNA purified from different fractions was further processed to make sequencing libraries. bi, Scatterplots of TPM values (biological replicates) derived from hepatocyte sequencing data for cytoplasmic (b,c), heavy polysome (d,e,), monosome (f,g), and light polysome (h,i) fractions prepared from chow- and DDC-fed mouse livers. Spearman correlation coefficients are indicated for each scatterplot. j, Scatterplot comparing ribosomal occupancies and gene expression changes in chow and DDC conditions for RNA-binding proteins and transcriptional factors. The sample size for bj was n = 2 biologically independent animals/condition.

Supplementary Figure 4 Reversal of hepatocellular damage and regeneration response after DDC recovery.

a, Representative histology and immunofluorescence analysis of wild-type mouse liver sections on chow (n = 6 biologically independent animals) after 4 weeks of the DDC diet (n = 4) (injury phase) followed by 4 weeks of regular chow diet (recovery phase) (n = 5). First column: yellow arrows point to porphyrrin plugs and black arrows point to hepatic necrosis and inflammatory cells. PV, portal vein. Second column: white arrows point to proliferating hepatocyte nuclei co-stained for Hnf4-a (green) and pHistone3 (red) and orange arrows point to proliferating non-hepatocytes stained with pHistone3 only. 3rd column: White arrows point to ductular hyperplasia (KRT19, red). bd, Quantifications of liver to body weight ratios (hepatosomatic index) (b), hepatocyte proliferation index (c), and ductular reaction (d) during the injury and recovery phases. In bd, each point indicates the value for a single field of view in the section (n = 5 fields/animal). Data plots are mean ± s.d.; t test (two-tailed) with Welch’s correction was used to determine P values, and P < 0.05 was considered significant. e, Western blot showing hepatic ESRP2 protein levels after different days of DDC injury. TBP served as a loading control. The experiment was repeated independently three times; a representative image is shown.

Supplementary Figure 5 Generation of tetracycline-inducible hepatocyte-specific ESRP2 transgenic mice.

a, The TRE-ESRP2 construct expressing mouse ESRP2 cDNA with an N-terminal FLAG tag is driven by a tetracycline response element (TRE) and CMV minimal promoter. TRE-ESRP2 hemizygous transgenic mice were mated with ApoE-rtTA hemizygous transgenic mice to generate TRE-ESRP2; ApoE-rtTA bitransgenic mice as shown. Eight-week-old adult bitransgenic and littermate control (ApoE-rtTA) mice were fed doxycycline (Dox)-containing diet for 2 weeks to induce FLAG-ESRP2 expression specifically in hepatocytes. b, Western blotting against FLAG shows that exogenous ESRP2 in the liver is only expressed when Dox is present in the diet. Western blotting against ESRP2 shows the relative amount of induction compared to littermate controls. TBP was used as a loading control. c, FLAG and ESRP2 western blots showing regulated and dose-dependent hepatic ESRP2 protein expression after different Dox titrations in the diet. d, Western blot for FLAG and ESRP2 showing relative expression in the liver after induction with 0.5 g/kg Dox under chow and DDC injury conditions. All blots were repeated independently three times.

Supplementary Figure 6 RT–PCR validations and analyses of alternative splicing for the core Hippo pathway genes in WT, Esrp2 KO and ESRP2 overexpression livers under chow and DDC conditions.

a, Reciprocal regulation of Arhgf10l and Lsm14b alternative exons in Esrp2 KO and ESRP2 overexpression (OE) livers under chow-fed conditions. Skipping of these alternative exons after DDC-induced liver injury is rescued upon ESRP2 overexpression. The numeral after the gene name signifies the size of the alternative exon. The bands corresponding to (+) indicate exon inclusion and (–) indicate exon skipping. Percent spliced in (PSI) data are shown as the mean ± s.d.; n = 3. b, A simplified schematic of the Hippo signaling pathway. The core genes within the pathway that harbor ESRP2-regulated alternative exons (Nf2, Csnk1d, Yap1, and Tead1) are highlighted in orange. c, RT–PCR validation of ESRP2-mediated alternative splicing for the core Hippo pathway genes under chow- and DDC-induced liver injury conditions. PSI data are shown as the mean ± s.d.; n = 3. d,e, Western blotting for ESRP2 and TBP (d) and RT–PCR analysis of exon inclusion levels of the four Hippo pathway genes (e) 48 h after siRNA-based ESRP2 depletion in AML12 hepatocytes. PSI data are shown as the mean ± s.d.; n = 3. f, Map of upstream and downstream introns of Nf2, Csnk1d, Yap1 and Tead1 alternatively spliced exons showing occurrences of the ESRP2 binding motif (Fig. 6a).

Supplementary Figure 7 Targeting design and validation of antisense oligonucleotide specificity in switching the splicing pattern of core Hippo pathway genes.

a, Schematic of antisense oligonucleotide (ASO) design for targeting the 45nt Nf2, 63nt Csnk1d, 48nt Yap1 and 12nt Tead1 alternative exons. The alternative exons are shown in black, and the upstream and downstream constitutive exons are shown in grey colors. Red comb indicates the site of ASO complementarity to the pre-mRNA. ss: splice site. b, RT-PCR analyses of three unrelated ESRP2 regulated alternative exons in AML12 cells show no difference following treatment with control or a mixture of Hippo targeting ASOs demonstrating their target specificity. Percent spliced in (PSI) data are mean ± s.d.; n = 3. c, Relative mRNA levels of indicated Hippo pathway genes Nf2, Csnk1d, Yap1, and Tead1 normalized to Tbp (qPCR) following treatment with control or a mixture of Hippo targeting ASOs. Data are mean ± s.d.; n = 3. d, ChIP-qPCR for Hnf4a and Birc5 promoter regions spanning TEAD binding sites. e, Western blot for YAP1 and TBP post control and Yap1 targeting siRNA treatment for 24 hours in AML12 cells. siRNA knockdown repeated independently confirmed twice. f, MTT assay for AML12 cells under various conditions. Each point represents a biological replicate (n = 5). All data plots are mean ± s.d.

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Bangru, S., Arif, W., Seimetz, J. et al. Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration. Nat Struct Mol Biol 25, 928–939 (2018). https://doi.org/10.1038/s41594-018-0129-2

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