Rib et al. Epigenetics & Chromatin (2018) 11:52 https://doi.org/10.1186/s13072-018-0222-0 Epigenetics & Chromatin RESEARCH Open Access Cycles of gene expression and genome response during mammalian tissue regeneration Leonor Rib1,2,3, Dominic Villeneuve1, Shilpi Minocha1, Viviane Praz1,2, Nouria Hernandez1, Nicolas Guex2*, Winship Herr1* and The CycliX Consortium Abstract Background: Compensatory liver hyperplasia—or regeneration—induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell prolifera- tion. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries. Results: During the frst 10–20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coor- dinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10–20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the frst internal exon, was preceded 5′ by H3K36me2; 3′ of the frst internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profles with abundant Pol II but little evident H3K4me3 or H3K36me3 modifcation, indicating that these modifcations are neither universal nor essential partners to Pol II transcription. Conclusions: PH and sham surgical procedures on mice reveal striking early post-operatory gene expression similari- ties followed by synchronized mRNA accumulation and epigenetic histone mark changes specifc to PH. Keywords: Gene expression, Transcription, Liver regeneration, Partial hepatectomy, Histone modifcation Background diferentiation-specifc genes and cell-proliferation genes In developing multicellular organisms, cells prolifer- are generally silenced as cells often exit the cell-division ate and diferentiate, and these processes are controlled cycle. Te resulting quiescent diferentiated cells possess by regulated gene expression. In embryonic develop- unique sets of active and repressed genes. Tese sets of ment, many cells proliferate via a cell-division cycle genes, however, can change as cells respond to physiolog- under the control of cell-proliferation genes; subse- ical changes such as feeding or circadian cycles. Moreo- quently, cells diferentiate through the activation of ver, cells can reenter the cell-division cycle as in the case of tissue regeneration. In eukaryotes, both active and repressed genes are *Correspondence: [email protected]; [email protected] 1 Center for Integrative Genomics, Génopode, University of Lausanne, packaged in nucleosome-containing chromatin in which 1015 Lausanne, Switzerland histones are reversibly modifed—often refecting the 2 Swiss Institute of Bioinformatics, Génopode, University of Lausanne, underlying gene transcription status. In this chroma- 1015 Lausanne, Switzerland Full list of author information is available at the end of the article tin context, we study how cyclical programs of gene © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rib et al. Epigenetics & Chromatin (2018) 11:52 Page 2 of 19 expression—i.e., circadian, nutrition and cell division— determination (ChIP-Seq) as listed in Additional fle 1: are regulated in diferentiated cells using the mouse liver Figure S1b. as model [1–4]. We focus on gene expression as meas- As illustrated in the hierarchical clustering dendrogram ured by RNA-transcript levels and study the relationship shown in Fig. 1a, the RNA-Seq analysis revealed that the between gene occupancy by RNA polymerase II (Pol II) triplicate livers had highly similar patterns of transcript and two specifc histone modifcations: histone H3 lysine abundance—in about three-quarters of cases the tripli- 4 trimethylation (H3K4me3) observed at active promot- cates were immediate neighbors. Even the three separate ers and histone H3 lysine 36 trimethylation (H3K36me3) sets of triplicate 0 h time points (C0), although clustering associated with the body of actively transcribed genes separately, displayed Pearson correlations of 0.94 or bet- (reviewed in [5]). Here, we describe how these gene- ter (Additional fle 1: Figure S1c). Globally, the clustering expression markers change during liver regeneration. dendrogram revealed three groups of samples (labeled By rapidly inducing proliferation of quiescent hepat- I, II, and III): Group I represents samples similar to the ocytes, the mammalian liver has a striking ability to resting 0 h C0 time point, Group II includes 4, 10, 20, compensate for cell loss caused by toxic substances or and 28 h post-PH samples along with 4- and 10-h sham surgical removal [6–8]. Tus, for example, removal of samples, and Group III represents 36–72 h post-PH sam- 70% of the liver mass via partial hepatectomy (PH) leads ples. Importantly, replicate samples never fell into dif- to synchronous cell-division-cycle reentry of most of ferent groups I–III, indicating that the PH protocol [10] the remaining hepatocytes. In mice, the frst round of was highly reproducible. Tus, in the ChIP-Seq analyses hepatocyte division is accomplished within 60 h post-PH; described below, where we pooled samples to have suf- subsequent cell-division cycles together with cell growth fcient material for analysis, the signals were probably not lead to regeneration of the complete mass of the liver— signifcantly blurred. compensatory hyperplasia—within 2–3 weeks [7–9]. We have used a characterized PH-induced mouse liver The post‑PH gene expression profle reveals two periodic regeneration protocol [10] to study how a program of cycles: an early cycle shared with sham‑surgery mice cell-division-cycle gene expression—dormant in the qui- followed by a PH‑specifc cycle escent liver—is re-activated in the context of a diferenti- To identify stage-specifc changes in gene expression, ated tissue. we used principal component analysis (PCA) to maxi- mally diferentiate patterns of transcript abundance Results among samples. Te PCA revealed frst (PC1) and sec- To analyze gene-expression changes associated with the ond (PC2) principal components with 0.30 and 0.19 cell-division cycle during liver regeneration, we inte- proportions of variances, respectively, thus accounting grated 70% PH into a dark/light and feeding-entrain- for nearly 50% of the variation in the samples. Figure 1b ment protocol described in [1] to study gene expression shows a PCA plot for the aggregated results of each through the circadian cycle. Prior to 70% PH, mice were time point and treatment (see Additional fle 1: Fig- entrained for 2 weeks on 12-h dark/12-h light cycles with ure S1d for standard deviations). Te analysis reveals a food provided only during the dark (waking) period (see robust initial response over 4 h that is shared by both Additional fle 1: Figure S1a; and Additional fle 2). PH the PH and sham samples (compare X1 to S1, and X4 was performed at Zeitgeber Time (ZT) 2, where ZT0 to S4), whereby both the PH and sham samples tran- represents the beginning of the light/fasting period, and sit from Group I to Group II of Fig. 1a. Te PH versus samples were collected at 1, 4, 10, 20, 28, 36, 44, 48, 60, sham similarity becomes progressively less pronounced and 72 h, as well as 1 and 4 weeks post-PH (labeled X). at 10–20 h (X10 vs. S10, and X20 vs. S20), yet there is To identify non-PH-related efects of the PH surgery, an evident shared clockwise trajectory—or “cycle”— we performed parallel sham surgeries in which all pro- for both the PH and sham samples. Te 20- and 48-h cedures but the liver resection were included and col- sham samples return to the resting state Group I lected samples at 1, 4, 10, 20 and 48 h post-sham surgery (Fig. 1a). In contrast, the PH samples form a new PH- (labeled S). specifc “cycle” progressing from Group II (X20 and We measured gene expression (1) at the transcript X28) to Group III (X36, X44, X48, X60, and X72) before level by ultra-high-throughput RNA-sequence determi- returning to Group I, where they remain at 1–4 weeks nation (RNA-Seq) of poly(A)-selected RNA from indi- post-PH (X1W and X4W). Te diference in change of vidual livers and (2) at the genomic level by measuring trajectory between the sham and PH samples is exem- Pol II density using chromatin-immunoprecipitation on plifed by the comparison of the S20-to-S48 vector pooled sets of the three livers used for RNA-Seq analy- (right to left) to the X20-to-X48 vector (left to right). ses followed by ultra-high-throughput DNA-sequence Tus, the PCA of the post-PH gene-expression profles Rib et al. Epigenetics & Chromatin (2018) 11:52 Page 3 of 19 Fig. 1 Gene-expression profles after sham and PH surgery. a Dendogram of hierarchical clustering of gene-expression profles for individual post-sham and post-PH samples.