Genome-wide analysis of histone marks identifying an epigenetic signature of promoters and enhancers underlying cardiac hypertrophy Roberto Papaita,b,1, Paola Cattaneoa, Paolo Kunderfrancoa, Carolina Grecoa, Pierluigi Carulloa,b, Alessandro Guffantic, Valentina Viganòc, Giuliano Giuseppe Stirparoa,d, Michael V. G. Latronicoa, Gerd Hasenfusse, Ju Chenf, and Gianluigi Condorellia,d,1 aHumanitas Clinical and Research Center, 20089 Rozzano, Italy; bInstitute of Genetics and Biomedical Research, National Research Council of Italy, 20098 Rozzano, Italy; cGenomnia srl, 20020 Lainate, Italy; dUniversity of Milan, 20098 Milan, Italy; eDepartment of Cardiology and Pneumology, Georg-August University, 37075 Göttingen, Germany; and fDepartment of Medicine, University of California, San Diego, La Jolla, CA 92093 Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 4, 2013 (received for review August 13, 2013) Cardiac hypertrophy, initially an adaptive response of the myo- Hitherto, most research on the epigenetics of heart failure has cardium to stress, can progress to heart failure. The epigenetic focused on the role of histone deacetylases (HDACs; e.g., HDAC5, signature underlying this phenomenon is poorly understood. Here, HDAC9, and HDAC2) and histone acetyl transferases (HATs; we report on the genome-wide distribution of seven histone mod- e.g., p300). These studies demonstrated a key role for these ifications in adult mouse cardiomyocytes subjected to a prohyper- enzymes in the cardiac hypertrophy program and, thus, suggest a trophy stimulus in vivo. We found a set of promoters with an role for histone acetylation in triggering the transcriptional changes epigenetic pattern that distinguishes specific functional classes occurring in cardiac hypertrophy and heart failure (11–14). How- fi of genes regulated in hypertrophy and identi ed 9,207 candidate ever, we still do not know which genes are regulated by histone active enhancers whose activity was modulated. We also analyzed modifications or how histone modifications influence transcrip- the transcriptional network within which these genetic elements fi tion in cardiac hypertrophy. act to orchestrate hypertrophy gene expression, nding a role Here, we describe epigenetic changes occurring in adult for myocyte enhancer factor (MEF)2C and MEF2A in regulating mouse cardiomyocytes subjected to a prohypertrophy stimulus enhancers. We propose that the epigenetic landscape is a key in vivo. determinant of gene expression reprogramming in cardiac hy- pertrophy and provide a basis for understanding the role of Results chromatin in regulating this phenomenon. Epigenetic Profile Changes in Hypertrophic Cardiomyocytes. To gain insight into the role epigenetics plays in heart failure, we gen- epigenetic regulation | histone acetylation | histone methylation erated genome-wide chromatin-state maps for normal and hy- pertrophic cardiomyocytes and compared them with the gene eart failure, a leading cause of mortality worldwide, is fre- expression profiles of those cells (Fig. 1A). To this end, we Hquently accompanied by cardiac hypertrophy, a process performed chromatin immunoprecipitation (ChIP) coupled with characterized by the expression of genes normally present during massively parallel DNA sequencing (ChIP-seq) on cardiomyocytes the fetal stage and the repression of certain genes expressed in isolated from the left ventricle of mice that had been subjected adults (1, 2). Although epigenetics is important in regulating to transverse aortic constriction (TAC), a surgical procedure that transcription, our understanding of its role in cardiac hypertro- phy remains scant (3). fi Histone marks, such as acetylation (ac) and methylation (me) Signi cance of lysine (K) residues of histone H3, play an important role in regulating transcription. Combinations of these marks create an Cardiac failure is a leading cause of mortality worldwide and “epigenetic code” that defines the transcriptional status of genes: a major financial burden for healthcare systems. New tools for high levels of monoacetylated (ac) Lys-9 and Lys-14 (H3K9ac understanding cardiovascular disease and developing better and H3K14ac) and trimethylated (me3) Lys-4 (H3K4me3) in therapeutic approaches are therefore needed. To this end, promoter regions, and trimethylated Lys-36 (H3K36me3) and di- transcriptional regulation has been extensively studied in car- methylated (me2) Lys-79 (H3K79me2) in the body of genes, are diac hypertrophy and failure, but there is still a lack of un- detected in transcriptionally active regions. In contrast, an ele- derstanding of the epigenetic framework in which transcrip- vated level of deacetylated histones and histone H3 trimethy- tion factors act. Our report adds significant knowledge to the lated on Lys-9 and Lys-27 (H3K9me3 and H3K27me3) are field because we demonstrate, in vivo, that a complex and associated with inactive regions (4–7). specific epigenetic signature regulates gene expression by Histone modifications also influence the activity of enhancers modulating promoters and enhancers, a large number of which involved in regulating gene expression during development and have been described here. These findings advance our un- cell differentiation. In human embryonic stem cells, specific derstanding of the mechanisms underlying this pathology. epigenetic signatures define the activity of enhancer elements involved in the early stages of embryogenesis: H3K27ac is a Author contributions: R.P. and G.C. designed research; R.P., P. Cattaneo, C.G., and P. Carullo performed research; G.H. and J.C. contributed new reagents/analytic tools; R.P., P. Cattaneo, mark of active (or class 1) enhancers, whereas a high level of P.K., C.G., A.G., V.V., G.G.S., and G.C. analyzed data; and R.P., M.V.G.L., and G.C. wrote H3K27me3 and the absence of H3K27ac are found in “poised” the paper. (or class 2) enhancers (8). The acetyltransferase and transcrip- The authors declare no conflict of interest. tional coactivator p300/CBP binds active enhancers in several This article is a PNAS Direct Submission. tissues and organs, including heart, during development (9). 1To whom correspondence may be addressed. E-mail: roberto.papait@humanitasresearch. Recently, a large set of enhancer elements involved in regulating it or [email protected]. cardiomyocyte gene expression during differentiation in vitro This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. was identified (10). 1073/pnas.1315155110/-/DCSupplemental. 20164–20169 | PNAS | December 10, 2013 | vol. 110 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1315155110 Downloaded by guest on October 1, 2021 Fig. 1. Genome-wide analysis of histone marks in adult cardiomyocytes isolated from pressure-overloaded and control mouse hearts. (A) Schematic of the experimental workflow. (B) Hierarchical clustering of H3K9ac (9ac), H3K27ac (27ac), H3K4me3 (4me3), H3K79me2 (79me2), H3K9me2 (9me2), H3K9me3 (9me3), and H3K27me3 (27me3) on the basis of their genomic distribution in normal (sham) and hypertrophic (TAC) cardiomyocytes. The heat map was generated with the DiffBind program. Also see Fig. S1B and Dataset S1. induces cardiac hypertrophy and then heart failure. Cardiomyocytes To obtain information on the biological role of genomic were isolated 1 wk after TAC, a time corresponding to the regions found with redistributed histone marks, we performed maximal activation of compensatory cardiac mechanisms (15, gene ontology (GO) analysis with the Genomic Regions En- 16). The purity of isolated cardiomyocytes was at least 95%, richment of Annotations Tool (21) (GREAT; default setting, 5 + as evaluated by counting the number of cells with and without 1 kb basal, up to 1 Mb extension) (Fig. S2B). The sites with the typical rod-shaped appearance (Fig. S1A). ChIP-seq was per- differential distribution of H3K9ac, H3K27ac, or H3K4me3 formed using antibodies against H3K9ac, H3K27ac, and H3K4me3, mapped preferentially to regulatory domains of genes involved in three marks associated with active regulatory regions; H3K79me2, heart function or epigenetic regulation of gene expression, a mark associated preferentially with transcribed genes (17); and whereas those with altered H3K79me2, H3K9me2, H3K9me3, or H3K9me2, H3K9me3, and H3K27me3, which are associated with H3K27me3 profiles were preferentially associated with genes repressed regions (4–7). regulating signal transduction (e.g., regulation of Rab-GTPase To identify the genomic regions undergoing a change of their activity and regulation of ARF GTPase activity) and organiza- histone mark profile on cardiac hypertrophy, we used two algo- tion and regulation of sarcomeric structure. Moreover, GO rithms, MACS (model-based analysis of ChIP-seq) (18) and SICER analysis for mouse phenotype revealed that sites with differential (spatial clustering approach for the identification of ChIP-enriched distribution of H3K9ac, H3K4me3, H3K79me2, or H3K27me3 regions) (19), to minimize the bias inherent in this procedure and were associated with hypertrophic heart phenotypes, such as compared the obtained peak datasets with DiffBind (differential thick ventricular wall, altered heart contraction, impaired skel- binding analysis of ChIP-seq peak data) (20). Hierarchical clus- etal muscle contractility, and abnormal cardiac stroke volume. tering analysis using affinity (read count) data revealed that the Together, these findings demonstrate that pressure-overload histone marks studied