Cell Death and Differentiation (2016) 23, 555–564 & 2016 Macmillan Publishers Limited All rights reserved 1350-9047/16 www.nature.com/cdd DOT1L-mediated H3K79me2 modification critically regulates gene expression during cardiomyocyte differentiation P Cattaneo1,2,3, P Kunderfranco1, C Greco1, A Guffanti4, GG Stirparo2,3, F Rusconi1, R Rizzi5,6, E Di Pasquale1,2, SL Locatelli2,3, MVG Latronico1, C Bearzi5,6, R Papait*,1,2 and G Condorelli*,1,2,3 Epigenetic changes on DNA and chromatin are implicated in cell differentiation and organogenesis. For the heart, distinct histone methylation profiles were recently linked to stage-specific gene expression programs during cardiac differentiation in vitro. However, the enzymes catalyzing these modifications and the genes regulated by them remain poorly defined. We therefore decided to identify the epigenetic enzymes that are potentially involved in cardiomyogenesis by analyzing the expression profile of the 85 genes encoding the epigenetic-related proteins in mouse cardiomyocytes (CMs), and then study how they affect gene expression during differentiation and maturation of this cell type. We show here with gene expression screening of epigenetic enzymes that the highly expressed H3 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) drives a transitional pattern of di-methylation on H3 lysine 79 (H3K79) in CMs at different stages of differentiation in vitro and in vivo. Through a genome-wide chromatin-immunoprecipitation DNA-sequencing approach, we found H3K79me2 enriched at genes expressed during cardiac differentiation. Moreover, knockdown of Dot1L affected the expression of H3K79me2-enriched genes. Our results demonstrate that histone methylation, and in particular DOT1L-mediated H3K79me2 modification, drives cardiomyogenesis through the definition of a specific transcriptional landscape. Cell Death and Differentiation (2016) 23, 555–564; doi:10.1038/cdd.2014.199; published online 19 December 2014 Cardiac morphogenesis is a complex process involving Here, we demonstrate that DOT1L H3K79 methyltransfer- rapid specification and differentiation of the cell types ase is required for CM gene transcription: Dot1L was highly – that form the heart.1 3 During embryonic development, the expressed in mouse CMs at embryonic and neonatal stages; activation of specific transcriptional programs defines the H3K79me2 marks mapped to lineage-specific CM genes in subsets of lineage-restricted genes that become expressed correlation with the transcriptional profile at distinct stages – while the rest of the genome is left in a repressed state.4 7 of differentiation; and a reduction in DOT1L interfered with Epigenetic regulation, such as via histone methylation, CM differentiation in vitro. establishes the global chromatin environment that stabilizes these transcriptional programs, and is thus a crucial phenom- enon for orchestrating these biological processes.8,9 Results Recent reports have shown that histone modification has a fundamental role in defining the transcription program of CM Dot1L is differentially expressed during differentiation differentiation in vitro. In fact, profiling of different methylation and maturation of CMs. To identify epigenetic enzymes marks on various lysines of H3, such as H3K4me1, H3K4me3, potentially involved in cardiomyogenesis, we analyzed the H3K27me3, and H3K36me3, revealed that distinct chromatin expression profile of 85 genes encoding epigenetic-related patterns are coordinated with stage-specific expression of proteins (15 histone acetyltransferases (HATs), 12 histone functionally relevant genes: programmed temporal alterations deacetylases (HDACs), 31 histone methyltransferases of chromatin structure distinguish the key regulator genes of (HMTs), 14 histone demethylases (HDMs), 2 DNA methyl- cardiovascular development from other genes.10,11 However, tranferases (DNMTs), and 11 chromatin-remodeling factors) the involvement of most epigenetic marks, and of the enzymes in two models: mouse embryonic stem cells (mES) at distinct that catalyze these modifications, remains poorly understood stages of differentiation (i.e., pluripotent cells, mesodermal during CM differentiation. precursors, cardiac progenitors, and differentiated CMs) and 1Laboratory of Cardiovascular Research, Humanitas Clinical and Research Center, Rozzano, Milan 20089, Italy; 2Operational Unit of Milan, Institute of Genetics and Biomedical Research, National Research Council of Italy (CNR), Rozzano, Milan 20089, Italy; 3Department of Biomedical Technologies and Translational Medicine, University of Milan, Milan 20133, Italy; 4GenOmnia, via Nerviano 31/b, Lainate, Milan, 20020, Italy; 5Casa di Cura Multimedica Istituto di Ricovero Cura Carattere Scientifico, Milan 20138, Italy and 6Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR), Rome 00100, Italy *Corresponding author: G Condorelli or R Papait, Cardiovascular Research, Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Milan, Italy. Tel: +39 02 8224521; Fax: +39 02 82245404; E-mail: [email protected] or [email protected] Abbreviations: ChIP-seq, chromatin immunoprecipitation-sequencing; CM, cardiomyocyte; CMa, adult cardiomyocyte; CMp, pup cardiomyocyte; DNMT, DNA methyltranferase; DOT1L, telomeric silencing-1-like H3K79 methyltransferase; GEO, Gene Expression Omnibus; H, histone; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDM, histone demethylase; HMT, histone methyltransferase; me, methylation; mES, mouse embryonic stem cell; TSS, transcription start site Received 23.6.14; revised 02.10.14; accepted 06.10.14; Edited by JC Marine; published online 19.12.14 DOT1L and cardiomyocyte differentiation P Cattaneo et al 556 mouse cardiac cells isolated at different stages of maturation Comparison of the methylation profiles across the genome (immature cells from E8.5, E10.5, E12.5, and E14.5 embryos; in mES, CMp, and CMa indicated that H3K4me3, H3K27me3, neonatal CMs from 1-day-old pups; and mature CMs purified and H3K79me2 became redistributed within various genomic from 2-month-old adult mice). The stages of cardiac regions during differentiation, whereas there was no signifi- differentiation and maturation were confirmed by measuring cant change in the distribution of H3K9me3 (Figure 2a). In the expression of markers of pluripotency (Pou5F1 and particular, H3K79me2 became increasingly more present Nanog), mesodermal populations (Eomes, Mesp1, and inside the gene body and less associated with the transcription Brachyury), cardiac progenitors or immature CMs (Nkx2-5, start site (TSS), whereas H3K4me3 shifted from the overlap Isl1, Gata4, and Tbx5), and mature CMs (Myh7 and Myh6; start region in mES to a region more proximal to the TSS, and Supplementary Figure 1). H3K27me3 decreased around the TSS, becoming localized We found that CM commitment and maturation are mainly up and downstream of the gene body in CMp and CMa. accompanied by a change in expression of several epigenetic These findings indicate that the histone methylation profile is enzyme genes already reported to be important for cardiac dynamic during CM differentiation. development (e.g., Jmjd3,12 Ezh213,14), as well as of HATs, To identify the genomic regions where these histone HDACs, HMTs, HDMs, and DNMTs not yet studied in the heart methylation marks became redistributed during cardiac differ- (Figures 1a and c). In particular, Dot1L, which encodes the entiation, we analyzed the genomic distribution of each mark only known H3K79 HMT, underwent a major differential with two algorithms (MACS (Model-based Analysis of ChIP- change in expression during CM differentiation and matura- seq) and SICER (Spatial Clustering Approach for the Identifica- tion. This finding was confirmed with conventional real-time tion of ChIP-Enriched Regions)) in order to minimize peak qPCR and immunoblotting (Figures 1b and d; Supplementary calling bias; peak data sets were then compared with 19 Figure 1). DiffBind. Hierarchical clustering analysis using affinity data In addition to the dynamic changes in Dot1L expression, we revealed that H3K79me2, like the other histone marks observed increased deposition of H3K79me2 subsequent to analyzed, was redistributed within the genome not only during the Dot1L expression peak (Figures 1b and d right panels, and CM specification (mES versus CMp) but also during CM Supplementary Figure 1). Interestingly, this epigenetic mark maturation (CMp versus CMa; Figure 2b). Interestingly, seems to be very stable, as it is abundantly found in adult CMs H3K79me2 in mES and CMp clustered with the clade contain- (CMa) even if these cells do not express high levels of DOT1L. ing H3K4me3, forming a branch with reduced similarity to the From these findings, we inferred that DOT1L is a key enzyme profile of H3K79me2 in CMa. This suggests that these two for CM differentiation and maturation. histone modifications mark similar regions in mES and CMp but not in CMa, and indicates that CM maturation is associated with redistribution of H3K79me2. This finding was corroborated by H3K792me2 regulates gene expression during CM differ- Principal Component Analysis, which indicated a higher entiation and maturation. To gain insight into the role of number of differential binding sites in mES versus CMa than DOT1L in cardiomyogenesis, we analyzed the genome-wide in mES versus CMp (Supplementary Figure 2). For each distribution of H3K79me2 in CMs during differentiation. histone modification