title: Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes.
abstract: Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial-mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome-wide epigenetic studies with important guidance such as the appropriate time window and loci to be utilized as positive and negative controls.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Ziqing
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Olivia
authors.initials: O
authors.lastname: Chen
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Zheng
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Chaoying
authors.initials: C
authors.lastname: Yin
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Jiandong
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, United States; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States. Electronic address: li_qian@med.unc.edu.
authors.forename: Li
authors.initials: L
authors.lastname: Qian
mesh_terms: D000818:Animals; D002460:Cell Line; D065150:Cellular Reprogramming; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000378:metabolism; D000071417:Twist-Related Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D006657:Histones; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D014157:Transcription Factors; D051879:Twist-Related Protein 1; D000071417:Twist-Related Protein 2
keywords: ChIP; DNA methylation; Epigenetics; H3K27me3; H3K4me3; Reprogramming; iCM