Genome-wide DNA methylation analysis reveals dynamic changes in the cardiac methylome during post-natal heart development (RNA-Seq)

Epigenetic modifications have emerged as central players in the coordination of gene expression networks during cardiac development. While several studies have investigated the role of histone modifications during heart development, relatively little is known about the role of DNA methylation. The purpose of the current study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the neonatal period, which is an important developmental window for cardiac maturation and cardiomyocyte cell cycle arrest. We used methyl binding domain protein sequencing (MBD-seq) and mRNA-seq to profile DNA methyation and gene expression respectively in neonatal hearts at P1 and P14 stages. Thousands of differentially methylated regions (DMRs) were identified between P1 and P14, the vast majority of which were hypermethylated. Gene ontology analysis revealed that these hypermethylated genes were associated with transcriptional regulation of important developmental signaling pathways, including Hedgehog, BMP, TGF beta, FGF and Wnt/b-catenin signaling. A significant enrichment for myogenic transcription factors and Smad2/3/4 binding sites was also noted among differentially methylated peaks at P14. This study provides novel evidence for widespread alterations in DNA methylation during post-natal heart maturation and suggests that DNA methylation plays an important role in cardiomyocyte cell cycle arrest during the neonatal period. Overall design: mRNA-seq to profile gene expression in neonatal hearts at P1 and P14 stages (post-natal day 1 and 14 respectively) in three biological replicates.

表观遗传修饰（epigenetic modifications）现已成为调控心脏发育过程中基因表达网络的核心环节。既往多项研究已探讨了组蛋白修饰在心脏发育中的作用，但关于DNA甲基化（DNA methylation）的功能，目前认知仍相对有限。本研究旨在明确DNA甲基化是否在新生期——这一心脏成熟与心肌细胞周期停滞的关键发育窗口——的转录组变化中发挥重要调控作用。
本研究分别采用甲基结合域蛋白测序（methyl binding domain protein sequencing, MBD-seq）与mRNA测序（mRNA-seq），对出生后第1天（P1）和第14天（P14）的新生小鼠心脏的DNA甲基化水平与基因表达谱分别进行了分析。研究在P1与P14样本间鉴定出数千个差异甲基化区域（differentially methylated regions, DMRs），其中绝大多数呈现高甲基化状态。
基因本体（Gene Ontology, GO）富集分析显示，这些高甲基化基因参与了多条重要发育信号通路的转录调控，包括Hedgehog、骨形态发生蛋白（BMP）、转化生长因子β（TGF-β）、成纤维细胞生长因子（FGF）以及Wnt/β-连环蛋白（Wnt/β-catenin）信号通路。此外，在P14样本的差异甲基化峰中，肌源性转录因子与Smad2/3/4结合位点亦存在显著富集。
本研究为出生后心脏成熟过程中广泛存在的DNA甲基化改变提供了全新的实验证据，并提示DNA甲基化在新生期心肌细胞周期停滞过程中发挥了关键调控作用。
实验整体设计：采用mRNA测序（mRNA-seq）对P1和P14阶段（分别对应出生后第1天与第14天）的新生小鼠心脏的基因表达谱进行检测，每组设置3个生物学重复。