Identification of genes regulated by REST in developing hearts

Selective suppression of cardiac gene expression by RE-1 silencing transcription factor (REST) in embryonic stage is essential for cardiogenesis and function; however, the underlying mechanism remains unclear. In this study, we show that REST suppression of cardiac genes during development is temporarily regulated through non-CpG methylation at REST binding sites. Gene expression analyses revealed that the expression of Hcn2 (hyperpolarization-activated cyclic nucleotide-gated ion channel 2), a REST-targeted gene, was developmentally upregulated, while DNMT3B level and REST expression was downregulated. Concurrently, bisulfate sequencing and chromatin immunoprecipitation (ChIP) analyses showed that non-CpG methylation of the REST site located in the Hcn2 intron 1 and its co-occupancy by REST and DNMT3B were diminished at E?. Further in vitro inhibition and in vivo myocardial deletion of Rest and Dnmt3b uncovered that non-CpG methylation of the REST site by DNMT3B promoted REST binding to suppress Hcn2 transcription in the embryonic stage. Moreover, using an integrated approach of transcriptomic analysis, ChIP assay, and bisulfate sequencing, we showed that the same mechanism regulated the expression of ~30% REST targeted genes. The expression of these genes was developmentally upregulated as Hcn2. Together, our results suggest that non-CpG methylation at a subset of transcription factor binding sites may regulate cardiac gene program critical for heart development and its dysregulation may contribute to heart disease. we performed RNA-seq analysis of E12.5 wild type (WT) and Rest KO ventricles to identify REST targets. Each group included three biological replicates, by pooling from 4 hearts for each replicate胚胎阶段中，RE-1沉默转录因子（RE-1 Silencing Transcription Factor, REST）对心脏基因表达的选择性抑制，对于心脏发生与心脏功能维持至关重要，但其背后的分子机制尚未明确。本研究发现，发育过程中REST对心脏基因的抑制作用，可通过REST结合位点处的非CpG甲基化进行暂时性调控。基因表达分析显示，作为REST靶向基因的Hcn2（超极化激活环核苷酸门控离子通道2，Hyperpolarization-activated Cyclic Nucleotide-gated Ion Channel 2）的表达随发育进程上调，而DNMT3B水平与REST的表达则随之下调。与此同时，亚硫酸氢盐测序与染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）分析结果显示，位于Hcn2内含子1的REST结合位点的非CpG甲基化，以及REST与DNMT3B对该位点的共同占据，在E?时期均有所减弱。进一步通过体外抑制实验与体内心肌Rest和Dnmt3b基因敲除实验发现，DNMT3B介导的REST结合位点非CpG甲基化，可在胚胎阶段促进REST结合以抑制Hcn2的转录。此外，通过整合转录组分析、ChIP实验与亚硫酸氢盐测序的联合分析方法，本研究证实该调控机制可影响约30%的REST靶向基因的表达。这些基因的表达均如Hcn2一般，随发育进程而上调。综上，本研究结果表明，部分转录因子结合位点处的非CpG甲基化，可调控心脏发育所必需的心脏基因表达程序，而该调控过程的失调或可诱发心脏疾病。本研究对E12.5野生型（Wild Type, WT）与Rest敲除（Rest KO）小鼠的心室组织开展了RNA测序（RNA-seq）分析，以筛选REST的靶向基因。每组设置3个生物学重复，每个重复由4个心脏组织样本混合制备。