H3K27me3 deposition over sarcomeric and cytoskeletal promoters is required for cardiomyocyte cytokinesis and wound invasion during zebrafish heart regeneration [RNA-seq]

We identify the global transcriptional changes that occur between homeostatic and proliferative cardiomyocytes in the zebrafish heart and uncover an essential role for H3K27me3 deposition in facilitating successful myocardial regeneration. Specifically, we learned that cardiomyocyte proliferation is accompanied by downregulation of sarcomeric and cytoskeletal components and upregulation of the polycomb methylase Ezh2. Using ChIPseq, we demonstrate that this transcriptional repression is associated with deposition of new H3K27me3 modifications over the promoters. Using new genetic zebrafish lines that allow for inducible and cardiomyocyte-specific expression of a mutant form of histone 3 that is unable to be tri-methylated on lysine 27 (H3.3K27M), we discovered that addition of H3K27me3 marks is essential for cardiac regeneration in vivo. Earlier in the regenerative window, we found that H3.3K27M–expressing wound edge cardiomyocytes aberrantly maintain homeostatic levels of sarcomeric and actomyosin gene expression and show significant retention of sarcomere structure. While DNA replication occurs normally in these H3.3K27M cardiomyocytes, we observed significant increases in cardiomyocyte nucleation, a phenotype indicative of cytokinesis failures. In addition, nuclear density at the wound edge increases as new cardiomyocytes fail to colonize the injured area. Together, our study reveals that production of new cardiomyocytes and their infiltration into the injured region relies on H3K27me3-mediated sarcomeric and actomyosin cytoskeletal gene repression. Overall design: Transcriptional profiling (RNA-seq) of regenerting versus uninjured FACS-purified cardiomyocytes from zebrafish ventricle apexes in triplicates

本研究鉴定了斑马鱼心脏内稳态心肌细胞与增殖性心肌细胞之间的全局转录组变化，并揭示了组蛋白H3赖氨酸27三甲基化（H3K27me3）沉积在促进有效心肌再生中的关键作用。
具体而言，本研究发现心肌细胞增殖过程伴随肌节与细胞骨架组分的表达下调，以及多梳家族甲基转移酶Ezh2的表达上调。
通过染色质免疫共沉淀测序（ChIP-seq），本研究证实该转录抑制现象与启动子区域上新的H3K27me3修饰沉积存在关联。
本研究构建了可诱导且心肌细胞特异性表达组蛋白H3赖氨酸27无法被三甲基化的突变体（H3.3K27M）的转基因斑马鱼品系，借此发现H3K27me3标记的添加对于体内心脏再生至关重要。
在再生早期阶段，表达H3.3K27M的创面边缘心肌细胞异常维持了肌节与肌动球蛋白基因的稳态表达水平，并显著保留了肌节结构。
尽管这些H3.3K27M心肌细胞的DNA复制过程正常，但本研究观察到心肌细胞成核现象显著增多，该表型提示胞质分裂失败。
此外，由于新生心肌细胞无法定植于损伤区域，创面边缘的细胞核密度出现升高。
综上，本研究揭示，新生心肌细胞的产生及其向损伤区域的浸润依赖于H3K27me3介导的肌节与肌动球蛋白细胞骨架基因的转录抑制。
实验设计：对取自斑马鱼心室尖部、经荧光激活细胞分选（FACS）纯化的再生与未损伤心肌细胞进行转录组分析（RNA测序，RNA-seq），实验设置三次生物学重复。