Transcriptome and Functional Analyses Reveal Roles For Regulators of Epigenetic States, Micro RNA Processing, And Long Non-Coding RNA In Myocyte Dedifferentiation: Insights Into Reprogramming A “Post-Mitotic” Cell

Purpose: The ability of adult zebrafish tissues to undergo dedifferentiation provides an opportunity to probe the molecular underpinnings of cell identity and reprogramming. Zebafish muscle regeneration utilizes dedifferentiation to reprogram mature multinucleated myocytes into dedifferentiated myoblast that re-enter the cell cycle. A unique advantage of this system is that the regenerating cell mass is large and fairly homogenous, facilitating genomics approaches to uncovering the underlying biology.Methods: To better understand cellular reprogramming of mature myocytes, we temporally analyzed the changing transcriptome leading up to the proliferative switch. RNA was obtained after Laser Micro-dissection (LMD) of Control, 9 hour post-injury (HPI) or 18 HPI using Trizol and micro column purification. Illumina's TruSeq Stranded mRNA Library Prep Kit and 0.1 - 4 µg total mRNA from pooled purified RNA samples were used for performing ribosomal-depletion (Ribo-Zero Gold rRNA Removal Kit, Illumina) and library preparation. Sequencing was performed by the UM DNA Sequencing Core, using an Illumina Hi-Seq 2000 (50-cycle, single end read) platform.Results: Clustering and functional annotation of differentially expressed genes highlighted the importance of catabolic and phagocytic processes upregulation at 9 and 18 hours post injury (hpi). Furthermore, genes encoding principle regulators of chromatin states were actively re-regulated during the reprogramming process. Utilizing the accessibility of these tissues in the zebrafish model, kKnockdown experiments enabled in vivo validation and phenotypic analysis of candidate genes and pathways for their roles in genomic and cellular reprogramming. Additionally, we found that despite of their low expression levels, lncRNAs were highly represented in gene clusters with dynamic, “switch-like” expression profiles, and that miRNA processing was also found important for reprogrammingConclusions: We conclude that reprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires both heritable yet nuanced epigenetic alterations and molecular switches that involve transcription factors, miRNA and lncRNA, while maintaining the lineage restriction of the cell of origin.

研究背景与目的：成年斑马鱼组织的去分化（dedifferentiation）能力为探究细胞身份与重编程的分子基础提供了绝佳契机。斑马鱼肌肉再生借助去分化过程，将成熟的多核肌细胞重编程为可重新进入细胞周期的去分化成肌细胞（dedifferentiated myoblast）。该系统的独特优势在于再生细胞团体量较大且均一性较好，便于通过基因组学方法揭示其背后的核心生物学机制。
研究方法：为深入解析成熟肌细胞的细胞重编程过程，我们对增殖转换前的动态转录组进行了时序分析。通过激光显微切割（Laser Micro-dissection, LMD）获取对照组、损伤后9小时（9 HPI）及损伤后18小时（18 HPI）的样本，采用Trizol法结合微量柱纯化提取总RNA。使用Illumina TruSeq链特异性mRNA文库制备试剂盒，取0.1~4 μg混合纯化后的总mRNA，经Ribo-Zero Gold核糖体RNA去除试剂盒（Ribo-Zero Gold rRNA Removal Kit, Illumina）去除核糖体RNA后完成文库构建。测序工作由密歇根大学DNA测序核心实验室（UM DNA Sequencing Core）完成，采用Illumina HiSeq 2000平台（50个循环，单端读长）进行测序。
研究结果：对差异表达基因进行聚类分析与功能注释后发现，损伤后9小时与18小时，分解代谢与吞噬相关的生物学过程显著上调，发挥了关键调控作用。此外，编码染色质状态主要调控因子的基因在重编程过程中被动态调控。依托斑马鱼模型组织的可操作性，我们通过基因敲降（knockdown）实验对候选基因及通路在基因组重编程与细胞重编程中的作用开展了体内验证与表型分析。此外，本研究发现，尽管长链非编码RNA（long non-coding RNA, lncRNA）的整体表达水平较低，但在具有动态“开关样”表达特征的基因簇中占比极高；同时，微小RNA（microRNA, miRNA）加工过程在重编程过程中也具有重要意义。
研究结论：本研究证实，将“有丝分裂后（post-mitotic）”肌细胞重编程为去分化成肌细胞，既需要可遗传且精细调控的表观遗传改变，也需要涉及转录因子、微小RNA与长链非编码RNA的分子开关，同时维持起源细胞的谱系限制性。