Tissue Regeneration Enhancer Element Discovery by Chromatin Accessibility Profiling of Regenerating Zebrafish Fins. Tissue Regeneration Enhancer Element Discovery by Chromatin Accessibility Profiling of Regenerating Zebrafish Fins

Mammalian genomes likely encode all gene products required to regenerate an amputated limb, yet they lack the correct instructions for strategically modulating those gene products to accomplish limb regeneration. Zebrafish and many other non-mammalian vertebrates possess these instructions, which exist as gene cis-regulatory elements. Here, to identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified thousands of DNA regions from each tissue type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding changes in RNA levels. To determine whether these profiles revealed bona fide TREEs, we tested the sufficiency of several sequences in stable transgenic and mutant lines. These experiments identified new regulatory sequences near several induced and/or required genes, including fgf20a, cx43, and mdka, with unique expression domains for each confirmed TREE. Deletion of TREE sequences from the zebrafish genome revealed requirements and redundancies surrounding cis-regulatory regions. Our study generates a novel resource for dissecting the regulatory mechanisms of appendage regeneration. Overall design: Samples were enzymatically dissociated with Liberase and collected in biological triplicates for RNA and ATAC library preparation. Corresponding sample pools include: whole fin 0 dpa and whole fin 4 dpa whole fin 0 dpa and whole fin 1 dpa, and fibroblast 0 dpa and fibroblast 4 dpa.

哺乳动物基因组大概率可编码截肢肢体再生所需的全部基因产物，但缺少用于策略性调控这些基因产物以实现肢体再生的精准调控指令。斑马鱼及众多其他非哺乳类脊椎动物则具备这类调控指令，其以基因顺式调控元件（cis-regulatory elements）的形式存在。本研究为鉴定对斑马鱼鳍再生至关重要的候选组织再生增强子元件（tissue regeneration enhancer elements, TREEs），对未损伤及正在再生的尾鳍的整体组织或纯化成纤维细胞开展了转座酶可及性染色质测序（assay for transposase-accessible chromatin sequencing, ATAC-seq）。我们从每种组织类型中鉴定出数千个在再生过程中染色质可及性发生动态变化的DNA区域，并将这些区域与RNA水平发生对应变化的邻近基因进行关联。为确认上述分析所揭示的区域是否为真实存在的TREEs，我们在稳定转基因与突变株系中检测了多条序列的调控充分性。本实验在多个受诱导且/或功能必需的基因（包括fgf20a、cx43及mdka）附近鉴定到了新的调控序列，且每个经验证的TREE均拥有独特的表达结构域。对斑马鱼基因组中的TREE序列进行敲除后，我们揭示了顺式调控区域的功能需求与冗余特性。本研究为解析附肢再生的调控机制提供了全新的研究资源。实验设计：样本采用Liberase进行酶解解离，设置生物学重复三份，分别用于RNA文库与ATAC测序文库的构建。对应样本组合包括：完整尾鳍截肢后0天（0 dpa，截肢后天数days post amputation的缩写）与截肢后4天（4 dpa）样本、完整尾鳍0 dpa与截肢后1天（1 dpa）样本，以及成纤维细胞0 dpa与4 dpa样本。