Chromatin states at homeoprotein loci distinguish axolotl limb segments prior to regeneration [ATAC-seq]. Chromatin states at homeoprotein loci distinguish axolotl limb segments prior to regeneration [ATAC-seq]

The salamander limb regenerates only the missing portion. Each limb segment can only form segments equivalent to- or more distal to their own identity, relying on a property termed “positional information”. How positional information is encoded in limb cells has been unknown. By cell-type-specific chromatin profiling of upper arm, lower arm, and hand, we found segment-specific levels of histone H3K27me3 at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. This means that, in the hand segment, the permissive chromatin state of the hand homeoprotein gene HoxA13 engages with regeneration regulatory elements, bypassing the upper limb program. Profiling chromatin accessibility (ACRs) of Axoltol limb connective tissue cells, and compare among developmental limb buds, and mature limb at different segments and regeneration time points. Overall design: We performed ACR profiling analysis using data obtained from ATAC-seq libraries of developmental limb buds (stage 40/41, stage 46 proximal and distal parts), and mature limb at different segments (mature UA vs LA vs hand). Therefore, we also performed libraries from the mature arm and 5, 9, 13 dpa (13 dpa blastema data have two different positions like proximal and distal parts) regenerating arm blastema.

蝾螈肢体仅能再生缺失的部分。每个肢体节段仅能形成与其自身身份等价或更远端的节段，这依赖于一种被称为“位置信息（positional information）”的特性。此前，肢体细胞中的位置信息如何编码的问题尚不明确。我们通过对上臂、前臂和手部开展细胞类型特异性染色质图谱分析，发现肢体同源框蛋白基因座上存在节段特异性的组蛋白H3K27三甲基化（histone H3K27me3）水平差异，而其上游调控因子并无此特征，进而构成了一套内在的节段信息编码体系。在再生过程中，再生特异性调控元件会在发育调控元件重新出现前被激活。这意味着，在手部节段中，手部同源框蛋白基因HoxA13的开放染色质状态会与再生调控元件结合，从而绕过上肢发育程序。本研究对美西钝口螈（Axoltol）肢体结缔组织细胞的染色质可及性（chromatin accessibility, ACRs）进行了图谱分析，并比较了不同节段的发育肢芽、成熟肢体以及不同再生时间点的样本。实验设计概述：我们利用发育肢芽（第40/41期、第46期近端与远端部分）、不同节段成熟肢体（成熟上臂（upper arm, UA） vs 前臂（lower arm, LA） vs 手部）的ATAC-seq文库数据，开展了染色质可及性图谱分析。此外，我们还构建了成熟肢体以及截肢后5天、9天、13天（13 dpa的芽基数据包含近端与远端两个不同位置的样本）再生肢体芽基的文库。