Axis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra. To gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity. The activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

中文翻译：

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九头蛇组织者形成过程中增强子元素的表观基因组学景观

在发育过程中轴模式伴随着大规模的基因表达变化。这些是由于组蛋白修饰的改变导致染色质结构的动态改变而引起的。顺式调节DNA元件也以依赖于上下文的方式在调节基因表达中起重要作用。九头蛇属于矢车菊门，在那里观察到人体计划中的第一个不对称并且起源于口腔轴。已经显示出Wnt信号传导决定了后生动物Hydra中的头部组织器功能。为了深入了解顺式调控元件和相关染色质特征的进化，我们在九头蛇中异位激活了Wnt信号通路，并监测了关键组蛋白修饰中的全基因组变化。九头蛇推定的基因间增强子元素的母题分析揭示了在发展中起关键作用的双侧顺式调节元件的保守性。在Wnt信号的异位激活后，鉴定到差异调节的增强子元件，并发现其可调节许多头部组织特异性基因。萤光素酶报告基因测定证实了许多已鉴定的顺式调控元件的增强子活性。Wnt信号激活后的定量染色质免疫沉淀分析进一步证实了H3K27ac在Hv_Wnt5a，Hv_Wnt11和头部组织基因Hv_Bra1，CnGsc和Hv_Pitx1的增强子元件上的富集。另外，使用特定的抑制剂对假定的H3K27me3橡皮擦活性的干扰影响了Wnt信号的异位激活，表明H3K27修饰的动态变化对调节涉及头部组织者活动的基因的重要性。与激活相关的组蛋白标记H3K4me3，H3K27ac和H3K9ac标记染色质的方式类似于在双语者中看到的方式。我们确定了基因间顺式调控元件，这些元件具有参与发育过程的关键转录因子的位点。差异调节的增强子表现出许多锌指，T-box和ETS相关TF的基序，它们的同源物在Hydra中具有头部特异性表达，并且可能是头部构图中先驱TF网络的一部分。