Chromatin dynamics enable transcriptional rhythms in the cnidarian Nematostella vectensis

In animals, circadian rhythms are driven by oscillations in transcription, translation, and proteasomal degradation of highly conserved genes, resulting in diel cycles in the expression of numerous clock-regulated genes. Transcription is largely regulated through the binding of transcription factors to cis-regulatory elements within accessible regions of the chromatin. Chromatin remodeling is linked to circadian regulation in mammals, but it is unknown whether cycles in chromatin accessibility are a general feature of clock-regulated genes throughout evolution. To assess this, we applied an ATAC-seq approach using Nematostella vectensis, grown under two separate light regimes (light:dark (LD) and constant darkness (DD)). Based on previously identified N. vectensis circadian genes, our results show the coupling of chromatin accessibility and circadian transcription rhythmicity under LD conditions. Out of 180 known circadian genes, we were able to list 139 gene promoters that were highly accessible compared to common promoters. Furthermore, under LD conditions, we identified 259 active enhancers as opposed to 333 active enhancers under DD conditions, with 171 enhancers shared between the two treatments. The development of a highly reproducible ATAC-seq protocol integrated with published RNA-seq and ChIP-seq databases revealed the enrichment of transcription factor binding sites (such as C/EBP, homeobox, and MYB), which have not been previously associated with circadian signaling in cnidarians. These results provide new insight into the regulation of cnidarian circadian machinery. Broadly speaking, this supports the notion that the association between chromatin remodeling and circadian regulation arose early in animal evolution as reflected in this non-bilaterian lineage.

在动物体内，昼夜节律（circadian rhythms）由高度保守基因的转录（transcription）、翻译（translation）及蛋白酶体降解（proteasomal degradation）的振荡所驱动，进而使众多时钟调控基因（clock-regulated genes）的表达呈现昼夜循环特征。转录过程主要通过转录因子（transcription factors）结合至染色质（chromatin）可及区域内的顺式调控元件（cis-regulatory elements）得以调控。在哺乳动物中，染色质重塑（chromatin remodeling）与昼夜节律调控存在关联，但目前尚不明确染色质可及性的周期变化是否是进化过程中时钟调控基因的普遍特征。为评估这一科学问题，本研究采用转座酶可及性测序（ATAC-seq）技术，以在两种不同光照制度下培养的星状海葵（Nematostella vectensis）为实验材料：光暗循环组（light:dark, LD）与持续黑暗组（constant darkness, DD）。基于此前已鉴定的星状海葵昼夜节律基因，本研究结果显示，在LD条件下，染色质可及性与昼夜转录节律存在显著耦合关联。在180个已注释的昼夜节律基因中，我们共筛选出139个相较于普通启动子具有高度可及性的基因启动子（gene promoters）。此外，在LD条件下我们鉴定得到259个活性增强子（active enhancers），而DD条件下的活性增强子数量为333个，两组处理共共享171个增强子。本研究通过建立高度可重复的ATAC-seq实验方案，并整合已发表的RNA测序（RNA-seq）与染色质免疫共沉淀测序（ChIP-seq）数据库，发现了一系列转录因子结合位点（transcription factor binding sites）的富集现象（如CCAAT增强子结合蛋白（C/EBP）、同源框（homeobox）以及MYB家族转录因子），而这些因子此前尚未被发现与刺胞动物（cnidarians）的昼夜节律信号通路相关。本研究结果为揭示刺胞动物昼夜节律机制的调控过程提供了全新视角。总体而言，本研究支持如下观点：染色质重塑与昼夜节律调控之间的关联早在动物进化早期就已出现，这一点可从该非两侧对称动物类群（non-bilaterian lineage）中得到印证。