Changes in chromatin accessibility ensure robust cell cycle exit in terminally differentiated cells

During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle–independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.在终末分化过程中，大多数细胞会退出细胞周期，进入持久或永久的G0期（G0），此时它们对有丝分裂原信号不敏感。细胞进入G0期通常通过形成由二聚化伴侣（dimerization partner, DP）、成视网膜细胞瘤（retinoblastoma, RB）样蛋白、E2F及MuvB组成的转录抑制复合物DREAM，来抑制细胞周期基因的表达，从而启动该过程。然而，在终末分化过程中，若DREAM复合物的抑制功能受损，则会启动额外的未知机制，以稳定抑制细胞周期进程，并确保细胞可靠地退出细胞周期。本研究以黑腹果蝇（Drosophila melanogaster）翅膀为模型，提供证据表明：在少数关键细胞周期基因位点上，发育程序调控的染色质可及性时序变化，可在终末分化过程中强制细胞退出细胞周期。我们观察到，在终末分化阶段，针对关键限速细胞周期调控因子细胞周期蛋白E（Cyclin E, cycE）、E2F转录因子1（E2F transcription factor 1, e2f1）与string（stg）的一组蛹翅增强子区域的染色质会发生闭合，这一染色质闭合过程与翅细胞进入稳定的有丝分裂后状态同步，此时细胞难以被重新激活进入细胞周期；且发生闭合的区域包含有丝分裂原信号通路效应因子（如约基（Yorkie）与Notch）的已知结合位点。当细胞周期退出过程被遗传干扰后，细胞周期基因位点的染色质可及性并未受到影响，且cycE、e2f1与stg远端增强子的闭合过程仍会正常进行，不受细胞周期状态的影响。与之相反，细胞周期退出过程的遗传干扰会导致参与终末分化进程的一类激素诱导型转录因子的可及性与表达发生改变。本研究结果揭示了一种不依赖于细胞周期的计时机制，该机制可在终末分化组织中限制细胞对有丝分裂原信号的响应以及异常细胞周期进程。此外，本研究还为变态过程中细胞周期退出与终末分化之间的交叉调控提供了全新的分子层面描述。