During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. In contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts reveals differential reactivation kinetics for key targets of Brn2 and Ascl1, with transcription onset detected in early (anaphase) versus late (early G1) phases, respectively. Moreover, by using a mitotic-specific dominant-negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin. Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition.

中文翻译：

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神经干细胞中 Brn2 和 Ascl1 在有丝分裂到 G1 转变过程中转录的分级再激活

在有丝分裂期间，染色质浓缩伴随着转录的全局停滞。最近的研究表明，有丝分裂退出时的转录重新激活发生在时间协调的波中，但潜在的监管原则尚未阐明。特别是，序列特异性转录因子 (TF) 的贡献仍然知之甚少。在这里，我们报告了神经干细胞身份的重要调节因子 Brn2 在整个细胞分裂过程中与浓缩染色质相关，正如通过增殖神经干细胞的活细胞成像所评估的那样。相反，神经元命运决定因子 Ascl1 与有丝分裂染色体分离。ChIP-seq 分析表明，Brn2 有丝分裂染色体结合在有丝分裂退出前不会导致序列特异性相互作用，主要依赖于静电力。然而，使用单分子 RNA-FISH 对未成熟转录本的活性转录进行调查揭示了 Brn2 和 Ascl1 关键靶标的不同再激活动力学，分别在早期（后期）和晚期（G1 早期）阶段检测到转录起始。此外，通过使用有丝分裂特异性显性失活方法，我们表明在有丝分裂退出期间与 Brn2 结合竞争会降低其靶基因的转录巢蛋白。我们的研究表明，TF 与有丝分裂染色体的差异结合（受其静电特性控制）在定义有丝分裂至 G1 过渡期间转录再激活的时间顺序方面具有重要作用。