Germier Thomas, Kocanova Silvia, Walther Nike, Bancaud Aurélien, Shaban Haitham Ahmed, Sellou Hafida, Politi Antonio Zaccaria, Ellenberg Jan, Gallardo Franck, Bystricky Kerstin
Laboratoire de Biologie Moléculaire Eucaryote (LBME), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Toulouse, France.
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Biophys J. 2017 Oct 3;113(7):1383-1394. doi: 10.1016/j.bpj.2017.08.014.
Genome dynamics are intimately linked to the regulation of gene expression, the most fundamental mechanism in biology, yet we still do not know whether the very process of transcription drives spatial organization at specific gene loci. Here, we have optimized the ANCHOR/ParB DNA-labeling system for real-time imaging of a single-copy, estrogen-inducible transgene in human cells. Motion of an ANCHOR3-tagged DNA locus was recorded in the same cell before and during the appearance of nascent MS2-labeled mRNA. We found that transcription initiation by RNA polymerase 2 resulted in confinement of the mRNA-producing gene domain within minutes. Transcription-induced confinement occurred in each single cell independently of initial, highly heterogeneous mobility. Constrained mobility was maintained even when inhibiting polymerase elongation. Chromatin motion at constant step size within a largely confined area hence leads to increased collisions that are compatible with the formation of gene-specific chromatin domains, and reflect the assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.
基因组动态与基因表达调控密切相关，而基因表达调控是生物学中最基本的机制，但我们仍然不知道转录过程本身是否驱动特定基因位点的空间组织。在这里，我们优化了ANCHOR/ParB DNA标记系统，用于对人类细胞中单个拷贝的雌激素诱导转基因进行实时成像。在新生的MS2标记mRNA出现之前和期间，在同一个细胞中记录了ANCHOR3标记的DNA位点的运动。我们发现，RNA聚合酶2引发的转录在几分钟内导致产生mRNA的基因结构域受到限制。转录诱导的限制在每个单细胞中独立发生，与初始的、高度异质的移动性无关。即使抑制聚合酶延伸，受限的移动性仍能维持。因此，在很大程度上受限区域内以恒定步长的染色质运动导致碰撞增加，这与基因特异性染色质结构域的形成相一致，并反映了在转录限速步骤中功能性蛋白质中心的组装和DNA加工过程。
