Individual transcription factors modulate both the micromovement of chromatin and its long-range structure [Hi-C]

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine High-resolution Diffusion mapping (Hi-D) and Dense Flow reConstruction and Correlation (DFCC) in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization. Hi-C in 3T3 with a doxycycline-inducible expression of CDX2, treated or not with doxycycline

真核基因表达的调控与高度动态的染色质结构密切相关。基因调控依赖于激活型与阻遏型转录因子（transcription factors, TFs），它们可介导局部染色质的开放与闭合。然而，目前尚不清楚全细胞核范围的染色质组织如何随特定转录因子的活性发生动态响应。本研究探讨了两种对局部染色质开放状态具有相反调控作用的转录因子，如何在全细胞核范围内调控染色质动态变化。我们将高分辨率扩散成像（High-resolution Diffusion mapping, Hi-D）与密集流重建及关联分析（Dense Flow reConstruction and Correlation, DFCC）技术应用于活细胞，实现了基于成像的纳米级分辨率分析，用以解析染色质与转录因子的局部扩散过程及长距离协同运动。研究发现，分别过表达具有大量结合位点的单个转录激活因子（CDX2）或阻遏因子（SIX6），均可提升全细胞核范围内的染色质流动性，但二者在微米尺度下诱导的染色质协同运动模式完全相反。对高阶染色质结构的Hi-C分析显示，先导转录因子CDX2的诱导表达不仅会改变局部染色质相互作用，还会影响A、B区室的分布，进而将染色质微运动与区室结构的变化建立关联。鉴于RNA聚合酶II（RNA Pol II）对转录起始与延伸的抑制仅对CDX2诱导的全局染色质动态变化产生部分影响，我们认为CDX2过表达可通过转录依赖与非依赖两种途径改变染色质结构动态。我们的生物物理分析表明，序列特异性转录因子可在多个结构层面调控染色质结构，这提示转录因子介导的局部染色质改变会影响长距离染色质流动性及其组织形式。本研究包含对经强力霉素诱导表达CDX2的3T3细胞进行的Hi-C测序，样本分别接受或不接受强力霉素处理。