Background

Aberrant expression of Zinc-finger E-box binding homeobox 1 (ZEB1), which remains repressed in normal cells, is frequently associated with cancer aggressiveness. However, transcriptional mechanism underlying such atypical ZEB1 expression in cancer is not yet well-understood.

Methods

ZEB1 promoter G-quadruplexes were studied and modeled extensively using circular dichroism, fluorescence spectroscopy, ITC and DMS protection assay. Luciferase assay, qPCR, FAIRE, ChIP, western blotting, confocal microscopy was used to access the regulation of ZEB1 transcription.

Results

Our study unravels the occupancy of nucleolin to ZEB1 promoter as a crucial determinant which facilitates the binding of SP1 transcription factor to chromatin, by locally remodelling the region. SP1, subsequently, recruits P300 acetyl transferase leading to enriched acetyl-histone H3 at promoter and activates ZEB1 transcription. ZEB1 promoter analysis identifies presence of four putative G-quadruplex (G4) forming motifs within 700 bp of TSS; each quadruplex is characterized structurally in details with an array of biophysical techniques. Surprisingly, stabilization of G4 with cationic porphyrin TMPyP4 represses its transcription and eventually impedes cell invasiveness.

Conclusions

TMPyP4 binding to a selected G4 motif (5′ -534/−511–3′ from TSS), where nucleolin/SP1/P300 co-occupies, prevents the association of nucleolin which consequently hinders SP1 binding, leading to chromatin compactness and transcriptional repression.

General significance

Our findings demonstrate an epigenetic mechanism of ZEB1 reactivation where dynamic occupancy of transcription regulators encompassing a G4 motif is crucial and thus, small molecule induced G-quadruplex stabilization may act as a potential molecular switch to turn-off gene expression.

中文翻译：

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启动子 G-四链体有利于表观遗传重编程诱导的 ZEB1 在癌细胞中的非典型表达

背景

锌指E - box 结合同源框 1 (ZEB1) 的异常表达，在正常细胞中仍然受到抑制，通常与癌症侵袭性有关。然而，癌症中这种非典型 ZEB1 表达的转录机制尚不清楚。

方法

使用圆二色性、荧光光谱、ITC 和 DMS 保护测定对 ZEB1 启动子 G-四链体进行了广泛的研究和建模。荧光素酶测定、qPCR、FAIRE、ChIP、蛋白质印迹、共聚焦显微镜用于访问 ZEB1 转录的调节。

结果

我们的研究揭示了核仁蛋白对 ZEB1 启动子的占据，这是通过局部重塑该区域促进 SP1 转录因子与染色质结合的关键决定因素。随后，SP1 招募 P300 乙酰转移酶，导致启动子处富含乙酰组蛋白 H3 并激活 ZEB1 转录。ZEB1 启动子分析确定在 TSS 的 700 bp 内存在四个推定的 G-四链体 (G4) 形成基序；每个四链体都通过一系列生物物理技术在结构上进行了详细表征。令人惊讶的是，用阳离子卟啉 TMPyP4 稳定 G4 会抑制其转录并最终阻碍细胞侵袭。

结论

TMPyP4 与选定的 G4 基序（来自 TSS 的 5'-534/-511-3'）结合，核仁蛋白/SP1/P300 共同占据，阻止核仁蛋白的结合，从而阻碍 SP1 结合，导致染色质致密和转录抑制.

一般意义

我们的研究结果证明了 ZEB1 重新激活的表观遗传机制，其中包含 G4 基序的转录调节因子的动态占据至关重要，因此，小分子诱导的 G-四链体稳定可能充当关闭基因表达的潜在分子开关。