Department of Biophysics, Bose Institute, Centenary Campus, P-1/12 C.I.T. Scheme VIIM, Kolkata 700054, India.
Barasat Cancer Research and Welfare Centre, Kolkata 700124, India.
Biochim Biophys Acta Gen Subj. 2021 Aug;1865(8):129899. doi: 10.1016/j.bbagen.2021.129899. Epub 2021 Apr 27.
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.
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.
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.
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.
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.
锌指 E 盒结合同源盒 1（ZEB1）的异常表达，在正常细胞中仍受到抑制，常与癌症的侵袭性有关。然而，癌症中这种非典型 ZEB1 表达的转录机制尚不清楚。
使用圆二色性、荧光光谱、ITC 和 DMS 保护测定法对 ZEB1 启动子 G-四链体进行了广泛研究和建模。使用荧光素酶测定、qPCR、FAIRE、ChIP、western blot、共聚焦显微镜来评估 ZEB1 转录的调控。
我们的研究揭示了核仁蛋白在 ZEB1 启动子上的占据是一个关键决定因素，它通过局部重塑该区域，促进 SP1 转录因子与染色质的结合。SP1 随后招募 P300 乙酰转移酶，导致启动子处富含乙酰化组蛋白 H3，并激活 ZEB1 转录。ZEB1 启动子分析确定在 TSS 附近 700bp 内存在四个潜在的 G-四链体（G4）形成基序；每个四联体都用一系列生物物理技术进行了详细的结构特征描述。令人惊讶的是，阳离子卟啉 TMPyP4 稳定 G4 会抑制其转录，最终阻碍细胞侵袭。
TMPyP4 与一个选定的 G4 基序（从 TSS 开始的 5'-534/-511-3'）结合，核仁蛋白/SP1/P300 共同占据该基序，阻止核仁蛋白的结合，从而阻碍 SP1 的结合，导致染色质的紧凑和转录抑制。
我们的研究结果表明了 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是至关重要的，因此，小分子诱导的 G-四链体稳定化可能作为一种潜在的分子开关来关闭基因表达。
