Modulation of NF-kB-Dependent Gene Transcription Using Programmable DNA Minor Groove Binders. Homo sapiens

Nuclear factor kappaB (NF-kB) is a transcription factor that regulates various aspects of immune response, cell death and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5' GGGWWW-3'. The compound is capable of binding to kB sites and reducing the expression of various NF-kB driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-kB inhibitor PS1145, identifying overlaps and differences in affected gene groups and showing that both affect comparable numbers of TNFa inducible genes. Inhibition of NF-kB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists. Overall design: A549 cells were treated with either a Py-Im polyamide or PS1145, subsequently induced with TNFa and compared with the untreated TNFa induced and the basal state by RNAseq. The NF-kB binding motif was derived by ChIP-seq

核因子κB（Nuclear factor kappaB, NF-κB）是一类转录因子，可调控免疫应答、细胞死亡与分化以及癌症发生等诸多生物学进程。本研究中我们介绍了Py-Im聚酰胺1（Py-Im polyamide 1），该化合物可优先结合5'-WGGWWW-3'与5'-GGGWWW-3'序列。该化合物能够靶向结合κB位点，并经定量逆转录PCR（qRT-PCR）验证可下调多种NF-κB驱动基因的表达，其中包括白细胞介素6（IL6）与白细胞介素8（IL8）。染色质免疫沉淀（Chromatin immunoprecipitation, ChIP）实验证实，上述基因的近端启动子区域内p65的结合占有率显著降低。通过RNA测序（RNA-seq）开展的全基因组表达分析，将该DNA结合型聚酰胺与已得到充分表征的NF-κB抑制剂PS1145进行对比，明确了二者所调控的基因群组的重叠与差异，并证实两者对肿瘤坏死因子α（TNF-α）诱导基因的影响数量相当。通过直接置换转录因子以抑制NF-κB的DNA结合能力，是现有拮抗剂之外的一种潜在替代策略。

总体实验设计：将A549细胞分别用Py-Im聚酰胺或PS1145处理，随后以TNF-α诱导，再通过RNA测序（RNA-seq）分别与未经处理的TNF-α诱导组以及基础状态组进行对照分析。本研究中的NF-κB结合基序通过染色质免疫沉淀测序（ChIP-seq）获取。