Shahabi Shandy, Biswas Tapan, Shen Yuting, Sanahmadi Rose, Zou Yaya, Ghosh Gourisankar
bioRxiv. 2025 Jun 18:2024.07.03.601930. doi: 10.1101/2024.07.03.601930.
Non-consensus binding sites of transcription factors are often observed within the regulatory elements of genes; however, their effect on transcriptional strength is unclear. Within the promoters and enhancers of NF-κB-responsive genes, we identified clusters of non-consensus κB DNA sites, many exhibiting low affinity for NF-κB in vitro. Deletion of these sites demonstrated their collective critical role in transcription. We explored how these "weak" κB sites exert their influence, especially given the typically low nuclear concentrations of NF-κB. Using proteomics approaches, we identified additional nuclear factors, including other DNA-binding TFs, that could interact with κB site-bound NF-κB RelA. ChIP-seq and RNA-seq analyses suggest that these accessory TFs, referred to as the TF-cofactors of NF-κB, facilitate dynamic recruitment of NF-κB to the clustered weak κB sites. Overall, the occupancy of NF-κB at promoters and enhancers appears to be defined by a collective contribution from all κB sites, both weak and strong, in association with specific cofactors. This congregation of multiple factors within dynamic transcriptional complexes is likely a common feature of transcriptional programs.
The NF-κB RelA dimers undergo rapid activation by cytokines and pathogens, driving expeditious expression of target genes upon binding to DNA elements known as κB sites, located in the regulatory regions. We find that promoter and enhancer regions of RelA target genes harbor multiple κB sites, most being non-consensus with minimal affinity to NF-κB in vitro. Recruitment of RelA dimer in vivo depend on these κB sites, weak and strong, and appears to be regulated by various accessory factors, including other DNA-binding transcription factors. Overall, this study points to a coordinated network of factors communicating with both weak and strong κB sites to recruit RelA dimers, enabling rapid gene activation.
转录因子的非一致性结合位点经常出现在基因的调控元件中；然而，它们对转录强度的影响尚不清楚。在NF-κB反应基因的启动子和增强子内，我们鉴定出非一致性κB DNA位点簇，其中许多在体外对NF-κB表现出低亲和力。删除这些位点证明了它们在转录中的集体关键作用。我们探讨了这些“弱”κB位点如何发挥其影响，特别是考虑到NF-κB通常在细胞核中的浓度较低。使用蛋白质组学方法，我们鉴定出了其他核因子，包括其他DNA结合转录因子，它们可以与结合在κB位点上的NF-κB RelA相互作用。ChIP-seq和RNA-seq分析表明，这些辅助转录因子，即NF-κB的转录因子辅助因子，促进了NF-κB向簇状弱κB位点的动态募集。总体而言，启动子和增强子处NF-κB的占据似乎由所有κB位点（包括弱位点和强位点）与特定辅助因子的共同作用所决定。动态转录复合物中多种因子的这种聚集可能是转录程序的一个共同特征。
NF-κB RelA二聚体被细胞因子和病原体快速激活，在与位于调控区域的称为κB位点的DNA元件结合后，驱动靶基因的快速表达。我们发现RelA靶基因的启动子和增强子区域含有多个κB位点，大多数在体外与NF-κB的亲和力最低且不一致。RelA二聚体在体内的募集依赖于这些κB位点，包括弱位点和强位点，并且似乎受各种辅助因子调节，包括其他DNA结合转录因子。总体而言，这项研究指出了一个协调的因子网络，该网络与弱κB位点和强κB位点进行通信以募集RelA二聚体，从而实现快速的基因激活。
