Although the transcriptional regulation of the programmed death ligand 1 (PD-L1) promoter has been extensively studied, the transcription factor residing in the PD-L1 super-enhancer has not been comprehensively explored. Through saturated CRISPR-Cas9 screening of the core region of the PD-L1 super-enhancer, we have identified a crucial genetic locus, referred to as locus 22, which is essential for PD-L1 expression. Locus 22 is a potential binding site for NFE2:MAF transcription factors. Although genetic silencing of NRF2 (NFE2L2) did not result in a reduction of PD-L1 expression, further analysis reveals that MAFG and NFE2L1 (NRF1) play a critical role in the expression of PD-L1. Importantly, lipopolysaccharides (LPS) as the major component of intratumoral bacteria could greatly induce PD-L1 expression, which is dependent on the PD-L1 super-enhancer, locus 22, and NFE2L1/MAFG. Mechanistically, genetic modification of locus 22 and silencing of MAFG greatly reduce BRD4 binding and loop formation but have minimal effects on H3K27Ac modification. Unlike control cells, cells with genetic modification of locus 22 and silencing of NFE2L1/MAFG failed to escape T cell-mediated killing. In breast cancer, the expression of MAFG is positively correlated with the expression of PD-L1. Taken together, our findings demonstrate the critical role of locus 22 and its associated transcription factor NFE2L1/MAFG in super-enhancer– and LPS-induced PD-L1 expression. Our findings provide new insight into understanding the regulation of PD-L1 transcription and intratumoral bacteria-mediated immune evasion.

尽管程序性死亡配体 1 (PD-L1) 启动子的转录调控已被广泛研究，但 PD-L1 超级增强子中的转录因子尚未得到全面探索。通过对 PD-L1 超级增强子的核心区域进行饱和 CRISPR-Cas9 筛选，我们鉴定了一个关键的遗传位点，称为位点 22，它对于 PD-L1 的表达至关重要。位点 22 是 NFE2:MAF 转录因子的潜在结合位点。尽管NRF2（NFE2L2）的基因沉默不会导致PD-L1表达减少，但进一步分析表明MAFG和NFE2L1（NRF1）在PD-L1的表达中发挥着关键作用。重要的是，脂多糖（LPS）作为瘤内细菌的主要成分可以极大地诱导PD-L1表达，这依赖于PD-L1超级增强子、基因座22和NFE2L1/MAFG。从机制上讲，基因座 22 的遗传修饰和 MAFG 的沉默极大地减少了 BRD4 结合和环形成，但对 H3K27Ac 修饰的影响很小。与对照细胞不同，具有基因座 22 基因修饰和 NFE2L1/MAFG 沉默的细胞未能逃脱 T 细胞介导的杀伤。在乳腺癌中，MAFG的表达与PD-L1的表达呈正相关。综上所述，我们的研究结果证明了基因座 22 及其相关转录因子 NFE2L1/MAFG 在超级增强子和 LPS 诱导的 PD-L1 表达中的关键作用。我们的研究结果为理解 PD-L1 转录调节和瘤内细菌介导的免疫逃避提供了新的见解。