ABSTRACT
Super-enhancers play prominent roles in driving robust pathological gene expression, but they are hidden in human genome at noncoding regions, making them difficult to explore. Leukemia inhibitory factor (LIF) is a multifunctional cytokine crucially involved in acute respiratory distress syndrome (ARDS) and lung cancer progression. However, the mechanisms governing LIF regulation in disease contexts remain largely unexplored. In this study, we observed elevated levels of LIF in the bronchoalveolar lavage fluid (BALF) of patients with sepsis-related ARDS compared to those with nonsepsis-related ARDS. Furthermore, both basal and LPS-induced LIF expression were under the control of super-enhancers. Through analysis of H3K27Ac ChIP-seq data, we pinpointed three potential super-enhancers (LIF-SE1, LIF-SE2, and LIF-SE3) located proximal to the LIF gene in cells. Notably, genetic deletion of any of these three super-enhancers using CRISPR-Cas9 technology led to a significant reduction in LIF expression. Moreover, in cells lacking these super-enhancers, both cell growth and invasion capabilities were substantially impaired. Our findings highlight the critical role of three specific super-enhancers in regulating LIF expression and offer new insights into the transcriptional regulation of LIF in ARDS and lung cancer.
Subject(s)
Leukemia Inhibitory Factor , Lung Neoplasms , Respiratory Distress Syndrome , Humans , Respiratory Distress Syndrome/metabolism , Respiratory Distress Syndrome/genetics , Respiratory Distress Syndrome/pathology , Leukemia Inhibitory Factor/metabolism , Leukemia Inhibitory Factor/genetics , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Bronchoalveolar Lavage Fluid/chemistry , Enhancer Elements, Genetic , Cell Proliferation , MaleABSTRACT
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.