Edward Mallinckrodt Department of Pediatrics, Division of Newborn Medicine, Washington University School of Medicine and St Louis Children's Hospital, St Louis, Missouri, USA.
Department of Pediatrics, University of California San Diego School of Medicine, San Diego, California, USA.
Pediatr Res. 2018 Sep;84(3):419-425. doi: 10.1038/pr.2018.30. Epub 2018 Apr 11.
Mutations in the NK2 homeobox 1 (NKX2-1) gene are associated with lung disease in infants and children. We hypothesize that disruption of normal surfactant gene expression with these mutations contributes to the respiratory phenotypes observed.
To assess transactivational activity, cotransfection of luciferase reporter vectors containing surfactant protein B or C (SFTPB or SFTPC) promoters with NKX2-1 plasmids was performed and luciferase activity was measured. To assess the binding of mutated proteins to target DNA, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein labeled with oligonucleotide probes representing NKX2-1 consensus binding sequences followed by gel electrophoresis. The effect of overexpression of wild-type (WT) and mutant NKX2-1 on SFTPB and SFTPC was evaluated with quantitative real-time PCR.
Decreased transactivation of the SFTPB promoter by both mutants and decreased transactivation of the SFTPC promoter by the L197P mutation was observed. EMSA demonstrated decreased DNA binding of both mutations to NKX2-1 consensus binding sequences. Transfection of A549 cells with NKX2-1 expression vectors demonstrated decreased stimulation of SFTPB and SFTPC expression by mutant proteins compared with that of WT.
Disruption of transcriptional activation of surfactant protein genes by these DNA-binding domain mutations is a plausible biological mechanism for disruption of surfactant function and subsequent respiratory distress.
NK2 同源盒 1（NKX2-1）基因突变与婴儿和儿童的肺部疾病有关。我们假设这些突变破坏了正常的表面活性物质基因表达，导致了观察到的呼吸表型。
为了评估转录激活活性，将含有表面活性蛋白 B 或 C（SFTPB 或 SFTPC）启动子的荧光素酶报告载体与 NKX2-1 质粒共转染，并测量荧光素酶活性。为了评估突变蛋白与靶 DNA 的结合，使用寡核苷酸探针标记的核蛋白进行电泳迁移率变动分析（EMSA），该探针代表 NKX2-1 共识结合序列，然后进行凝胶电泳。通过定量实时 PCR 评估野生型（WT）和突变型 NKX2-1 对 SFTPB 和 SFTPC 的过表达效应。
观察到两种突变体均降低了 SFTPB 启动子的转录激活，而 L197P 突变降低了 SFTPC 启动子的转录激活。EMSA 表明两种突变均降低了 DNA 与 NKX2-1 共识结合序列的结合。用 NKX2-1 表达载体转染 A549 细胞，与 WT 相比，突变蛋白对 SFTPB 和 SFTPC 的表达刺激减少。
这些 DNA 结合域突变破坏了表面活性蛋白基因的转录激活，这是表面活性功能破坏和随后呼吸窘迫的一种合理的生物学机制。
