NF1, Sp1 and HSF1 are synergistically involved in sulfide-induced sqr activation in echiuran worm Urechis unicinctus.

Sulfide is a well-known environmental toxic substance. Mitochondrial sulfide oxidation is a main mechanism of sulfide detoxification in organisms, and sulfide: quinone oxidoreductase (SQR) is a key enzyme which is involved in transferring electrons from sulfide to ubiquinone and converting sulfide into thiosulfate. Previous studies have revealed the SQR-mediated mitochondrial sulfide oxidation exists in the echiuran worm Urechis unicinctus, and its sqr mRNA level increased significantly when the worm is exposed to sulfide. In this study, we attempt to reveal the synergistic regulation of transcription factors on sulfide-induced sqr transcription in U. unicinctus. ChIP and EMSA were used to identify the interactions between sqr proximal promoter (from -391 to +194bp) and transcription factors NF1 (nuclear factor 1) and Sp1 (specificity protein 1). Site-directed mutation and transfection assays further revealed their binding sites and synergistic roles of HSF1, NF1 and Sp1 in the sqr transcription. When U. unicinctus were exposed to 150μM sulfide, the expression levels and nuclear contents of NF1 and Sp1 were examined by Western blotting, and the binding contents between NF1 or Sp1 and the sqr promoter were also detected by ChIP. Transcription factors NF1 and Sp1 were confirmed to interact with the sqr proximal promoter, and their binding sites were identified in -75 to -69bp for NF1 and -210 to -201bp for Sp1. Transfection assays showed mutation of NF1 or Sp1 binding site significantly decreased the sqr promoter activity by 50% or 73%, respectively. Moreover, we demonstrated three transcription factors NF1, Sp1 and HSF1 enhanced synergistically the activity of sqr transcription. Furthermore, contents of NF1 or Sp1 binding to the sqr proximal region increased significantly in the hindgut when the worms were exposed to 150μM sulfide. Similar changes of NF1 or Sp1 levels and nuclear NF1 or Sp1 levels were also presented. Transcription factors NF1, Sp1 and HSF1 are all involved in sulfide-induced sqr transcription. Sulfide can activate sqr transcription by not only increasing their expression levels, but also promoting them entering nucleus and binding to the sqr promoter. NF1 and Sp1 participate in both basal and sulfide-induced sqr transcription, while HSF1 functions mainly in sulfide-induced sqr transcription.