In Silico Analysis of the Regulatory Architecture of the HSP70 Promoter in Samia ricini: Insights into Transcription Factor Binding and CpG Features

Abstract

Temperature is a critical environmental factor shaping physiological performance and survival in ectothermic organisms, including insects. The heat shock protein 70 (HSP70) plays a central role in cellular protection against thermal stress; however, the regulatory architecture governing its expression in Samia ricini remains poorly understood. This study aimed to characterize the promoter region of the HSP70 gene and explore the integration of transcriptional and epigenetic regulatory mechanisms using an in silico approach. The HSP70 genomic locus was identified through homology-based mapping using BLASTn against the S. ricini genome. The upstream regulatory region was extracted and analyzed using Neural Network Promoter Prediction (NNPP), MEME Suite, and CpGplot. Cis-regulatory elements and transcription factor binding sites (TFBSs) were identified, followed by protein–DNA interaction analysis using HDOCK to evaluate Heat Shock Factor (HSF) binding to Heat Shock Elements (HSEs). Promoter analysis revealed a complex regulatory architecture consisting of conserved core elements, including TATA box, CAAT box, GC box, and a predicted transcription start site (TSS). Two HSE motifs were identified in close proximity (−283 and −278 bp), suggesting a potential regulatory hotspot for HSF binding. Notably, a CpG island was detected within the region spanning 1650–1850 bp, overlapping with HSE-associated sequences, indicating a possible interaction between DNA methylation and transcription factor accessibility. Docking analysis supported stable HSF–DNA interaction, with the top-ranked model showing a strong binding affinity (docking score: −276.36) and high confidence. These findings suggest that HSP70 regulation in S. ricini involves a coordinated interplay between conserved promoter elements, transcription factor binding, and epigenetic features. This integrative regulatory framework may underlie adaptive responses to thermal stress and provides a foundation for future experimental validation of gene regulation mechanisms in Lepidoptera