Identification and Functional Analysis of the Regulatory Elements in the pHSPA6 Promoter.

Shuyu Jiao,Chuheng Zhao,Hongsheng Ouyang,Xiaochun Tang,Feng Li,Zicong Xie,Chunyan Bai,Kang Yang,Lanxin Hu,Chunyun Qi,Daxin Pang,Heyong Wu

doi:10.3390/genes13020189

Abstract

Functional and expressional research of heat shock protein A6 (HSPA6) suggests that the gene is of great value for neurodegenerative diseases, biosensors, cancer, etc. Based on the important value of pigs in agriculture and biomedicine and to advance knowledge of this little-studied HSPA member, the stress-sensitive sites in porcine HSPA6 (pHSPA6) were investigated following different stresses. Here, two heat shock elements (HSEs) and a conserved region (CR) were identified in the pHSPA6 promoter by a CRISPR/Cas9-mediated precise gene editing strategy. Gene expression data showed that sequence disruption of these regions could significantly reduce the expression of pHSPA6 under heat stress. Stimulation studies indicated that these regions responded not only to heat stress but also to copper sulfate, MG132, and curcumin. Further mechanism studies showed that downregulated pHSPA6 could significantly affect some important members of the HSP family that are involved in HSP40, HSP70, and HSP90. Overall, our results provide a new approach for investigating gene expression and regulation that may contribute to gene regulatory mechanisms, drug target selection, and breeding stock selection.

Highlights

The heat shock protein 70 (HSP70) family is a set of highly conserved proteins that are known for increased synthesis, exposing organisms to a mass of cellular stresses
A cell density assay was performed using primary kidney cells (PK) cells at F0 and three cell culture densities (30%, 80%, and 130%), which showed the highest expression of porcine HSPA6 (pHSPA6) in low-density cell culture (30%) and significantly reduced expression of pHSPA6 when the cell density reached 80%, which may have been due to inhibition of pHSPA6 expression when the cell density exceeded 68%
The results indicated that GS and DNAJB1 had a positive correlation with downregulated pHSPA6 in the three clones, and DNAJC3 had a positive correlation with downregulated pHSPA6 in clones heat shock elements (HSEs)-1 and HSE-2, whereas DNAJC3 was uncorrelated with conserved region (CR) (Figure 5a,d–f)

Summary

Introduction

The heat shock protein 70 (HSP70) family is a set of highly conserved proteins that are known for increased synthesis, exposing organisms to a mass of cellular stresses. HSPA6 is a poorly characterized member of the HSP70 family and is induced after severe cellular stress [2,3]. HSPA6 was identified to have higher expression in cattle and goats under heat stress [5,6,7]. This may be because severe stress conditions cause HSPA6 to evolve into a gene that maintains basic biological functions [8]. HSPA6 was strongly induced by MG132 treatment of human nerve cells [13,14]. Knockdown experiments showed that HSPA6 could protect human nerve cells from stress [15]. The results implied that human nerve cells treated with

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