TRUNCATED HS3ST4 EXPRESSED DOWNSTREAM OF CHRONIC PAIN- ASSOCIATED SINGLE NUCLEOTIDE POLYMORPHISMS REGULATES TRANSCRIPTIONAL PROMOTER ACTIVITY
Authors: Ohka *Seii , Kawaji Hideya , Yamada Souichi , Fukui Yoshiko , Hasegawa Junko , Nishizawa Daisuke , Fukushi Shuetsu , Ebihara Hideki , Ikeda Kazutaka 
Published: February 2025

Abstract

Background
The pathogenic mechanism of post-herpetic neuralgia (PHN), a chronic pain that occurs after herpes zoster caused by reactivation of varicella-zoster virus (VZV), has not been fully understood. By statistical analysis of the genome-wide association study (GWAS) data of patients with chronic pain including PHN patients and healthy subjects, we previously found that single nucleotide polymorphisms (SNPs) located in heparan sulfate 3-O-sulfotransferase 4 (HS3ST4) gene were significantly associated with PHN or chronic pain (Nishizawa et al., 2021), and full-length HS3ST4 induces cell fusion in the presence of VZV glycoproteins (Ohka et al., 2021). The chronic pain-associated HS3ST4 SNPs exist in the intronic region of HS3ST4 gene. However, the impact of this intronic region on the expression/function of the HS3ST4 remains unclear.

Aims & Objectives
We aim to clarify the functions of the intronic region where chronic pain-associated SNPs reside.

Method
Public cap-analysis gene expression (CAGE) and poly-A RNA fragment databases were searched. 5’ rapid amplification of cDNA end (RACE) of HS3ST4 mRNA derived from the human primitive neuroectodermal tumor cell line FU-RPNT-1 was performed. The plasmids expressing truncated HS3ST4 and GFP or NanoLuc luciferase under the cytomegalovirus (CMV) promoter or human elongation factor 1-α (EF1α) promoter were introduced in the human neuroblastoma cell line SK-N-SH. Expression of the GFP or luciferase was monitored by the fluorescent microscope or the luciferase assay, respectively.

Results
Public CAGE databases showed transcription start site (TSS) downstream of the chronic pain-associated HS3ST4 SNPs in FU-RPNT-1 cells and the adult human brain. Moreover, a poly-A RNA fragment had been reported from over 10 kbp downstream of the SNPs. 5’ end of a novel mRNA just downstream of the SNPs was identified by 5’ RACE using mRNA from FU-RPNT-1 cells. The novel mRNA had an open reading frame of a truncated traditional HS3ST4 protein. Plasmid transfection experiments revealed that the truncated HS3ST4 protein enhanced the GFP and luciferase expression in neural SK-N-SH cells. These data suggest that the truncated HS3ST4 protein enhanced the transcriptional activity of CMV and EF1α promoters.

Discussion & Conclusion
Novel mRNA from downstream of the chronic pain-associated SNPs encoded the open reading frame of the truncated traditional HS3ST4. The truncated HS3ST4 could manipulate the promoter activities. It is assumed that the truncated HS3ST4 upregulates pain-related protein transcriptions, contributing to chronic pain expression. These facts may contribute to a novel target of therapeutic agents.

References
Nishizawa, D. et al. (2021) Genome-wide association study identifies candidate loci associated with chronic pain and postherpetic neuralgia, Mol Pain, 17: 1–21. Available at: DOI: 10.1177/1744806921999924
Ohka, S. et al. (2021) Heparan sulfate 3-O-sulfotransferase 4 is genetically associated with herpes zoster and enhances varicella-zoster virus– mediated fusogenic activity, Mol Pain, 17: 1–10. Available at: DOI: 10.1177/17448069211052171