Back pain is among the most common musculoskeletal disorders worldwide, and roughly 40 percent of cases are attributed to degenerative disc disease, a progressive breakdown of the cushions between the vertebrae. About 10 percent of people with back pain are forced to change jobs or stop working entirely because of severe symptoms. While disc degeneration has traditionally been linked to genetics, loading history, and inadequate metabolite transport, growing evidence points to cellular senescence as a major biological driver. Senescent cells are those that have permanently stopped dividing, often as a protective response to excessive DNA damage, but they secrete a cocktail of pro-inflammatory factors known as the senescence-associated secretory phenotype, or SASP. In the intervertebral disc, this SASP cocktail includes inflammatory cytokines, matrix-degrading proteases, and pain-related growth factors that erode the disc’s structure and sensitize nerves, creating a vicious feedback loop of degeneration and pain.

The research team first identified ZNF865 in genome-wide CRISPR perturbation screens originally designed to find cell-engineering tools that could enhance tissue deposition in engineered discs. Bioinformatic analysis revealed that ZNF865, also known as BLST, encodes a 1059-amino-acid C2H2 zinc finger protein containing 20 unique zinc finger domains, two transactivation domains, and two TGEKP linkers. The transactivation domains indicate that ZNF865 likely functions as a transcription factor, stimulating gene expression by interacting with other transcriptional regulators. Despite broad expression across virtually all human cell types, no publication had ever functionally characterized its biological role. Adding to the intrigue, a rare disease mutation in ZNF865 has been associated with a striking range of symptoms in human patients, including global developmental delay, joint hypermobility, muscle hypertonia, loose inelastic skin, hand tremors, and abnormalities of the eyes, ears, and brain, hinting at a fundamental role across many tissues.

To probe the gene’s function, the investigators used a CRISPR activation system, CRISPRa, built on the dCas9-VPR platform, to upregulate ZNF865 in three distinct human cell types: embryonic kidney cells, adipose-derived stem cells, and degenerative human nucleus pulposus cells, the disc cells themselves. RNA sequencing revealed thousands of significantly differentially expressed genes in each cell type, and pathway analysis showed a remarkable convergence on the same molecular processes regardless of cell type. Cell cycle regulation, DNA replication, cellular senescence, mismatch repair, p53 signaling, mTOR signaling, base excision repair, and homologous recombination were all consistently enriched. This shared signature suggested that ZNF865 acts as a core regulator of cell division, DNA repair, and senescence, rather than a gene with cell-type-specific duties.

The team then measured ZNF865 expression in disc cells from real patients. In healthy nucleus pulposus cells obtained from trauma patients with no history of back pain, ZNF865 expression showed an extraordinarily strong correlation with age, with an R-squared of 0.95 and a p-value of 0.0054. In degenerative disc cells from back pain patients at matched ages, expression was significantly lower. Immunofluorescence staining confirmed that ZNF865 protein, localized to the nucleus as expected for a transcription factor, was markedly reduced in the degenerative samples. Only 16.8 percent of degenerative cells stained positive for ZNF865 compared with 28.7 percent of healthy cells, indicating that the gene is not expressed by all cells at detectable levels at any given time. Single-cell RNA sequencing of mouse intervertebral discs at 4, 12, and 24 months of age showed the same pattern in vivo: both the average expression of the mouse orthologue ZFP865 and the percentage of cells expressing it declined with age, with a near-perfect negative correlation between the proportion of expressing cells and age.

To establish causality rather than mere correlation, the researchers used CRISPR interference, CRISPRi, with a dCas9-KRAB repression system to knock down ZNF865 in healthy human disc cells. Within days, proliferative capacity dropped. After ten days, the cells showed dramatic increases in three canonical senescence markers: senescence-associated beta-galactosidase rose 5.4-fold, p16 rose 4.7-fold, and p21 rose 6.9-fold. Alkaline comet assays, which quantify DNA strand breaks by measuring the migration of damaged DNA through an electrophoretic field, revealed significantly elevated DNA damage three weeks after knockdown. The same experiment in rat disc cells produced conserved results, with reduced proliferation, increased senescence staining, elevated DNA damage, and increased secretion of the inflammatory proteins MCP-1 and VEGF-A. In short, removing ZNF865 from a healthy disc cell was enough to push it into the senescent, DNA-damaged state seen in degenerative disease.

The reverse experiment proved even more striking. When ZNF865 was upregulated in degenerative disc cells from chronic back pain patients using the CRISPRa system, cell growth increased significantly within three days, and doubling time fell from 4.09 days to 2.49 days, matching the 3.2-day doubling time of healthy cells. Senescence marker staining dropped to levels statistically indistinguishable from healthy controls. Comet assays showed that DNA damage decreased in all seven patient samples, with five reaching statistical significance individually, and donor-mean damage in the ZNF865-upregulated cells matched the asymptomatic controls. A Luminex assay of ten SASP-related proteins in the culture supernatant showed that nine of them, including the pro-inflammatory cytokines IL-1 beta, IL-6, TNF-alpha, and IL-8, the matrix-degrading proteases MMP-1, MMP-3, and MMP-12, and the pain-related growth factors NGF-beta and VEGF-A, fell significantly after ZNF865 upregulation. In three-dimensional pellet culture, the treated cells produced significantly more collagen, a key structural component of the disc’s extracellular matrix, and a composite anabolic score rose significantly for both patient samples tested.

To understand how these phenotypic changes were encoded at the level of gene regulation, the team performed ATAC-seq, a sequencing method that maps regions of open, transcriptionally accessible chromatin. Comparing healthy and degenerative disc cells revealed 1163 sites of differential chromatin accessibility associated with 1705 genes, with functional enrichment in pathways including glycosaminoglycan synthesis, MAPK signaling, and cellular senescence. After ZNF865 upregulation in the degenerative cells, only 70 sites of differential accessibility remained when compared to healthy cells, a 94 percent reduction, with no enriched pathways at all. Principal component analysis of both the ATAC-seq and RNA-seq data showed that the degenerative cells shifted toward the healthy molecular state upon ZNF865 upregulation. Hypergeometric tests confirmed statistically significant overlap between genes changing in expression and genes changing in chromatin accessibility, with more than six-fold enrichment over chance for upregulated genes in the treated cells. Notable individual examples included COL6A2, a Type VI collagen chain recently implicated as a marker of disc degeneration, which was upregulated with more open chromatin in both healthy and ZNF865-treated cells, and USP12, an inflammatory mediator involved in MMP production, which was downregulated with closed chromatin in both groups.

The most consequential experiment came in vivo. The researchers verified a rat orthologue of ZNF865, validated CRISPRi knockdown in rat disc cells in vitro, and then injected ZNF865-targeting or non-targeting CRISPRi lentiviral vectors directly into the L4-L5 and L5-L6 intervertebral discs of Sprague Dawley rats under fluoroscopic guidance. Over eight weeks, behavioral testing revealed a significant increase in mechanical pain sensitivity in the knockdown group compared with both naive and non-target controls by five weeks, as measured by the von Frey assay, and increased thermal pain sensitivity by eight weeks on the Hargreaves assay. Disc height fell significantly at the L4-L5 level, and histological scoring showed significantly increased degeneration across both injected levels. Immunofluorescence staining of the injected discs revealed significant increases in p16, VEGF, IL-6, and the DNA damage marker H2AX in both the nucleus pulposus and annulus fibrosus regions. Critically, the degree of pain behavior correlated strongly with the degree of molecular damage: paw withdrawal threshold showed R-squared values of 0.983 with H2AX and 0.929 with p16 in the nucleus pulposus, and 0.814 with H2AX and 0.902 with p16 in the annulus fibrosus.

What makes this in vivo result particularly significant is that pain was induced purely through transcriptional downregulation, without any needle puncture, enzymatic digestion, or biomechanical overload. Most rodent models of discogenic pain rely on acute structural damage that may confound the interpretation of pain mechanisms. Here, molecular dysfunction alone was sufficient to drive both degeneration and nociceptive sensitization, and the gradual progression of pain behavior over weeks mirrors the slow course of human disc disease. The findings position ZNF865 as a fundamental guardian of genomic stability in disc tissue, whose loss with age permits the DNA damage and senescence cascade that culminates in degeneration and pain. The broader implications extend well beyond the spine: ZNF865 has been independently associated with better prognosis in esophageal cancer, identified as a potentially relevant target in Parkinson’s disease, and listed among the top pathways affected in ALS and Alzheimer’s disease in the team’s own sequencing data. The researchers caution that key questions remain, including the identification of upstream regulators and downstream effectors through DNA binding studies, the development of blocking antibodies, inhibitors, or activators that could modulate ZNF865 outside of CRISPR-based therapies, and larger clinical studies with broader patient populations. Still, this first functional characterization of a previously unknown gene opens a genuine new frontier in the biology of aging, senescence, and the molecular origins of chronic pain.

Subject of Research: ZNF865 (BLST) a Novel Regulator of DNA Damage and Cell Senescence in Back Pain

Article Title: ZNF865 (BLST) a Novel Regulator of DNA Damage and Cell Senescence in Back Pain

Article References: Lewis, C., Levis, H., Holbrook, J., Polaski, J. T., Jacobsen, T. D., Mizels, J., Czachor, M., Gullbrand, S. E., Diekman, B., Iatridis, J. C., Gertz, J., Lawrence, B., & Bowles, R. D. (2026). ZNF865 (BLST) a Novel Regulator of DNA Damage and Cell Senescence in Back Pain. Aging Cell, 25(10), Article e70754. https://doi.org/10.1111/acel.70754

Image Credits: AI Generated

DOI: 10.1111/acel.70754

Keywords: ZNF865, BLST, Novel, Regulator, Damage, Cell, Senescence, Back, Pain, scientific research