Aging Cells Ratchet Up a Gene Called EYA4, and Silencing It Delays Senescence

Scientists probing the molecular machinery of aging have identified a surprising new player in the drive toward cellular senescence. A team at Sun Yat-Sen University, working with colleagues at Southwest Medical University, reports that EYA4, a gene long known for its roles in organ development and DNA damage repair, is consistently upregulated as human tissues and cells grow old. When the researchers silenced EYA4 in aging fibroblasts, the cells resisted both replicative senescence and chemically induced stress senescence, retaining the ability to divide far longer than untreated controls. The finding, published in Advanced Biotechnology, positions EYA4 as a potential lever for delaying the aging process at the cellular level.

The investigation began with a large-scale survey of human gene expression. The team mined the Genotype-Tissue Expression database, known as GTEx, which catalogues RNA sequencing data from hundreds of donors. Comparing individuals aged 20 to 49 with those aged 60 and above across 25 tissues, they found that EYA4 expression was significantly elevated in 12 aging tissues, while remaining unchanged in the other 13. This pattern suggested that EYA4 is not a passive bystander in aging but a gene whose expression is actively remodeled as organisms grow older, echoing a growing appreciation that senescence-associated genes can feed back to regulate the aging trajectory itself.

To connect that tissue-level observation to a cellular mechanism, the researchers turned to primary human diploid fibroblasts, the classic workhorse of senescence research. They generated replicatively senescent HFF-1 and BJ fibroblasts by culturing them beyond 50 population doublings, roughly double the lifespan of young control cells at about 25 doublings. They also induced senescence chemically, treating young fibroblasts with bleomycin, a DNA-damaging agent, for three days. In both models, senescence was confirmed by elevated senescence-associated beta-galactosidase activity and increased p21, a cyclin-dependent kinase inhibitor that serves as a canonical senescence marker. Crucially, EYA4 rose in parallel, at both the messenger RNA and protein levels, in every senescent culture examined.

The functional test followed. Using small interfering RNAs to knock down EYA4 in fibroblasts approaching replicative senescence, the team observed a striking rescue: levels of p21 and senescence-associated beta-galactosidase dropped, and EdU incorporation assays showed that many cells retained their capacity to synthesize DNA and divide. The same held true in bleomycin-induced senescence, where EYA4 depletion again reduced senescence markers and preserved proliferative capacity. Together, these experiments demonstrated that EYA4 is not merely correlated with senescence but actively promotes it, and that removing it can delay both the replicative and stress-induced forms of the process.

What made this result mechanistically intriguing is that EYA4 is an unusual protein with two distinct biochemical faces. Its C-terminal Eya domain carries tyrosine phosphatase activity and mediates protein interactions, while its N-terminal domain harbors threonine phosphatase activity and transcriptional co-activation functions. To determine which activity drives p21 upregulation, the researchers engineered phosphatase-deficient mutants targeting each domain separately. Remarkably, both mutants promoted p21 expression just as strongly as wild-type EYA4. The senescence-promoting function of EYA4 therefore depends not on its enzymatic phosphatase activity but on its transcriptional activation capacity, a distinction that matters because it narrows the search for the relevant molecular partners.

That search led to SIX2, a transcription factor from the SIX family, whose members are famous for partnering with EYA proteins during development. Because EYA proteins lack any intrinsic DNA-binding ability, they depend on SIX partners to be escorted into the nucleus and tethered to specific promoters. Mining GTEx data through the GEPIA platform, the team found that SIX1, SIX2 and SIX4 expression correlated positively with EYA4 across tissues, and that SIX2 was the most abundantly expressed of the three in the 12 tissues where EYA4 rises with age, as well as in HeLa cells used for mechanistic assays. Co-immunoprecipitation experiments then confirmed that EYA4 and SIX2 physically interact, with the Eya domain of EYA4 serving as the primary binding interface.

The partnership proved functionally essential. Immunofluorescence microscopy showed that SIX2 overexpression drove EYA4 into the nucleus, while SIX2 knockdown diminished EYA4’s nuclear localization. Overexpressing SIX2 alone increased p21, but silencing SIX2 abolished EYA4’s ability to boost p21, and conversely, silencing EYA4 negated SIX2’s effect. Chromatin immunoprecipitation followed by quantitative PCR revealed that SIX2 binds to the P21 promoter within roughly one kilobase upstream of the transcription start site, and dual-luciferase reporter assays confirmed that this binding region is sufficient to activate P21 transcription. The model that emerges is one of mutual dependence: SIX2 recruits EYA4 to DNA, and EYA4 supplies the transcriptional activation power that neither protein can provide alone.

The p53 connection added a final layer of complexity. p53, the famed tumor suppressor, binds the P21 promoter about 2.4 kilobases upstream of the transcription start site and is classically regarded as p21’s master regulator. When the researchers generated p53-knockout HeLa cells using CRISPR, p21 expression fell to undetectable levels, and neither EYA4 nor SIX2 overexpression could restore it. Rescuing p53 in those cells brought p21 back to normal, and EYA4 knockdown then reduced p21 and senescence-associated secretory phenotype factors as before. Importantly, further experiments showed that p53 does not disrupt the EYA4-SIX2 interaction itself, nor does it prevent SIX2 from binding the P21 promoter, and the promoter remained responsive to p53-independent activation by an AKT inhibitor. This suggests p53 acts as a permissive gatekeeper for transcriptional initiation while the EYA4-SIX2 complex fine-tunes expression levels, though the authors caution that the precise mechanism requires further study.

The study’s implications extend in several directions. Because p21 is persistently activated in replicative and DNA damage-induced senescence, and elevated p21 has been linked to metabolic and cardiovascular diseases, the EYA4-SIX2 axis offers a candidate target for interventions aimed at delaying age-related pathology. The researchers also note that EYA4 plays dual roles in cancer, promoting some tumors through its phosphatase activity while suppressing others, so any therapeutic strategy would need to account for context. Open questions remain, including whether EYA4 partners with other SIX family members in different tissues, whether the axis also governs developmentally programmed senescence, and whether the complex preferentially regulates early-stage rather than late-stage senescence. The authors acknowledge the absence of endogenous co-immunoprecipitation data, owing to antibody limitations, but point to structural studies of related EYA-SIX complexes as strong support for a direct interaction. As senescence research moves from correlation toward mechanism, EYA4’s emergence as a p53-dependent, phosphatase-independent driver of p21 transcription offers a fresh and testable entry point into the biology of growing old.

Subject of Research: The role of EYA4 and SIX2 in regulating p21 transcription and cellular senescence during aging

Article Title: EYA4 promotes cellular senescence by enhancing P21 transcription through interaction with SIX2

Article References: Li, X., Mao, P., Chen, D., Li, L., Fang, H., Huang, J., & Liu, H. (2026). EYA4 promotes cellular senescence by enhancing P21 transcription through interaction with SIX2. Advanced Biotechnology, 4(2), Article 17. https://doi.org/10.1007/s44307-026-00109-8

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00109-8

Keywords: EYA4, SIX2, p21, cellular senescence, p53, aging, GTEx, transcriptional regulation, phosphatase, fibroblasts, gene expression, senescence markers