IntroductionAging is broadly defined as functional decline that occurs throughout the whole body. The accumulation of senescent cells is considered a hallmark of aging and is thought to contribute to various aging pathologies1,2,3. Cellular senescence is a state of cell cycle arrest accompanied by the secretion of chemokines, cytokines, matrix-remodeling proteases and other molecules4,5. This hypersecretory phenotype is known as the senescence-associated secretory phenotype (SASP)6. The SASP is a highly heterogeneous program, and its composition is influenced by a range of intrinsic and extrinsic factors7,8. The SASP has been hypothesized to link cellular senescence and inflammation9 and to participate in tissue dysfunction. The accumulation of senescent cells with the SASP is associated with increased susceptibility to several age-related diseases10, including neurodegenerative diseases, cardiovascular diseases, metabolic disorders, and immune system diseases11. In normal cells, SASP genes are usually highly inhibited to prevent the inappropriate activation of inflammatory signals, whereas in senescent cells, the SASP genes are highly expressed12. How SASP gene expression is activated to trigger cellular senescence, inflammation and the aging process remains to be actively studied.The Mediator complex is a master transcriptional cofactor comprising ~30 subunits in mammalian cells13 and links extracellular signals to the basal transcriptional machinery for selective target gene expression14. In response to various environmental or developmental cues, distinct transcription factors interact with different Mediator subunits to control diverse biological processes15. MED1, for example, interacts with PPARγ2 to regulate adipogenesis16, and MED12 interacts with β-catenin to relay the Wnt signaling17 and gastrulation18. The interaction between MED16 and NRF2 plays a crucial role in ROS production and redox homeostasis19,20. We previously reported that MED23 controls lipid metabolism and obesity21 and participates in the RoRα-regulated liver inflammatory response and liver fibrosis22. A single-site mutation of MED23 (R617Q) leads to intellectual disability by altering the selective chromatin conformation and enhancer activities23. MED15 modulates transforming growth factor β (TGFβ)/Smad signaling during development24 and breast cancer cell metastasis25; MED15 deficiency attenuates TGFβ-targeted gene expression and relieves TGFβ-mediated growth inhibition and metastasis25. Secreted TGFβ is considered a SASP factor and plays a pivotal role in cellular senescence and age-related diseases, including Alzheimer’s disease, muscle atrophy, fibrosis, and obesity26,27,28,29. Despite that much has been know about various interactions between transcription factors and Mediator components, less is known about how signaling directly hinges upon the Mediator complex per se; and specifically, how exactly TGFβ signaling impacts on the Mediator to regulate the associated transcription factors and the selective biological processes remains to be further investigated.In this study, we found that the MED15 T603 phosphosite plays a crucial role in controlling the SASP production and aging. Cellular senescence and the SASP are relieved by the T603A mutation in MED15 but accelerated by the T603D mutation. An integrated analysis revealed that MED15 T603 dephosphorylation delays aging processes through an increased interaction with forkhead box protein A1 (FOXA1) to suppress downstream SASP gene expression. Notably, we generated Med15 T604A mutant mice using a gene editing technique, and these mice exhibit alleviated tissue aging and resistance to cognitive decline, accompanied by decreased serum levels of SASP factors in aging mice compared to wild-type (WT) control mice. Overall, our study revealed a crucial phosphorylation switch in the Mediator complex that controls cellular senescence and tissue aging through the modulation of SASP production.ResultsMED15 T603 phosphorylation participates in TGFβ-inhibited cell growthTo understand the mechanism and regulation of TGFβ signaling hinging upon the Mediator complex, we performed immunoprecipitation-mass spectrometry (IP-MS) analysis and identified a novel phosphorylated threonine residue (T603) in MED15 (Fig. 1a, b). The PhosphoSitePlus database also showed that T603 site is a major high-abundance phosphorylation site of MED15 (Fig. 1c). The multiple sequence alignment of amino acids revealed that the T603 site of MED15 is conserved across species (Fig. 1d). We utilized CRISPR-Cas9 gene editing to generate an endogenous MED15 T603A (threonine to alanine) single-site mutant to mimic the MED15 T603 dephosphorylation state in HaCaT cells (Fig. 1e), an immortalized epithelial cell line often used for studying TGFβ/Smad signaling, and to verify the function and relevance of this phosphorylation site to TGFβ signaling. Cell proliferation assays revealed that MED15 T603A mutant cells significantly relieved TGFβ-induced inhibition of cell growth compared to WT cells treated with TGFβ (Fig. 1f). The flow cytometry analysis further revealed that compared to WT cells, the MED15 T603A mutant increased the proportion of cells in the S phase after TGFβ treatment for 96 h (Fig. 1g; Supplementary Fig. S1), suggesting that the MED15 T603A mutation confers resistance to TGFβ-induced G1/S cell cycle arrest.Fig. 1: MED15 T603 testified as a phosphorylation site involved in TGFβ-inhibited cell growth.a Endogenous co-IP assay using HA-beads to pull down HA-MED15 from 293T cells. The HA tag was knocked into the N-terminus of MED15. b Tandem mass spectrometry analysis of the MED15 peptides modified by phosphorylation of the T603 residue. c Posttranslational modification analysis of MED15 by PhosphoSitePlus database. d Sequence alignment of MED15 proteins across different species. The blue bold T indicates the MED15 T603 site. e Endogenous MED15 T603A mutant genotyping via PCR and Sanger sequencing in HaCaT cells. f CCK-8 assays were used to assess the proliferation rates of WT and T603A mutant HaCaT cells treated without or with 5 ng/mL TGFβ. The values are presented as means ± SD. ***P