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The m6Am methyltransferase PCIF1 promotes osteogenic differentiation of mesenchymal stem cells through stabilization of Wnt-related transcripts [1]

['Wei Song', 'State Key Laboratory Of Oral Diseases', 'National Center For Stomatology', 'National Clinical Research Center For Oral Diseases', 'West China Hospital Of Stomatology', 'Sichuan University', 'Chengdu', 'Sichuan', 'Department Of Oral Implantology', 'Kuan-Jui Su']

Date: 2026-04 

Osteogenesis depends on the self-renewal and differentiation of mesenchymal stem cells (MSCs). Emerging research underscores the regulatory functions of RNA methylation on bone homeostasis. Here, we show PCIF1, the N6,2′-O-dimethyladenosine (m 6 Am) methyltransferase, is essential for maintaining bone mass and promoting osteogenic differentiation of MSCs. Multiple complementary analyses—including GWAS, TWAS, and single-cell transcriptomics—collectively point to PCIF1 as a regulator of human bone mineral traits and early-stage mesenchymal differentiation. Global or MSC-specific Pcif1 deletion elicits osteoporotic pathology in mice, although myeloid cell-specific Pcif1 knockout does not induce femur bone alterations. Mechanistically, Pcif1 knockout decreases m 6 Am signals of Wnt-related genes (Wnt11, Fzd4, and Fgfr2) and accelerates mRNA degradation. This down-regulates active β-Catenin protein, and thus impairs osteogenic function of MSCs. Additionally, the WNT agonist attenuates the osteoporosis-like phenotype induced by Pcif1 deletion. These findings highlight the crucial role of PCIF1-mediated m 6 Am modification in regulating osteogenesis and suggest potential therapeutic implications for bone disorders.

Funding: This research was supported by grants from the National Natural Science Foundation of China (Grant No. 82125006 to QY; https://www.nsfc.gov.cn/ ), West China Hospital of Stomatology (Grant No. RCDWJS2025-2 to QY; https://hxkq.org/ ), and the National Institutes of Health (NIH) (R01AR069055, U19AG055373, and P20GM109036 to HWD, R01AG061917 to HS; https://www.nih.gov/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All data are available in the main text or the supplementary materials. The RNA-seq data have been deposited into the NCBI database under the accession number GSE324917, and the scRNA-seq data of human head bone tissue have been deposited under accession number GSE324998. The custom code for single-cell analysis generated in this study is publicly available and archived at Zenodo (DOI: https://doi.org/10.5281/zenodo.19026412 ). We also downloaded the m6Am-Exo-Seq data of murine testes of Pcif1-HET and Pcif1-KO mice from GEO database (GSE151229).

Here, we demonstrate PCIF1 is required for maintenance of bone mass and osteogenic functions of MSCs by regulating WNT signaling. Multi-omics approaches suggest PCIF1 is associated with osteoporosis pathogenesis and might control the lineage commitment of MSCs. Whole-body or MSC-specific Pcif1 deletion decreases trabecular bone mass and impairs osteogenic differentiation of MSCs. Mechanistically, MSC osteogenic potential is regulated through the m 6 Am-Wnt signaling, featured by the accelerated degradation rates of Wnt-related mRNA transcripts and decreased active β-Catenin level by Pcif1 deficiency. Thus, these results highlight the significance of m 6 Am on bone formation, with potential strategies for bone repair and remodeling.

Bone homeostasis is primarily a dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption [ 20 – 22 ]. MSCs have the capacity to differentiate into osteoblasts, chondrocytes, and adipocytes in response to changes in the microenvironment and cell–cell interactions [ 23 , 24 ]. During osteogenesis, MSCs convert to osteoblastic progenitors, migrate to the bone surface, and participate in osteoblast maturation as well as the formation of mineralized bone tissues [ 25 ]. This process is mediated by coordinated regulation of multiple signaling pathways and transcriptional factors [ 26 ]. MSC dysfunction leads to bone developmental diseases such as osseous dysplasia, osteoporosis, and osteogenesis imperfecta [ 27 , 28 ]. Dissecting the regulatory molecular mechanisms of MSC biological traits could provide valuable ideas for understanding the onset and progression of skeletal diseases.

RNA epigenetics has been extensively implicated in the modulation of bone homeostasis, underscoring its pivotal role in maintaining skeletal integrity and orchestrating bone remodeling [ 1 – 5 ]. N6,2′-O-dimethyladenosine (m 6 Am) modification occurs on the first 2′-O-methyladenosine (Am) nucleotide at the 5′-end of capped mRNAs [ 6 , 7 ]. m 6 Am dynamics are synergistically regulated by the “writer” Phosphorylated CTD Interacting Factor 1 (PCIF1), the “eraser” Fat Mass and Obesity-associated Protein (FTO), and the “reader” Premature Cleavage Factor II (PCF11) [ 8 – 12 ]. m 6 Am modification has been closely associated with tumor cell aggressiveness, macrophage activation, and CD8 + T cell ferroptosis [ 13 – 17 ]. Currently, there is limited research on the effects of m 6 Am modification on bone health. Our team has first reported that Pcif1 deletion could regulate murine periodontal inflammation and bone loss by inhibiting macrophage function [ 16 ]. Mesenchymal stem cell (MSC)-specific knockout of Methyltransferase like 3 (Mettl3), methyltransferase responsible for N6-methyladenosine (m 6 A), results in skeletal disorders and aberrant adipogenic differentiation of MSCs [ 18 ]. Furthermore, FTO, a common demethyltransferase for m 6 Am and m 6 A, is involved in regulating bone mass and osteoblast functions [ 19 ]. Given the structural similarity and functional consistency between m 6 A and m 6 Am, we propose that m 6 Am modulation has similar effects on bone development.

To test the effect in vivo, we performed weekly intraperitoneal injection of rhRSPO2 (4 mg/kg) to 3-week-old Prrx1-Cre;Pcif1 fl/fl male mice for five weeks. Notably, the bone deformities caused by Pcif1 deficiency were alleviated ( Fig 7D ). The trabecular parameters of Prrx1-Cre;Pcif1 fl/fl mice, including BMD, BV/TV, Tb.N, and Tb.Th, demonstrated notable improvement following rhRSPO2 injection ( Fig 7E ). As well, the increased Tb.Sp by Pcif1 deletion was effectively ameliorated by rhRSPO2 treatment ( Fig 7E ).

(A) Representative images and measurements of ALP and ARS staining of MSCs (n = 3–4). (B) qRT-PCR analysis of osteogenic markers in MSCs after induction for 7 days (n = 3). (C) Western blot showing expression of Wnt-related proteins. (D) Representative Micro-CT images of distal femur metaphysis and midshaft cortical bone after administration of WNT agonist. Scale bar, 200 μm. (E) Quantitation of bone volume (n = 6). (F) Schematic diagram of the manuscript created using selected icons from BioRender. Song, W. (2025) https://BioRender.com/lihviy1 . The data underlying panels A, B, and E can be found in S1 Data (Sheet Fig 7).

R-spondin-2 (RSPO2) has been widely identified as an activator of canonical WNT signaling [ 43 – 46 ]. We treated MSCs from Prrx1-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl littermates with recombinant RSPO2 (rhRSPO2) (100ng/mL). After 7 days of osteoblast induction, rhRSPO2 treatment enhanced the ALP activity and mineralization of Pcif1-wildtype cells ( Fig 7A ). In detail, rhRSPO2 partially restored the impaired osteogenic capacity of Pcif1-deleted cells, although the recovery did not reach the original level observed in Pcif1-wildtype cells ( Fig 7A ). qRT-PCR results showed the decreased expression of Runx2, Alp, and Bglap by Pcif1 deficiency was elevated by rhRSPO2 ( Fig 7B ). Besides, the compromised expression of WNT11, FZD4, FGFR2, and active β-Catenin in Pcif1-knockout cells were up-regulated by rhRSPO2 treatment ( Fig 7C ).

To directly assess the role of m 6 Am in regulating WNT11 mRNA stability, we generated a dual-luciferase WNT11 reporter plasmid, a wild-type PCIF1 overexpression plasmid (PCIF1-WT), and a catalytic-dead PCIF1 mutant plasmid (PCIF1-MUT) [ 13 , 15 ]. Overexpression of wild-type PCIF1 restored the compromised WNT11 expression induced by PCIF1 knockdown ( Fig 6G ). Catalytically inactive PCIF1 could not increase the WNT11 level in the siPCIF1 group ( Fig 6G ). Likewise, similar results were also observed for the expression of FZD4 and FGFR2 ( Fig 6H and 6I ), suggesting that the catalytic activity of PCIF1 is essential for mRNA stability of these genes.

As for Fzd4, potential m 6 Am modifications were modeled within the first 100 nucleotides (position 53, 55, 56, and 77) ( Fig 6C ). Similarly, by examining this region, we confirmed that loss of Pcif1 in MSCs led to an over 50% reduction of m 6 Am level on Fzd4 mRNA ( Fig 6D ). While the +400:+500 sequence of Fgfr2 transcript was predicted to have a much higher probability than the 0:+150 region ( Fig 6E ), MeRIP-qPCR results revealed a completely opposite conclusion. The 0:+150 region (Primer 1), rather than the +400:+500 region (Primer 2), was identified as the m 6 Am-modified site ( Fig 6F ).

(A) The calculated likelihood ratio of m 6 Am modification sites on mouse Wnt11 mRNA predicted by m6AmPred database. (B) MeRIP-qPCR analysis of m 6 Am enrichment at two predicted sites within Wnt11 mRNA in MSCs. (C) The calculated likelihood ratio of m 6 Am modification sites on mouse Fzd4 mRNA predicted by m6AmPred database. (D) Detection of the m 6 Am level in Fzd4 mRNA by MeRIP-qPCR. (E) The calculated likelihood ratio of m 6 Am modification sites on mouse Fgfr2 mRNA predicted by m6AmPred database. (F) MeRIP-qPCR–based quantification of m 6 Am enrichment at predicted sites in Fgfr2 mRNA. (G–I) Relative luciferase activity of WNT11 (G) , FZD4 (H) , and FGFR2 (I) in 293T cells co-transfected with PCIF1 siRNA and PCIF1 overexpression plasmid. The data underlying panels B, D, F, G, H, and I can be found in S1 Data (Sheet Fig 6).

To examine whether these Wnt-related genes are regulated by m 6 Am modifications in MSCs, we predicted the m 6 Am distribution using the m6AmPred database [ 42 ]. Mouse Wnt11 transcript revealed a high likelihood of m 6 Am modification within the first 200 nt downstream of the TSS, particularly at position 56, 59, 65, 177, and 194 ( Fig 6A ). We further performed MeRIP-qPCR to assess m 6 Am enrichment on the Wnt11 mRNA using two primer sets (Primer 1, 0:+125 distance; Primer 2, +125:+250 distance). Primer 1 detected a prominent m 6 Am peak near the 5′-UTR, and the m 6 Am level was markedly reduced upon Pcif1 deficiency ( Fig 6B ). In contrast, Primer 2 showed low enrichment in the corresponding region ( Fig 6B ). Collectively, these results indicated that m 6 Am modification is present on the adenosine located downstream of the Wnt11 TSS.

To further assess the effect of m 6 Am modulation on the aforementioned mRNA transcripts, we examined their decay rates. Following a 7-day osteoblastic induction period, Pcif1-deficient cells and Pcif1-wildtype cells were treated with actinomycin D. After 2, 4, or 6 hours of treatment, Wnt11 mRNA in Pcif1-deleted cells exhibited greater degradation compared to that in the control cells ( Fig 5F ). The mRNA levels of Fzd4 and Fgfr2 exhibited similar trends ( Fig 5G and 5H ).

By integrating our transcriptome profiles, the m 6 Am database (GSE151229), and the Wnt signaling pathway gene set (mmu04310) ( S4C Fig ), we proposed that PCIF1-mediated m 6 Am modification was prevalent in the canonical and non-canonical WNT signaling pathways. Specifically, we prioritized several genes for a detailed analysis, focusing on those with m 6 Am peaks present at the transcription start sites (TSS) ( Fig 5E ). Wnt family member 11 (Wnt11) has been recognized as one of the predominant non-canonical WNT ligands [ 34 , 35 ]. Frizzled class receptor 4 (Fzd4) primarily participates in the canonical WNT signaling pathway, activating the β-Catenin-dependent signaling cascade, thereby initiating the expression of genes associated with bone development [ 36 – 38 ]. Fibroblast growth factor receptor 2 (Fgfr2) and the WNT signaling pathway act synergistically, particularly in skeletal and craniofacial development [ 39 – 41 ]. Loss of Pcif1 decreased m 6 Am modification levels on the Wnt11, Fzd4, and Fgfr2 mRNAs ( Fig 5E ). In addition, Pcif1 knockout reduced the expression of active β-Catenin, but no significant difference was observed in β-Catenin levels ( Fig 5D ).

(A) GO analysis of differentially expressed genes by RNA-seq of MSCs from Prrx1-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl controls after 7 days of osteogenic induction. (B) KEGG analysis of significantly down-regulated genes. (C) Heatmap of representative down-regulated genes associated with ossification and Wnt signaling. (D) Western blot showing expression of Wnt-related proteins. (E) IGV images showing down-regulated m 6 Am peaks of Wnt11, Fzd4, and Fgfr2 mRNAs by Pcif1 deletion. (F–H) RNA decay analyses of Wnt11, Fzd4, and Fgfr2 in MSCs after 7-day osteogenic induction in vitro (n = 3). The data underlying panels F, G, and H can be found in S1 Data (Sheet Fig 5).

To uncover the potential mechanism of PCIF1 in regulating osteogenesis, we performed bulk RNA-seq using MSCs from Prrx1-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl littermates after 7 days of osteoblast induction. A total of 370 down-regulated genes and 162 up-regulated genes were identified (|log 2 FoldChange| ≥ 1, padj ＜ 0.05) ( S4A Fig ). Gene ontology (GO) analysis revealed that ossification, osteoblast differentiation, and canonical Wnt signaling pathway were the most significantly down-regulated gene sets in Pcif1-deficient cells ( Fig 5A ). Kyoto Encyclopedia of Genes and Genomes (KEGG) assessment suggested Pcif1 deficiency may mainly affect Wnt signaling pathway, calcium signaling pathway, and cGMP-PKG signaling pathway ( Fig 5B ). We found that Pcif1 knockout reduced the expression of osteogenic marker genes, such as Dlx5, Alpl, Sp7, Bmp4, and Bmp5, as well as Wnt-related genes, including Wnt11, Wnt4, Fgfr2, Fzd4, Snai2, Lgr5, Pth1r, and Rspo2 ( Fig 5C ). Western blot confirmed a decrease in WNT11, FZD4, and FGFR2 protein levels by Pcif1 deficiency ( Fig 5D ). Additionally, we examined expression levels of Wnt-related transcripts in MSCs under basal conditions. Pcif1 deletion markedly reduced Wnt11 and Fzd4 expression in undifferentiated MSCs, though Fgfr2 remained unchanged ( S4B Fig ).

(A) Representative Micro-CT images of femurs from 6-week-old male LysM-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl littermates. Scale bar, 200 μm. (B) Quantitation of bone parameters (n = 8). (C) Representative Von Kossa images of undecalcified femoral sections. Scale bar, 500 μm. (D) Representative TRAP images of distal trabecular bone. Scale bar, 50 μm. (E) Quantitation of osteoclast and osteoblast numbers of trabecular bone (n = 8). (F) Representative in-vitro TRAP images and osteoclast quantification from BMDMs of LysM-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl controls (n = 3). Scale bar, 200 μm. (G) qRT-PCR analysis of osteoclast markers in vitro (n = 3). The data underlying panels B, E, F, and G can be found in S1 Data (Sheet Fig 4).

Bone homeostasis relies on the equilibrium between osteogenesis and osteoclastogenesis [ 31 – 33 ]. Tartrate-resistant acid phosphatase (TRAP) staining showed reduced bone resorption occurring in femoral trabecular bone of Pcif1 −/− mice ( Fig 2D and 2E ). Although the change in osteoclast numbers was smaller than that in osteoblast numbers ( Fig 2E ), we investigated whether Pcif1 deletion could directly induce osteoclast impairment in vivo by generating myeloid cell-specific Pcif1 knockout mice (LysM-Cre;Pcif1 fl/fl mice) [ 16 ]. Femurs from 6-week-old mice exhibited comparable bone parameters between LysM-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl littermates, regardless of sex, as supported by BMD, BV/TV, Tb.N, Tb.Th, Tb.Sp, and Ct.Th ( Figs 4A , 4B , and S3 ). Histomorphometric measurements did not show notable difference by LysM-driven deletion of Pcif1 ( Fig 4C – 4E ). Although in-vitro induction of BMDMs derived from LysM-Cre;Pcif1 fl/fl mice displayed decreased osteoclast numbers and sizes ( Fig 4F ), the expression of osteoclast markers did not differ between two groups ( Fig 4G ).

(A) Representative Micro-CT images of femurs from 3-week-old or 6-week-old male Prrx1-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl littermates. Scale bar, 200 μm. (B) Quantitative measurements of trabecular bone parameters in the distal femur and cortical bone thickness at the femoral midshaft (n = 8). (C) Representative images demonstrating Von Kossa staining of undecalcified femoral sections. Scale bar, 500 μm. (D) Representative TRAP images of trabecular bone at the distal end of femurs. Scale bar, 50 μm. (E) Quantitation of osteoblast and osteoclast numbers of trabecular bone (n = 8). (F) Representative images showing calcein double-labeling of trabecular bone. Scale bar, 20 μm. (G) Quantitation of MAR and BFR/BS of trabecular bone from the femoral metaphysis (n = 8). (H) Representative ALP and ARS images with quantification of MSCs isolated from Prrx1-Cre;Pcif1 fl/fl mice and Pcif1 fl/fl controls (n = 3–4). (I) qRT-PCR analysis of osteogenic gene expression in MSCs following 7 days of culture in osteogenic medium in vitro (n = 3). The data underlying panels B, E, G, H, and I can be found in S1 Data (Sheet Fig 3).

To determine whether the low bone mass of Pcif1 −/− mice specifically results from osteogenic defects, we generated the MSC-specific Pcif1 knockout mice model (Prrx1-Cre;Pcif1 fl/fl mice). Prrx1-Cre;Pcif1 fl/fl mice were born alive at an expected mendelian ratio. Micro-CT revealed Prrx1-Cre;Pcif1 fl/fl mice had significantly reduced bone mass compared to Pcif1 fl/fl controls at both 3 and 6 weeks of age ( Fig 3A and 3B ). Quantitative results showed that ablation of Pcif1 in Prrx1 + MSCs resulted in a reduction in BMD, BV/TV, Tb.N, and trabecular thickness (Tb.Th), along with an increase in Tb.Sp ( Fig 3B ). Ct.Th remained unchanged ( Fig 3B ). Pcif1 deletion diminished the mineralized trabecular bone ( Fig 3C ), as well as MAR and BFR/BS of femoral trabecular bone ( Fig 3F and 3G ). Although the number of osteoclasts was slightly decreased, we also observed a further weakened state of osteoblasts in Prrx1-Cre;Pcif1 fl/fl mice ( Fig 3D and 3E ).

(A) Representative Micro-CT images of femurs from 6-week-old male Pcif1 −/− knockout mice and Pcif1 +/+ littermates. Scale bar, 200 μm. (B) Quantitative analyses of distal trabecular bone and midshaft cortical thickness of femurs (n = 8). (C) Representative images of Von Kossa staining of undecalcified femoral sections. Scale bar, 500 μm. (D) Representative TRAP images of trabecular bone from the femoral metaphysis. Scale bar, 50 μm. (E) Quantitation of osteoblast and osteoclast numbers of trabecular bone (n = 8). (F) Representative images demonstrating calcein double-labeling patterns in trabecular bone from the femoral metaphysis. Scale bar, 20 μm. (G) Quantitation of mineralization apposition rate (MAR) and bone formation rate (BFR/BS) of trabecular bone (n = 8). (H) Representative images and quantitative analyses of ALP and ARS staining of MSCs isolated from Pcif1 −/− knockout mice and Pcif1 +/+ controls (n = 3–4). (I) qRT-PCR analysis of osteogenic markers in MSCs cultured with osteogenic medium for 7 days in vitro (n = 3). The data underlying panels B, E, G, H, and I can be found in S1 Data (Sheet Fig 2).

To discern the role of Pcif1 in osteogenesis, we first assessed the bone characteristics using whole-body Pcif1 knockout mice (Pcif1 −/− ) and control littermates (Pcif1 +/+ ). Micro-computed tomography (micro-CT) analysis revealed a reduction in the trabecular bone mass of the distal femur of male Pcif1 −/− mice, as indicated by decreased BMD and bone volume (BV/TV) ( Fig 2A and 2B ). Extended research showed Pcif1 deletion reduced trabecular number (Tb.N) and increased trabecular separation (Tb.Sp), while midshaft cortical thickness (Ct.Th) was unaffected ( Fig 2B ). Von Kossa staining also confirmed the impaired mineralization of distal femoral metaphysis ( Fig 2C ). Histomorphometric analyses suggested that Pcif1 −/− mice exhibited a significant reduction in osteoblast numbers (N.Ob/B.Pm) and a marginal decrease in osteoclast numbers (N.Oc/B.Pm) ( Fig 2D and 2E ). Loss of Pcif1 contributed to the decreased mineral apposition rate (MAR) and bone formation rate (BFR/BS) as assessed by calcein double-labeling ( Fig 2F and 2G ). Female Pcif1 −/− mice exhibited a bone loss phenotype similar to that observed in male mice ( S2 Fig ).

To reconstruct the osteogenic differentiation continuum, we applied Monocle 2 pseudotime analysis, which revealed a continuous, unbranched trajectory extending from MSCs to mature osteoblasts ( Fig 1H ). Cells at the earliest pseudotime points overlapped with those exhibiting the highest CytoTRACE scores, reinforcing their identity as early progenitors. MSC_Fibro and OB cells occupied intermediate states, while Mature OBs were enriched at the terminus of the trajectory. Notably, PCIF1 expression was highest at early pseudotime stages and progressively declined along the osteogenic trajectory ( Fig 1H ), further supporting its functional involvement in early-stage mesenchymal differentiation.

To assess the cellular context and transcriptional pattern of PCIF1 in human bone, we performed single-cell RNA sequencing (scRNA-seq) on femoral head-derived cells collected from patients undergoing total hip arthroplasty for osteoarthritis ( Fig 1C ). Following stringent quality control, 40,821 high-quality cells were retained for downstream analysis, with a median of 1,030 genes and 2,228 unique molecular identifiers (UMIs) detected per cell. Unsupervised graph-based clustering partitioned the transcriptomes into 11 distinct clusters ( Fig 1D ), encompassing both hematopoietic and non-hematopoietic populations. These included B cells, T/NK cells, neutrophils, macrophages, red blood cells (RBCs), vascular smooth muscle cells, endothelial cells, and mesenchymal lineage cells such as MSCs, fibroblast-like MSCs (MSC_Fibro), osteoblast precursors (OB), and mature osteoblasts (Mature OB). The key cell lineage marker genes are presented in S1 Fig .

In the transcriptome-wide association studies (TWAS), PCIF1 was found to have 139 associations with various diseases ( S3 File ). Notably, it was identified as associated with osteopenia using the GIFT method (p = 2.77 × 10⁻⁷) and showed significant associations with M65-M68 disorders of the synovium and tendon (p = 6.11 × 10⁻⁸) and spinal injury (p = 1.74 × 10⁻⁷). Additional associations included joint disorders (p = 5.02 × 10⁻⁸), M25 joint disorders (p = 9.69 × 10⁻⁹), and gouty arthropathy (p = 5.33 × 10⁻⁷), suggesting a potential role for PCIF1 in osteoporosis, joint diseases, and musculoskeletal health ( Fig 1B ).

We examined genetic associations of key m 6 Am regulators ( S1 File ). The m 6 Am eraser FTO was strongly associated with fracture risk and BMD, consistent with previous reports [ 19 , 29 ]. The m 6 Am reader PCF11 also showed associations with estradiol levels and BMD. Given PCIF1’s central role as the m 6 Am writer and its impact on murine bone physiology, we therefore focused this study on elucidating PCIF1’s function in bone homeostasis.

(A) GWAS analysis for PCIF1 gene. a: P-value for gene-based association test; b: Female samples only; %: the number of SNPs. Skull BMD: Head skull BMD; Osteoclast eQTL. TB-BMD: Total body BMD. (B) TWAS analysis for PCIF1 gene identified by (Gene-based Integrative Fine-mapping through conditional TWAS; GIFT) method. (C) Flowchart of the scRNA-seq pipeline. This schematic diagram was Created in BioRender. Song, W. (2025) https://BioRender.com/w178r3g and https://BioRender.com/vhpjizr . (D) UMAP projection of all cell types identified from femoral head scRNA-seq data. Major immune, vascular, and skeletal cell populations were annotated, including neutrophils, macrophages, NK/T cells, endothelial cells, red blood cells (RBC), vascular smooth muscle cells (VSMCs), and stromal osteolineage populations: mesenchymal stem cells (MSC), fibroblast-like MSCs (MSC_Fibro), osteoblast precursors (OB), and mature osteoblasts (Mature OB). (E) PCIF1 expression mapped onto the UMAP projection. (F) Violin plot showing PCIF1 expression among human femoral head-derived bone-resident cell types. (G) CytoTRACE analysis of osteolineage cells. Differentiation potential predicted by CytoTRACE demonstrates a gradual loss of stemness from MSC and MSC_Fibro populations toward mature osteoblasts. (H) Monocle 2 pseudotime trajectory of osteoblast differentiation. Cells are arranged along a pseudotime axis showing a continuous transition from MSC_Fibro through OB to Mature OB.

To investigate the impact of PCIF1 gene on skeletal homeostasis, we first conducted a gene-based association study involving 11 published genome-wide association studies (GWAS) focused on BMD across various skeletal sites, alongside traits such as fracture susceptibility, osteoclast activity, and estrogen levels ( S1 Table ). A total of 31 unique SNPs showed associations with PCIF1 across the analyzed traits ( S1 File ). Four significant associations for PCIF1 were identified in gene-based analyses of bone-related traits ( Fig 1A and S1 File ). The strongest associations were detected for estimated bone mineral density (eBMD) (p = 2.0 × 10⁻⁸, rs8119032), skull bone mineral density (BMD) (p = 0.034, rs183625591), and osteoclast-related eQTL traits (p = 0.023, rs8114050), suggesting a consistent role of PCIF1 in bone regulation.

Discussion

Emerging evidence has elaborated the role of m6Am modification in tumorigenesis, anti-tumor immunity, anti-infection responses, and various biological processes [15–17,47–49]. Since PCIF1 has been identified as the methyltransferase for editing m6Am peaks [50,51], it has become a prominent research topic in the field of epigenetic regulation. Existing studies have mainly focuses on the immunological functions of m6Am modification, with limited attention given to its role in bone elongation. Moreover, m6A and m6Am exert similar functions in bone physiology, probably because of their structural similarity. Our previous work has unveiled that m6A methyltransferase METTL3 modulates MSC differentiation, tooth root formation, calvarial ossification, cartilage development and periodontal inflammation [2,5,18,52,53]. In contrast, current research on m6Am has predominantly centered on its immunological functions. Our group remains the only one to provide evidence that m6Am regulates periodontal bone loss, a biological effect that parallels the role of METTL3-mediated m6A modification in periodontal inflammation-associated bone loss. In this study, we further demonstrate that m6Am-Wnt signaling regulates femoral bone mass, expanding the known scope of m6Am function in skeleton development, and suggesting its potential as a therapeutic target for osteoporosis.

We used a combination of human gene-based association genetic studies from large population-based cohorts and scRNA-seq data to investigate the role of PCIF1 in bone-related traits. GWAS and TWAS studies identified the strong associations between PCIF1 and eBMD or bone disorders. Notably, to the best of our knowledge, previous genome-wide and transcriptome-wide studies have not directly implicated PCIF1 in bone-related traits. Our current gene-based results revealed that PCIF1 harbors multiple SNPs associated with bone phenotypes, with the lead SNP rs8119032 showing genome-wide significance (p = 2.0 × 10⁻⁸). Annotation of the lead PCIF1-associated SNPs (rs8119032, rs18362559, and rs8114050) using Ensembl showed that they lie in intronic or proximal intergenic regions relative to PCIF1, a genomic context where many regulatory elements reside. Although such noncoding variants frequently act through regulatory mechanisms, none of the lead SNPs appeared as significant cis-eQTLs for PCIF1 in GTEx v8 single-tissue datasets. This suggests that any regulatory effects may be tissue-specific, modest in magnitude, or restricted to bone-related or disease-associated cellular contexts that are not represented in GTEx. Thus, while our genetic analyses robustly implicate the PCIF1 locus in bone traits, the precise regulatory elements and mechanisms linking these noncoding variants to PCIF1 activity remain to be determined by future functional studies. In line with our previous report [16], PCIF1 also showed significant correlation with osteoclasts, indicating that it may act as a broader regulator of bone homeostasis. These findings nominate PCIF1 as a previously unrecognized genetic contributor to bone health and provide functional insight into its role.

Our scRNA-seq analysis of femoral head-derived bone-resident cells revealed that PCIF1 is selectively expressed in MSCs and progressively declines along the osteogenic differentiation trajectory, suggesting a role in early osteolineage programming. This expression pattern implies that PCIF1 may contribute to the maintenance of MSC identity or the regulation of early differentiation events. The use of femoral head tissue from total hip arthroplasty remains a practical and ethically appropriate approach for obtaining human bone cells. Since our scRNA-seq data were derived from femoral head tissue of patients with osteoarthritis, we acknowledge that disease-associated inflammatory factors might influence the transcriptional profiles or composition of bone-resident MSCs. Although evidence suggests that osteoarthritis has limited effects on deep trabecular bone [30], the potential impact of disease-related changes on PCIF1 expression and MSC heterogeneity cannot be fully excluded. We note that our findings may not fully represent the transcriptional landscape of healthy bone. Nevertheless, this dataset provides a valuable foundation for future comparative studies aimed at validating PCIF1’s role in normal bone development and homeostasis. The elevated expression of PCIF1 in undifferentiated MSCs suggests that PCIF1-mediated m6Am methylation may help sustain gene expression programs essential for stemness and inhibit premature lineage commitment.

Notably, cells with high PCIF1 expression also exhibited gene expression profiles characteristic of early-stage progenitors, while more differentiated osteoblasts showed minimal PCIF1 activity. This graded expression pattern supports the idea that PCIF1 may act as a molecular checkpoint in osteogenic differentiation, helping to regulate the transition from stemness to commitment. Similar functions for RNA methylation machinery have been described in other stem cell systems, such as the role of METTL3-mediated m6A methylation in promoting differentiation in embryonic stem cells [54]. While PCIF1 catalyzes the related m6Am modification, its expression pattern in MSCs suggests it may similarly modulate stemness and lineage progression in skeletal tissue. Our findings extend the regulatory role of RNA methylation in cell fate decisions to the context of bone development. Based on these data, we hypothesized that PCIF1 was strongly correlated with bone mass, and might regulate bone homeostasis via osteoblast–osteoclast balance.

To verify this hypothesis, we performed extensive studies using several Pcif1-knockout mouse models. We first observed global Pcif1 ablation mice had shorter size and reduced trabecular bone measurements, while cortical bone thickness was unaffected by Pcif1 deficiency. Notably, loss of Pcif1 led to a decrease in osteoblast and osteoclast numbers, with the more pronounced downregulation of osteoblasts compared with osteoclasts. The observed reduction in osteoclasts may reflect altered RANKL/OPG signaling secondary to compromised osteoblast function, as osteoblasts are a major source of RANKL and OPG [55,56]. Hence, we introduced Prrx1-Cre and LysM-Cre for conditional Pcif1 knockout in MSC and myeloid cells, respectively, to further explore whether PCIF1 targets at osteogenesis or osteoclastogenesis. Prrx1-driven Pcif1 knockout induced delayed bone formation in murine femurs, resembling the phenotype observed in systemic knockout mice. However, LysM-driven Pcif1 deletion did not cause changes of trabecular volumetric BMD, though in-vitro culture showed compromised osteoclast differentiation. We propose that the effects of Pcif1 on osteoclasts may be masked or compensated by other factors in vivo, and unable to produce measurable changes. Moreover, osteoclast formation is mainly driven by RANKL in vitro, and could not fully recapitulate the complex microenvironment found in vivo. In this paper, we prioritized MSCs as the principal cell population affected by Pcif1 deficiency during skeleton development.

WNT signaling facilitates MSC renewal and osteoblast differentiation and inhibits osteoblast apoptosis [57–59]. Accumulating reports have confirmed the crucial role of WNT signaling in skeletal development and bone regeneration [60–62]. Our RNA-seq of osteogenic MSCs results showed the most significant downregulation pathways including ossification and Wnt signaling. Wnt-related genes exhibited widespread downregulation, suggesting PCIF1 could broadly affect WNT pathway rather than targeting a single gene or a limited subset of genes. By combing analyses of a published m6Am-exo-seq database, we noticed the TSS regions of Wnt11, Fzd4, and Fgfr2 mRNA had PCIF1-mediated m6Am peaks. WNT11 is a potent ligand of non-canonical WNT signaling [63,64], regulating osteoblast maturation and bone formation through RSPO2-LGR5 pathway [65,66]. Overexpression of FZD4 restores the inhibited bone formation by mechanical unloading [67]. Inducible FGFR2 activation enhances the osteogenesis by activating Wnt/β‐catenin signaling after bone marrow ablation [68]. FGFR2 mutations cause lethally systemic skeletal malformation in human subjects [69]. Our previous research has reported that FGFR2 ligand FGF2 controls BMSC fate by modulating rDNA chromatin architecture through phase separation [70]. Existing literature has illustrated that PCIF1-mediated m6Am regulation controls mRNA stability [8,14,16,47]. Here, we found Pcif1 deficiency accelerated decay rates of Wnt11, Fzd4, and Fgfr2 mRNAs, indicating that PCIF1 manipulates WNT signaling by stabilizing mRNAs. We validated the down-regulated expression of WNT11, FZD4, and FGFR2 proteins, as well as the decreased levels of active β-Catenin.

Given the epitranscriptomic landscape is highly cell-type and context-specific, we integrated m6AmPred predictions with MeRIP-qPCR for further validation. We subjected the Pcif1-deleted MSCs and WT cells to m6Am enrichment, and designed the primers according to the prediction scores by m6AmPred. Mouse Wnt11 and Fzd4 transcripts showed significant m6Am distribution within the first ~150 nt downstream of TSS, which were consistent with previous reports [10,51]. The pronounced loss of these signals in the Pcif1-deleted cells strongly supported that the detected modification corresponds to m6Am. Interestingly, mouse Fgfr2 transcript got higher score in the+400:+500 region, whereas MeRIP-qPCR results identified the m6Am peaks within the 0:+150 region. This might be ascribed to the limitations of the prediction model, the complexity of the sequence context, and cell-type-specific TSS selection. Dual-luciferase reporter assays also showed that exogenous expression of PCIF1 with m6Am catalytic activity was able to restore the expression of WNT11, FZD4, and FGFR2 in PCIF1-knockdown cells. A limitation is that, although m6Am was detected, its precise modification sites remain to be determined. Further investigations are needed to elucidate the broader landscape of m6Am-mediated regulation.

We next evaluated whether the WNT activator RSPO2 could function as potential therapeutic intervention for Pcif1 knockout-induced osteoporosis. Prior research has identified RSPO2 mutations in fetuses exhibiting severe limb disorders, emphasizing its critical role in skeletal development [71]. Conditional Rspo2 ablation in Osteocalcin+ cells could lead to decreased body size and femoral trabecular bone in either male or female mice [72]. Overexpression of Rspo2 promotes BMP-induced osteoblast mineralization in vitro [65]. In vitro and in vivo functional studies revealed administration of rhRSPO2 partially rescued the bone formation disruption and the restrained osteoblast differentiation of MSCs. The partial rescue suggests that PCIF1 may affect other signaling cascades related to bone biology. For example, RNA-seq results indicates that Pcif1 deletion also downregulates the Calcium signaling and cGMP–PKG pathways. Besides, Pcif1 deletion reduces the expression of multiple genes related to osteoblast differentiation and mineralization, e.g., Bmp4, Bmp5, Alpl, and Sp7. Moreover, RSPO2 efficacy is likely influenced by the abundance of downstream receptors and WNT ligands [46]. It is also possible that some developmental defects are not fully reversible when established during the early postnatal period in Pcif1-deficient mice.

It should be noted that osteoporosis in humans is predominantly an age-related condition, whereas our study primarily employed young mouse models (3- and 6-week-old). Future studies using aged or osteoporotic animal models will be essential to determine whether PCIF1-mediated m6Am modification similarly contributes to age-associated bone loss.

In sum, our findings demonstrate PCIF1 as a candidate associated with osteoporosis-related traits. Pcif1 deletion impairs trabecular bone formation by suppressing osteogenic differentiation. Mechanically, PCIF1-mediated m6Am modulation enhances the mRNA stability of Wnt-related genes. Our findings unveil a pivotal role of PCIF1-mediated m6Am modulation in skeletal homeostasis, highlighting its potential for therapeutic intervention.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003739

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