Epigenetic Regulator Ptip Governs Skeletal Stem Cells Maintenance Via Regulating Glycolysis For Maintaining Skeletal Development

Aims:Skeletal stem cells（SSCs）are recently identified as a class of stem cells with specific cell markers and cell lineages in growth plates of long bone.Studies have shown that these cells play important roles in physiological and pathological fracture repair,distraction osteogenesis and cartilage regeneration.However,the regulatory mechanism of SSCs lineage differentiation and its role in maintaining skeletal homeostasis remain unclear.Methods and Results:In this study,we firstly analyzed the public single-cell RNA-seq data,flow cytometry analysis,and histological analysis,and found that the proportion of SSCs decreased with skeletal development and maturity under physiological conditions.RNA-seq of SSCs lineage revealed that the function of characteristic genes in SSCs lineage was consistent with the process of skeletal development and maturation.These results suggest that SSCs lineage differentiation plays an important role in maintaining skeletal homeostasis.Secondly,we plotted the epigenetic landscape of SSCs lineages by integrated analyzing Ch IP-seq（H3K4me1,H3K4me3,H3K27ac）and RNA-seq data,and found that SSCs lineages differentiation was mainly regulated by activity of enhancers.In addition,by analyzing the super enhancers in each population,we found that Ptip was the gene with the highest activity in SSCs,and its expression decreased with the differentiation of SSCs lineage.Thus,this indicates Ptip may be a key regulatory molecule that determines the fate of SSCs.Ptip is a subunit of the MLL3/4complex,which is mainly involved in the methylation and acetylation of histones and plays an important role in a variety of stem cells such as embryonic stem cells and hematopoietic stem cells.However,its role in SSCs lineage differentiation is not clear.In order to explore the role of Ptip in SSCs lineage differentiation,we applied rainbow tracing system with knock of Ptip mice and flow cytometry to study the function of SSCs,and found that Ptip deletion would lead to abnormal SSCs lineage differentiation and an increased number of SSCs derived clones.In order to explore the molecular mechanism of Ptip,we performed RNA-seq and Ch IP-seq and integrated analysis,and found that loss of Ptip activates Pgk1 and increases the glycolysis activity in SSCs.Further,we found knockdown of Pgk1 rescued abnormal glycolytic function of SSCs due to loss of Ptip.To verify the role of glycolysis in SSCs,we used glycolysis inhibitors（2-DG and 2-Me OE）restored the abnormal proliferation and differentiation of SSCs mediated by Ptip deletion.To investigate the role of Ptip-mediated SSCs dysfunction in bone development,we breed Ptip^f/f;Col2-cre mice and found SSCs lineage were disturbed by loss of Ptip analyzed by flow cytometry.The skeletal phenotype was further systematically examined by histological techniques,and it was found that Ptip^f/f;Col2-cre mice exhibited dwarf,disordered growth plates,increased bone mass,and cartilage tumor formation.Finally,to explore the clinical significance of PTIP in differentiation of human chondrocytes and progression of cartilage tumors,we detected the expression of PTIP at the histological level,and found that the expression of PTIP was increased in undifferentiated chondrocytes,but decreased in differentiated chondrocytes.In human cartilage tumors,the expression of PTIP was increased in poorly differentiated cells,but decreased in highly differentiated cells.Furthermore,we explored the role of Ptip in tooth development and found that Ptip deletion in Sp7⁺cells leads to abnormal tooth mineralization.More importantly,we identified putative SSCs population in periodontal and pulp tissues of mice and humans by using surface markers of mouse and human SSCs.Conclusions:Ptip regulates SSCs lineage differentiation through Pgk1-mediated glycolysis,thus maintaining skeletal homeostasis under physiological conditions.Significance:This study revealed the role and mechanism of epigenetic molecules in regulating SSCs lineage differentiation through metabolism and affecting skeletal development,providing a new mechanism for skeletal development and a new target for clinical treatment of abnormal skeletal development diseases.The functional exploration of Ptip and the identification of SSCs population in tooth tissues lay a theoretical foundation for elucidating the mechanism of tooth development.