OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases

Yan Lu,Xiangshu Jin,Xinqi Wang,Wenhong Xu,Yantong Guo,Yiyang Jia,Huinan Qu,Hairi Li,Chengshi Quan,Da Qi,Peiye Song,Yulin Li,Shutong Liu

doi:10.1186/s13287-018-1120-x

Abstract

BackgroundSelf-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved.MethodsA lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCsOCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected.ResultsThe overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCsOCT4 and hHFMSCsEGFP, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCsOCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCsOCT4, and triggered cell senescence.ConclusionsOCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Highlights

Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is regulated by OCT4. p21, a gene negatively associated with selfrenewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition
Establishment of the human hair follicle mesenchymal stem cells (hHFMSCs) cell line with ectopic expression of OCT4 The proliferation, cell cycle, and differentiation potential were inhibited in hHFMSCs, and they entered into a state of replicative senescence after a certain length of cell culture (Additional file 1: Figure S1 & S2)
HHFMSCs infected with scrambled lentiviruses are referred to as hHFMSCsEGFP, and hHFMSCs transduced with lentiviruses encoding OCT4 are referred to as hHFMSCsOCT4

Summary

Introduction

Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is regulated by OCT4. p21, a gene negatively associated with selfrenewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is regulated by OCT4. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved. Fully understanding the molecular mechanisms involved in the regulation of hHFMSC selfrenewal and senescence (i.e., maintaining stem cell properties) would provide a great advancement in the application of these cells. The regulation of stem cell self-renewal and its properties resides in conserved transcriptional regulatory networks [5, 6] and epigenetic modifications, such as DNA methylation, that work together to repress developmental genes and activate stemness genes [7]

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