SP1 regulates KLF4 via SP1 binding motif governed by DNA methylation during odontoblastic differentiation of human dental pulp cells

Zheyi Sun, Shuaitong Yu, Shuo Chen, Huan Liu, Zhi Chen

Research output: Contribution to journalArticle

Abstract

Objective: DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process, however, its detailed molecular mechanism during odontoblastic differentiation remains elusive. We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor (TF) on its target gene. Methods: DNA methyltransferase (DNMTs) inhibition assay and following odontoblastic differentiation assay were performed to evaluate the effect of DNA methylation inhibition on odontoblastic differentiation. Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation. The enriched target sites and motifs were further analyzed by methylation-specific polymerase chain reaction (MS-PCR) and sequencing. The functional target sites were validated in vitro with Luciferase assay. The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay. Results: Inhibition of DNMTs in preodontoblast cells increased the expression level of Klf4 as well as marker genes of odontoblastic differentiation including Dmp1 and Dspp, and enhanced the efficiency of odontoblastic differentiation. SP1/KLF4 binding motifs were found to be highly enriched in the promoter regions and showed demethylation during odontoblastic differentiation. Mutation of SP1 binding site at -75 within KLF4′s promoter region significantly decreased the luciferase activity. The in vitro methylation of KLF4′s promoter decreased the transactivation of SP1 on KLF4. Conclusion: We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4′s promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1′s binding and transcriptional regulation on KLF4.

Original languageEnglish (US)
Pages (from-to)14688-14699
Number of pages12
JournalJournal of Cellular Biochemistry
Volume120
Issue number9
DOIs
StatePublished - Sep 1 2019

Fingerprint

Dental Pulp
DNA Methylation
Pulp
Assays
Methylation
Genetic Promoter Regions
Methyltransferases
Luciferases
Transcriptional Activation
Transcription Factors
Genes
Binding Sites
Nucleotide Motifs
CpG Islands
Deciduous Tooth
Polymerase chain reaction
DNA
Microarrays
Oligonucleotide Array Sequence Analysis
Epigenomics

Keywords

  • DNA methylation
  • human dental pulp cells
  • Klf4
  • odontoblastic differentiation
  • SP1

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

SP1 regulates KLF4 via SP1 binding motif governed by DNA methylation during odontoblastic differentiation of human dental pulp cells. / Sun, Zheyi; Yu, Shuaitong; Chen, Shuo; Liu, Huan; Chen, Zhi.

In: Journal of Cellular Biochemistry, Vol. 120, No. 9, 01.09.2019, p. 14688-14699.

Research output: Contribution to journalArticle

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abstract = "Objective: DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process, however, its detailed molecular mechanism during odontoblastic differentiation remains elusive. We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor (TF) on its target gene. Methods: DNA methyltransferase (DNMTs) inhibition assay and following odontoblastic differentiation assay were performed to evaluate the effect of DNA methylation inhibition on odontoblastic differentiation. Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation. The enriched target sites and motifs were further analyzed by methylation-specific polymerase chain reaction (MS-PCR) and sequencing. The functional target sites were validated in vitro with Luciferase assay. The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay. Results: Inhibition of DNMTs in preodontoblast cells increased the expression level of Klf4 as well as marker genes of odontoblastic differentiation including Dmp1 and Dspp, and enhanced the efficiency of odontoblastic differentiation. SP1/KLF4 binding motifs were found to be highly enriched in the promoter regions and showed demethylation during odontoblastic differentiation. Mutation of SP1 binding site at -75 within KLF4′s promoter region significantly decreased the luciferase activity. The in vitro methylation of KLF4′s promoter decreased the transactivation of SP1 on KLF4. Conclusion: We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4′s promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1′s binding and transcriptional regulation on KLF4.",
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T1 - SP1 regulates KLF4 via SP1 binding motif governed by DNA methylation during odontoblastic differentiation of human dental pulp cells

AU - Sun, Zheyi

AU - Yu, Shuaitong

AU - Chen, Shuo

AU - Liu, Huan

AU - Chen, Zhi

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N2 - Objective: DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process, however, its detailed molecular mechanism during odontoblastic differentiation remains elusive. We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor (TF) on its target gene. Methods: DNA methyltransferase (DNMTs) inhibition assay and following odontoblastic differentiation assay were performed to evaluate the effect of DNA methylation inhibition on odontoblastic differentiation. Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation. The enriched target sites and motifs were further analyzed by methylation-specific polymerase chain reaction (MS-PCR) and sequencing. The functional target sites were validated in vitro with Luciferase assay. The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay. Results: Inhibition of DNMTs in preodontoblast cells increased the expression level of Klf4 as well as marker genes of odontoblastic differentiation including Dmp1 and Dspp, and enhanced the efficiency of odontoblastic differentiation. SP1/KLF4 binding motifs were found to be highly enriched in the promoter regions and showed demethylation during odontoblastic differentiation. Mutation of SP1 binding site at -75 within KLF4′s promoter region significantly decreased the luciferase activity. The in vitro methylation of KLF4′s promoter decreased the transactivation of SP1 on KLF4. Conclusion: We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4′s promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1′s binding and transcriptional regulation on KLF4.

AB - Objective: DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process, however, its detailed molecular mechanism during odontoblastic differentiation remains elusive. We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor (TF) on its target gene. Methods: DNA methyltransferase (DNMTs) inhibition assay and following odontoblastic differentiation assay were performed to evaluate the effect of DNA methylation inhibition on odontoblastic differentiation. Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation. The enriched target sites and motifs were further analyzed by methylation-specific polymerase chain reaction (MS-PCR) and sequencing. The functional target sites were validated in vitro with Luciferase assay. The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay. Results: Inhibition of DNMTs in preodontoblast cells increased the expression level of Klf4 as well as marker genes of odontoblastic differentiation including Dmp1 and Dspp, and enhanced the efficiency of odontoblastic differentiation. SP1/KLF4 binding motifs were found to be highly enriched in the promoter regions and showed demethylation during odontoblastic differentiation. Mutation of SP1 binding site at -75 within KLF4′s promoter region significantly decreased the luciferase activity. The in vitro methylation of KLF4′s promoter decreased the transactivation of SP1 on KLF4. Conclusion: We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4′s promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1′s binding and transcriptional regulation on KLF4.

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KW - SP1

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