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

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.

Epigenetic Regulation of Dental Pulp Stem Cell Fate

“…Inhibition of DNMTs causes demethylation of the Klf4 promoter region, leading to enhanced binding of SP1, a transcriptional factor that upregulates the expression of Klf4 . Krüppel like factor 4 (KLF4) has been proved to be vital for odontogenic differentiation [ 99 ]. Besides, the myogenic differentiation is also improved after treatment with 5-Aza-CdR [ 100 ].…”

LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site

“…Given the fact that many genes regulated in GCs by the LH surge have Sp1 binding sites and exhibit Sp1-dependent transactivation [ 27 , 31 ], the presence of multiple Sp1 binding sites in the −715/−402 bp region of the Klf4 promoter suggested participation of Sp1 in the upregulation of Klf4 gene expression in GCs. Indeed, overexpression of Sp1 transactivated Klf4 promoter activity in a dose-dependent manner in preovulatory GCs ( Figure 5 A) as in non-gonadal cells [ 8 , 32 , 33 ].…”

“…ChIP analysis confirmed direct binding of Sp1 to the endogenous Klf4 promoter containing the three Sp1 binding sites in the −715/−500 bp region in intact chromatin in response to LH treatment ( Figure 6 ). On the other hand, the Sp1 binding sites located at −145 or −75 bp from the TSS have been reported to play a role in regulating Klf4 expression in non-gonadal cell [ 25 , 33 ]. Thus the critical regions of the Klf4 promoter for interaction with Sp1 may be different depending on the type of cells.…”

“…We also found that a conserved Sp1 binding site at −698/−688 bp in the Klf4 promoter was indispensable for basal as well as LH-stimulated Klf4 transcription in GCs ( Figure 7 A). In contrast, one/three consensus Sp1 binding sites between −150/+1 bp of the Klf4 promoter have shown to be important for PDGF- or butyrate-induced Klf4 expression in VSMCs [ 32 ], colon cancer cells [ 25 ], or odontoblast [ 33 ]. As stated above, the location of Sp1 binding domains required for the increased expression of Klf4 in GCs was different from in non-gonadal cells, but Sp1 binding sites seem to be critical in transcriptional control of Klf4 in diverse cells.…”

Hsa-miRNA-143-3p Regulates Differentiation of Human Stem Cells From The Apical Papilla Through Targeting Nuclear Factor I-C

“…The reason might be the synergistic effect of miR-143-3p on DSPP and DMP1 by directly acting on NFIC. The function of KLF4 as a transcription factor is relative complex, and it is responsible for promoting the differentiation of mouse pre-odontoblast and the expression of Dmp1 and Dspp, but shows inhibitory effect on some embryonic stem cells to maintain their stemness [28] . ALP and OCN expression was not affected by miR-143-3p, which may be because they are more related to the dentin mineralization in the late-stage of root development [29] , or associated with regulation by root, leading to the absent root phenotype in Nfic null mouse [30] .…”