Figure 8: Klf5 binding to its motifs of Dspp and Dmp1 regulatory regions in vivo. | Scientific Reports

Figure 8: Klf5 binding to its motifs of Dspp and Dmp1 regulatory regions in vivo.

From: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

Figure 8

(a) The diagram shows that six pairs of primers were designed to amplify the Klf5 binding sites in the first intron 1 of mouse Dspp gene from Dspp-site 1 to Dspp-site 9 in vivo for ChIP assay. The position number was stated as Dspp-primer 1: 50 bp to 257 bp; Dspp-primer 2: 1,219 bp to 1,422 bp; Dspp-primer 3: 1,885 bp to 2,074 bp; Dspp-primer 4: 2,177 bp to 2,468 bp; Dspp-primer 5: 2,574 bp to 2,861 bp; Dspp-primer 6: 3,025 bp to 3,265 bp. (b–g) ChIP assay showed that endogenous Klf 5 interacted with its motifs in the Dspp regulatory regions while Klf 5 overexpression significantly increased binding to its motifs in Dspp regulatory regions from Dspp site 1 to Dspp site 9 in vivo. (h). Two pairs of primers were used for the Klf5 binding sites in human Dmp1 promoter from Dmp1-site 1 to Dmp1-site 2 for ChIP assay. ChIP assay was performed with chromatin from iMDP-3 cells with transfection of Dspp-5.7 kb reporter construct (b–g) and Dmp1–2.6 kb promoter construct (i and j) with either pcDNA3-Klf5 or pcDNA3 plasmid. The results revealed that Klf5 binds to the Dspp regulatory regions encompassing the CACCC/GGGTG boxes between +73 to +2.8 kb and the Dmp1 promoter between −2.6 kb to −1,656 bp in vivo and exhibited an increasing transcription in Klf5-stimulated groups in iMDP-3 cells.

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