Study On Enhancers In Odontoblastic/osteogenic Differentiation

1.Landscape of open chromatin regions during the terminal differentiation of mouse odontoblasts Objective: Enhancers are short regions of DNA,serving as cis-regulatory elements to recruit cell-specific transcription factors and initiate transriptional regulation.Odontoblast differentiation is an essential step for tooth development and is governed by a complex gene regulatory network(GRN).However,enhancers interacting with transcription factors during odontoblast differentiation remain largely unknown.In present study,we utilized next generation sequencing to identify transcription factor families regulating odontoblastic commitment and differentiation,and its underling epigenetic mechanism.Methods: We established an in vitro model for odontoblast differentiation using dental papillary cells isolated from first lower molars of embryonic day 18.5(E18.5)mouse.We performed ATAC-seq before and after the induction,marked by significant upregulation of Dmp1 and Dspp.Anti-H3K27 ac ChIP-seq at the same time points were also performed to annotate the tissue-specific active enhancers among all open chromatin regions identified by ATAC-seq.Besides,we utilized time-series ATAC-seq to analyze the dynamic change of odontoblast-specific enhancers.In the end,immunohistochemical assay was used to detect the expression pattern of transcription factor ATF5 in vivo.And lentivirus mediated overexpression was used to identify the function of ATF5 during odontoblastic differentiation.Results: Overall,we identified 19755 active enhancers before the differentiation,with a majority of their target genes associated with tooth/dentin morphology,indicating odontoblast lineage commitment is already established at or before E18.5.However,5422 active enhancers were specifically enriched at the end of induction,correlating with significant up-regulation of Dspp and Dmp1.Also,genes associated with these enhancers were significantly enriched with mineralization and hard tissue formation,such as Dmp1,Mepe,and Col1 a.Multidimensional scaling and k-mean clustering analysis for the differential enhancers during different time points of odontoblast differentiation revealed two significant clusters of odontoblast-specific enhancers,one of which gradually gained enhancer activity while the other lost activity.Motif analysis revealed that transcription factors belonging to Zinc finger,Runx,Forkhead and MADS families governed odontoblast lineage commitment while b-ZIP family uniquely regulated odontoblastic terminal differentiation.Among b-ZIP family,ATF5 was increased during odontoblastic differentiation and overexpression of ATF5 accelerated calcified nodules formation.Dual luciferase assay also confirmed that ATF5 activated an enhancer of Dmp1.Conclusion: Taken together,our present study illustrates that Zinc finger,Runx,Forkhead and MADS families govern odontoblast lineage commitment while b-ZIP family uniquely regulates odontoblastic terminal differentiation.ATF5,belongs to b-ZIP family,promotes Dmp1 gene expression through targeting NFRs in cluster 3 and finally enhances terminal odontoblast differentiation.2.RUNX2 co-operates with EGR1 to regulate osteogenic differentiation through Htra1 enhancersObjective: Runt-related transcription factor 2(Runx2)has been shown to regulate osteoblast differentiation by directly or indirectly regulating numerous osteoblast-related genes.However,our understanding of the transcriptional mechanisms of RUNX2 are mainly restricted to its transactivation,while the mechanism underlying its inhibitory effect during osteoblast differentiation remains largely unknown.We aimed to find the downstream genes that inhibited by RUNX2 during osteogenic differentiation.Methods: we incorporated the anti-RUNX2 chromatin immunoprecipitation(Ch IP)sequencing in MC3T3-E1 cells and RNA-sequencing of parietal bone from Runx2 heterozygous mutant mice,to identify the putative genes negatively regulated by RUNX2.We identified Htr A serine peptidase 1(Htra1)as a target gene and Htra1 enhancers potentially modulated by RUNX2 was confirmed dual luciferase assays.Furthermore,we investigated the motifs in the vicinity of RUNX2-binding sites to identify a potential partner transcriptional factor(TF)and early growth response 1(Egr1)was selected.ReCh IP assays was used to confirm the co-occupancy of RUNX2 and EGR1 in Htra1 enhancers.Function of RUNX2 cooperates with EGR1 was confirmed by Alizarin red staining and ALP staining.Results: RUNX2 and EGR1 co-repressed Htra1 and increased the expression levels of other osteoblast marker genes,such as osterix(Osx),osteocalcin(Ocn),and osteoprotegerin(Opn)at the m RNA and protein level.Moreover,Alizarin red staining combined with alkaline phosphatase(ALP)staining showed decreased calcified nodules and ALP activity in siRUNX2+si EGR1 group compared with siRUNX2 group.Conclusion: Taken together,our findings reveals that reciprocal interaction of RUNX2 and EGR1 promotes Osx,Ocn and Opn expression,and represses Htra1 enhancers to boost osteoblast differentiation in MC3T3-E1.