Screening Of Shox2 Tissue-specific Enhancers And Target Genes Involved In Osteoblast Differentiation During Palate Development In Mice

Cleft palate is the most common craniofacial developmental disorder in humans,which is usually attributed to genetic or environmental factors,affecting patients’speeching,eating and hearing.Cleft palate is classified several types clinically,including cleft lip and palate,cleft palate,complete cleft,incomplete cleft,cleft uvula,submucosal cleft.Mice and human gene homology is up to 99%,the patterns of palate development are similar in human and mice,transgenic mice is the best animal to study the molecular mechanisms of cleft palate.The process of palate development is complex and delicate.Before the fusion of palate,gene differentially expressed in the anterior and posterior palate,subsequently the mesenchymal cells of anterior differentiate into osteoblasts forming hard palate,while the posterior differentiate into muscle forming soft palate.A number of genes specifical expression in the anterior and posterior palate,such as Msx1,Bmp4,Bmp2,Shh,Spry2,Fgf10,Fgf7,Shox2 expressed specifically in the anterior,Meox2,Tbx22,Mn1,Barx1expressed specifically in the posterior.These gene regulate the pattern formation of the hard palate and soft palate.As a member of the homeodomain family,Shox2 is an important tissue-specific transcription factors,expressing in the anterior palate,sinoatrial node and the proximal of limb.The precise regulation of transcription factors in mammals’organ development is becoming a biological research hotspot.The tissue-specific transcription factors bind to the enhancer,regulating the downstream signaling pathway,subsequently directing cell differentiation and determining organ development.We previously found that Shox2binding to limb bud-related enhancer regulate limb bud pattern formation,performing the ChIP-Seq of Shox2^HA-DsRed mice.But Shox2 how to regulate the pattern formation of the palate has not been elucidated.The present study will screen and verify the tissue-specific enhancer binding with Shox2 performing the ChIP-Seq of the palate of Shox2^HA-DsRedA-DsRed mice,then analysis the chromatin open region in Shox2⁺lineages mesenchymal cells during osteogenic differentiation,performing RNA-Seq and ATAC-Seq of Shox2^cre/+mice.Finally,screening and verifying target genes related osteogenic differentiation,which is directly regulated by Shox2.This study will lay the foundation for the prevention and treatment of cleft palate in human.Firstly,to investigate the transcription factor characteristics of Shox2 before palate fusion in mice.We dissect the anterior and posterior palatal tissue of Shox2^HA-DsRed mice to perform the ChIP-Seq of Shox2 and H3K27ac,respectively using anti-HA-antibodies and anti-H3K27ac antibodies.Data analysis show that Shox2-binding site enrich near the transcription start site of anterior palate-specific gene（such as Msx1）30Kbp-100Kbp,revealed Shox2,as a key transcription factors,bind to the anterior tissue-specific enhancers regulating the anterior and posterior palate development.Then,we screen one enhancer from ChIP-Seq results existing near Meis1 35Kbp.To identify the activity of this enhancer,we successfully construct Meis1-enhancer-Hsp68-LacZ plasmid and harvest Meis1-enhancer-Hsp68-LacZ mice by prokaryotic microinjection technology.We collect the embryo of 12.5,by X-Gal stain and eosin stain of paraffin slice,finding that the anterior palate stain with distinct blue,but not in the posterior palate.Result proves that this enhancer is anterior palate tissue-specific active enhancers.Secondly,to explore the regulation mechanism of Shox2 in the osteogenic differentiation of mesenchymal cells after palate fusion,we perform the immunofluorescent staining of GFP and Runx2 in the E14.5,E15.5,E16.5Shox2^Cre/+;Nkx2.5^F/F;R26R^mTmGTmG mice and Shox2^Cre/-;Nkx2.5^F/F;R26R^mTmG mice,result shows that osteoblast in anterior part of palatine process of maxilla decrease significantly,elaborating Shox2 is involved in the osteogenic differentiation of mesenchymal cells in the anterior palate.To further understand the function of Shox2,as a transcription factor,in the osteogenic differentiation of palatal mesenchymal cells,we use fluorescence activated cell sorting（FACS）to collect the GFP-positive palatal mesenchymal cells from the palate tissues of E14.5Shox2^Cre/+;Nkx2.5^F/F;R26R^mTmGand Shox2^Cre/-;Nkx2.5^F/F;R26R^mTmG mice to perform ATAC-Seq.The analysis shows that numbers of chromatin accessible regions in palatal mesenchymal cells lacking Shox2indicated closed.In order to analyze the direct target genes regulated by Shox2,we compare the chromatin accessible regions in palatal mesenchymal cells that Shox2normally expresses,seeking out genes within 50Kbp of the differential region,then combining with the differentially expressed genes of RNA-Seq of palatal mesenchymal cells lacking Shox2 and having Shox2,we screen some gene located at the transcription start site 50kbp near the target genes regulated by Shox2,we preform real-time PCR to identify the expression of five of target genes（Alk3,Alk5,Col2a1,Dlx2,Smoc1）,result is that the expression of these genes are down-regulated,we conducted GO analysis on this five gene revealed they are pattern formation and osteogenic differentiation associated genes,proving that Shox2 being a transcription factor regulate chromatin accessible regions opening or closing to derecte osteogenic differentiation of mesenchymal cells.In sum,Shox2,as a key transcriptional factor,involve in the patterning formation of palate by binding the anterior palate-specific distal active enhancers,by the data of ChIP-Seq,we screen and identify one anterior palate-specific distal active enhancer.Then,comprehensively analyzing the data of mice lacking Shox2 of ATAC-Seq and RNA-Seq,we screen taget gene,directly regulating by Shox2,related to osteogenic differentiation of mesenchymal cells.Our study will provide novel knowledge for understanding the function of Shox2 in the pattern formation of palate and the osteogenic differentiation of hard palate.