Research On The Relationship Between HOXD13 And Idiopathic Congenital Clubfoot And Its Mechanism In Pathogenesis

IntroductionIdiopathic congenital clubfoot(CCF,MIM119800)is a type of congenital limb deformity with an estimated incidence of 1～4.5‰live births.The mechanism by which CCF develops remains unclear even though the mechanical,neurological,muscular, bony,connective tissue,and vascular mechanisms have been proposed.Whilst both genetic and environmental factors are implicated,no specific genes have been identified.At present,investigations on human CCF mainly focus on the environmental factors at early stage of pregnancy and many syndromes with clubfoot malformations. Candidate genes and regions for CCF,such as COL9A1,CASP10,WNT7A,HOXD13 have been identified,respectively.However,little is known about the pathogenesis of human CCF.Our previous studies using the transmission disequilibrium test showed that the HOXD13 gene in the 5′HOXD clusters at chromosome 2q31 is associated with CCF. The HOX genes encode a highly conserved family of transcrption factors which play a fundamental role in embryonic morphogenesis.The most 5'members of the HOXD clusters(HOXD9～HOXD13)are particularly important in vertebrate limb development. It is known that Hoxd13 regulates many key cellular events such as adhesion,apoptosis, proliferation and migration during limb morphogenesis.The genetic pathways through which it functions,however,are poorly characterized.In particular,the identity of the downstream effector genes of these pathways remains elusive.Williams et al.have found that in mouse embryonic fibroblasts Slim1 is strongly activated by Hoxd13, which indicated that Hoxd13 might regulate the expression of Slim1.In this study we researched the relationship between HOXD13 and CCF and its mechanism in pathogenesis.MethodsSamples:Veinous blood of 84 CCF patients and 15 CCF muscle tissues were obtained from Department of Pediatric Orthopedic Surgery,Second Affiliated Hospital, China Medical University.Adult SD rats were obtained from the experimental animal center of our university;E12.5 day rat embryos showing high Hoxd13 gene expression level were dissected from pregnant rats.All procedures were carried out in accordance with an approved animal handling protocol.1.Mutation in the coding region of HOXD13 in 84 CCF patients was detected by denaturing gradinent electrophoresis.The total 2 exons of HOXD13 gene were PCR from 84 CCF patients,20%～80% DGGE was used to detect the mutation in these two exons.2.The mRNA and protein levels of HOXD13 and SLIM1 were evaluated by RT-PCR,immunohistochemistry and Western-blot, respectively.The protein and RNA were prepared from CCF patients and normal control, RT-PCR,Western-blot and immunohistochemistry were used to detect the expression of HOXD13 and SLIM1 gene.3.Western-blot and immunofluorescence were utilized to detect Hoxd13 and Slim1 expression at E12.5d rat embryo.Western-blot was used to detect the expression of Hoxd13 and Slim1 in the developing limbs in 12.5E rat embryo,Immunofluorescence was performed to analyze the tissue localization of Hoxd13 and Slim1 in the developing limbs in 12.5E rat embryo. 4.Analysis Slim1 expression in L6GNR4 cell after transfected HOXD13 expression vector.We constructed HOXD13 expression vector,pcDNA-HOXD13,and transfected the expression vector in L6GNR4 cell.After 48h,the expression of Sliml level was detected.5.P-Match software was used to analyze the sequence upstream of the transcription start site of the Slim1 gene.The 1500bp sequence upstream of Slim1 gene was obtained from http://www. ensembl.org,we used P-Match software to predict the binding sites of transcriptional factors on the sequence.6.Luciferase report vector assay.The promoter sequence in Slim1 were obtained by polymerase chain reaction,we constructed pGL3-Slim1 vectors.L6GNR4 cell were transfected pGL3-Slim1 and/or pcDNA-HOXD13 vectors.After 48h,we analyzed HOXD13 regulates the transcription of Slim1.7.Electrophoretic mobility shift assay.Nucleoprotein was extracted from E12.5 rat embryonic limbs and incubated with the Slim1 upstream region-containingbinding site 2 labeled using a Biotin 3'End DNA ing Kit for 60 min at room temperature in a gel shift buffer.Reactions were examined for nucleoprotein binding by electrophoretic mobility shift assays(EMSA)on a 10% nondenaturing polyacrylamide,transfered to the memberane,cross-linked.DNA binding bands were detected using a chemiluminescence system.8.Chromatin immunoprecipitation assay.The chromatin was extracted from L6GNR4 cells,limb buds,and brain tissue dissected from E12.5 wild-type embryos,sheared with an Enzymatic Shearing Kit to obtain 500-1000bp fragments.Hoxd13 antibody was used at the immunoprecipitation step.Eluted DNA from the sample and control were assessed for the presence of Slim1 DNA region by PCR.Results1.No mutation was found in the coding region of HOXD13 in 84 samples from patients with CCF.2.Both HOXD13(5/15,33.3%)and SLIM1(7/15,46.6%)were down regulated in CCF muscle tissue.3.Both Hoxd13 and Sliml expressed within interdigital tissues at E12.5 rat embryo.4.The exogenous expression of HOXD13 up-regulated Slim1 transcription in L6GNR4 cells.5.The 5'region of the rat Slim1 gene contained two potential binding sites for the Hoxd13 protein,designated Hoxd13 binding site 1(-1164～-1158)and Hoxd13 binding site 2(-1140～-1134).6.HOXD13 activated transcription via the site 2 in Slim1 promoter region.L6GNR4 cells were transiently cotransfected with pGL3-Slim1(-1185)and increasing amounts of constructs expressing HOXD13.HOXD13 significantly increased the basal reporter activity.We then generated a deletion construct pGL3-Slim1(-1154)containing a fragment from-1154 to+15 bp of the Slim1 promoter, which included site 2.HOXD13 could efficiently activate the pGL3-Slim1(-1154) reporter basal activity to levels similar to those obtained with the pGL3-Slim1(-1185) reporter.Finally,we mutated the sequence of site 2 within the context of the pGL3-Slim1(-1154)reporter(pGL3-Slim1(-1154M)),HOXD13 had virtually no effect on the pGL3-Slim1(-1154M)reporter basal activity when site 2 was mutated, indicating that site 2 might be the binding site of HOXD 13.7.Hoxd13 directly binds to site 2 in Slim1 promoter region.Strong DNA binding was observed in the presence of the Hoxd13 protein.A competition experiment and supershift existence in the presence of the Hoxd13 antibody demonstrated the specificity of such binding.The result indicated Hoxd13 is bound to site 2 in vitro.8.Hoxd13 binds with site 2 in Slim1 promoter region in the developing limb in 12.5E rat.To verify the binding in vivo of Hoxd13 to binding site 2 within the Slim1 promoter,we used the chromatin formaldehyde cross-linking and immunoprecipitation (ChIP)technique.The immunoprecipitated L6GNR4 cell chromatin showed a substantial enrichment only of the sequence containing site 2,indicating that Hoxd13 efficiently bound only to site 2 in vivo.No enrichment was detected for the control site. In order to determine whether endogenous Hoxd13 also binds to site 2 of the Slim1 promoter in vivo in the developing limb,chromatin was prepared from E12.5 rat hindlimbs and immunoprecipitated using a Hoxd13 antibody.The immunoprecipitated limb chromatin showed a significant enrichment of the fragment containing site 2.No enrichment of the site 2-containing sequence was observed in the control brain chromatin;additionally,there was no enrichment of the control sequence.Conclusion1.HOXD13 gene mutation in coding region was not involved in outbreak in idiopathic congenital clubfoot.2.Changes of HOXD13 and SLIM1 gene expression related to the development of clubfoot malformation.3.Hoxd13 directly controls the expression of Slim1 through Hoxd13 binding dite 2 in the developing limb in rat embryo.