HOXD13与先天性马蹄内翻足的相关研究及其作用机制探讨
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摘要
前言
先天性马蹄内翻足(idiopathic congenital clubfoot,CCF)是常见的严重危害儿童健康的先天畸形之一,发病率在1～4.5‰。遗传因素在CCF的发病过程中发挥重要作用,遗传度为65%。但其遗传方式、外显率等均不清楚,易感基因尚未确定。目前研究较多的与CCF发病相关的基因主要集中在与足踝部的骨骼、软骨、肌肉和神经发育相关的基因,如COL9A1,CASP10,WNT7A,HOXD13等。
我室前期应用ETDT方法对84个CCF核心家系HOXD基因簇内的4个多态位点进行分析,提示HOXD13基因可能是先天性马蹄内翻足的易感基因。HOX基因家族是一个高度保守的转录因子家族,该家族在胚胎发育阶段基本体轴和次级体轴的形成中起作用,所有的HOX蛋白都是通过一个60个氨基酸的DNA结合基序来结合特定的DNA序列而发挥其转录调控作用。Hoxd13参与的遗传通路,特别是受其影响的下游靶基因目前还不清楚。有研究表明,Hoxd13可以激活小鼠成纤维细胞中Slim1基因的表达,提示Hoxd13可能调控Slim1的表达。本实验进一步探讨HOXD13,SLIM1与先天性马蹄内翻足的关系以及Hoxd13在大鼠胚胎肢体发育中调控Slim1的机制。
方法
标本:84例CCF患者外周静脉血和15例CCF患者肌肉组织标本由中国医科大学附属第二临床医院小儿外科提供,3例同年龄组的正常人足部肌肉组织由中国医科大学法医学院提供。所有标本使用均经患者知情并同意。成年SD大鼠购自中国医科大学实验动物中心,所有的实验过程都遵照动物保护条例。
1、变性梯度凝胶电泳技术检测HOXD13基因编码区的突变
PCR扩增84例患者HOXD13基因全部2个外显子,应用20%～80%的变性聚丙烯酰胺凝胶电泳筛查2个外显子的突变情况。
2、半定量RT-PCR,免疫组织化学和Western-blot方法研究HOXD13和SLIM1基因在CCF患者肌肉组织中的表达
分别提取CCF患者及正常人肌肉组织的RNA和蛋白质,应用半定量RT-PCR和Western-blot检测HOXD13和SLIM1基因的表达;分别制备CCF患者及正常人肌肉组织的常规石蜡包埋切片,应用免疫组织化学方法检测HOXD13和SLIM1基因的表达。
3、Western-blot和免疫荧光检测孕12.5天的大鼠胎鼠下肢肢芽中Hoxd13和Slim1的表达
应用Western-blot技术检测孕12.5天的大鼠胎鼠下肢肢芽中Hoxd13和Slim1的表达,应用免疫荧光检测12.5天的大鼠胎鼠下肢肢芽中Hoxd13和Slim1基因表达的组织定位。
4、在大鼠L6GNR4细胞系中外源表达HOXD13后观察Slim1基因表达
构建HOXD13的真核细胞表达载体pcDNA-HOXD13,转染大鼠L6GNR4细胞后观察Slim1基因表达水平的改变。
5、应用P-MATCH软件预测了大鼠Slim1基因5'上游序列转录因子的结合位点
获取Slim1基因5'上游1500bp序列信息(http://www.ensembl.org),应用P-MATCH软件预测其转录因子结合位点。
6、荧光素酶报告基因系统
PCR扩增Slim1基因启动子序列,构建荧光素酶报告载体pGL3-Slim1。pGL3-Slim1和pcDNA-HOXD13转染大鼠L6GNR4细胞,观察HOXD13对Slim1的转录调控作用。
7、凝胶阻滞实验
孕12.5天胎鼠下肢肢芽组织核蛋白和3'生物素标记的探针(含有位点2)在凝胶阻滞缓冲液中室温结合1小时,10%的聚丙烯酰胺凝胶电泳,转膜、紫外交联后,应用化学发光检测系统检测结果。
8、染色质免疫沉淀实验
提取L6GNR4细胞和孕12.5天胎鼠下肢肢芽和脑组织的染色质,甲醛交联、酶切后应用Hoxd13抗体进行沉淀,沉淀下来的染色质通过PCR扩增检测结果。
结果
1、在84例CCF患者外周静脉血中未发现HOXD13基因的编码区突变。
2、CCF患者肌肉组织中存在HOXD13(5/15,33.3%)和SLIM1(7/15,46.7%)基因的表达下调。
3、在孕12.5天的胎鼠下肢肢芽中Hoxd13和Slim1共表达在趾间组织。
4、外源表达HOXD13可明显激活大鼠成肌细胞L6GNR4中Slim1基因的表达。
5、在大鼠Slim1基因启动子区域存在2个Hoxd13的可能结合位点,分别命名为Hoxd13结合位点1(-1164～-1158)和Hoxd13结合位点2(-1140～-1134)。
6、Hoxd13通过和Hoxd13结合位点2结合发挥转录调控作用。
L6GNR4细胞瞬时转染了pGL3-Slim1(-1185)荧光素酶报告基因表达载体和HOXD13表达载体后,HOXD13可以明显提高荧光素酶活性。我们又构建了缺失型的pGL3-Slim1(-1154)荧光素酶报告基因的表达载体,转染后的荧光素酶活性与转染pGL3-Slim1(-1185)比较,HOXD13同样可以明显提高荧光素酶活性。我们改变了Hoxd13结合位点2的核心序列,构建了pGL3-Slim1(-1154m)表达载体,转染L6GNR4细胞后发现当Hoxd13结合位点2突变后HOXD13对荧光素酶活性无影响。综合以上结果,说明Hoxd13结合位点2可能是HOXD13的结合位置。
7、在体外Hoxd13蛋白可以和Slim1基因启动子区位点2直接结合。
EMSA结果表明当Hoxd13蛋白质存在时出现阻滞的DNA-蛋白质复合体,当加入Hoxd13抗体时出现超阻滞条带。证明在体外Hoxd13和预测的Hoxd13结合位点2可以直接结合。
8、在孕12.5天大鼠胚胎肢体发育过程中Hoxd13可以和Slim1基因启动子区Hoxd13结合位点2直接结合。
我们应用染色质免疫沉淀技术验证在体内Hoxd13和Slim1基因上游序列的结合作用。沉淀的L6GNR4细胞染色质中有Hoxd13结合位点2的扩增,无对照位点的扩增。为进一步验证在胚胎肢体发育过程中Hoxd13是否和Slim1启动子结合,我们以孕12.5天的大鼠下肢肢芽组织为研究对象进行了染色质免疫沉淀实验,沉淀的肢芽染色质有Hoxd13结合位点2的扩增,无对照位点的扩增,而在对照脑组织中没有Hoxd13结合位点2的扩增。实验结果表明在大鼠胚胎肢体发育过程中Hoxd13蛋白可以和Slim1启动子区的Hoxd13结合位点2结合发挥其转录调节作用。
结论
1、HOXD13基因的编码区突变可能不是CCF发病的原因。
2、HOXD13基因和SLIM1基因的表达下调可能与CCF畸形的发生有关。
3、在大鼠胚胎肢体发育过程中Hoxd13直接结合Hoxd13结合位点2调控Slim1基因的表达。
Introduction
Idiopathic 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.
Methods
Samples: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.
Results
1.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.
Conclusion
1.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.
先天性马蹄内翻足(idiopathic congenital clubfoot,CCF)是常见的严重危害儿童健康的先天畸形之一,发病率在1～4.5‰。遗传因素在CCF的发病过程中发挥重要作用,遗传度为65%。但其遗传方式、外显率等均不清楚,易感基因尚未确定。目前研究较多的与CCF发病相关的基因主要集中在与足踝部的骨骼、软骨、肌肉和神经发育相关的基因,如COL9A1,CASP10,WNT7A,HOXD13等。
我室前期应用ETDT方法对84个CCF核心家系HOXD基因簇内的4个多态位点进行分析,提示HOXD13基因可能是先天性马蹄内翻足的易感基因。HOX基因家族是一个高度保守的转录因子家族,该家族在胚胎发育阶段基本体轴和次级体轴的形成中起作用,所有的HOX蛋白都是通过一个60个氨基酸的DNA结合基序来结合特定的DNA序列而发挥其转录调控作用。Hoxd13参与的遗传通路,特别是受其影响的下游靶基因目前还不清楚。有研究表明,Hoxd13可以激活小鼠成纤维细胞中Slim1基因的表达,提示Hoxd13可能调控Slim1的表达。本实验进一步探讨HOXD13,SLIM1与先天性马蹄内翻足的关系以及Hoxd13在大鼠胚胎肢体发育中调控Slim1的机制。
方法
标本:84例CCF患者外周静脉血和15例CCF患者肌肉组织标本由中国医科大学附属第二临床医院小儿外科提供,3例同年龄组的正常人足部肌肉组织由中国医科大学法医学院提供。所有标本使用均经患者知情并同意。成年SD大鼠购自中国医科大学实验动物中心,所有的实验过程都遵照动物保护条例。
1、变性梯度凝胶电泳技术检测HOXD13基因编码区的突变
PCR扩增84例患者HOXD13基因全部2个外显子,应用20%～80%的变性聚丙烯酰胺凝胶电泳筛查2个外显子的突变情况。
2、半定量RT-PCR,免疫组织化学和Western-blot方法研究HOXD13和SLIM1基因在CCF患者肌肉组织中的表达
分别提取CCF患者及正常人肌肉组织的RNA和蛋白质,应用半定量RT-PCR和Western-blot检测HOXD13和SLIM1基因的表达;分别制备CCF患者及正常人肌肉组织的常规石蜡包埋切片,应用免疫组织化学方法检测HOXD13和SLIM1基因的表达。
3、Western-blot和免疫荧光检测孕12.5天的大鼠胎鼠下肢肢芽中Hoxd13和Slim1的表达
应用Western-blot技术检测孕12.5天的大鼠胎鼠下肢肢芽中Hoxd13和Slim1的表达,应用免疫荧光检测12.5天的大鼠胎鼠下肢肢芽中Hoxd13和Slim1基因表达的组织定位。
4、在大鼠L6GNR4细胞系中外源表达HOXD13后观察Slim1基因表达
构建HOXD13的真核细胞表达载体pcDNA-HOXD13,转染大鼠L6GNR4细胞后观察Slim1基因表达水平的改变。
5、应用P-MATCH软件预测了大鼠Slim1基因5'上游序列转录因子的结合位点
获取Slim1基因5'上游1500bp序列信息(http://www.ensembl.org),应用P-MATCH软件预测其转录因子结合位点。
6、荧光素酶报告基因系统
PCR扩增Slim1基因启动子序列,构建荧光素酶报告载体pGL3-Slim1。pGL3-Slim1和pcDNA-HOXD13转染大鼠L6GNR4细胞,观察HOXD13对Slim1的转录调控作用。
7、凝胶阻滞实验
孕12.5天胎鼠下肢肢芽组织核蛋白和3'生物素标记的探针(含有位点2)在凝胶阻滞缓冲液中室温结合1小时,10%的聚丙烯酰胺凝胶电泳,转膜、紫外交联后,应用化学发光检测系统检测结果。
8、染色质免疫沉淀实验
提取L6GNR4细胞和孕12.5天胎鼠下肢肢芽和脑组织的染色质,甲醛交联、酶切后应用Hoxd13抗体进行沉淀,沉淀下来的染色质通过PCR扩增检测结果。
结果
1、在84例CCF患者外周静脉血中未发现HOXD13基因的编码区突变。
2、CCF患者肌肉组织中存在HOXD13(5/15,33.3%)和SLIM1(7/15,46.7%)基因的表达下调。
3、在孕12.5天的胎鼠下肢肢芽中Hoxd13和Slim1共表达在趾间组织。
4、外源表达HOXD13可明显激活大鼠成肌细胞L6GNR4中Slim1基因的表达。
5、在大鼠Slim1基因启动子区域存在2个Hoxd13的可能结合位点,分别命名为Hoxd13结合位点1(-1164～-1158)和Hoxd13结合位点2(-1140～-1134)。
6、Hoxd13通过和Hoxd13结合位点2结合发挥转录调控作用。
L6GNR4细胞瞬时转染了pGL3-Slim1(-1185)荧光素酶报告基因表达载体和HOXD13表达载体后,HOXD13可以明显提高荧光素酶活性。我们又构建了缺失型的pGL3-Slim1(-1154)荧光素酶报告基因的表达载体,转染后的荧光素酶活性与转染pGL3-Slim1(-1185)比较,HOXD13同样可以明显提高荧光素酶活性。我们改变了Hoxd13结合位点2的核心序列,构建了pGL3-Slim1(-1154m)表达载体,转染L6GNR4细胞后发现当Hoxd13结合位点2突变后HOXD13对荧光素酶活性无影响。综合以上结果,说明Hoxd13结合位点2可能是HOXD13的结合位置。
7、在体外Hoxd13蛋白可以和Slim1基因启动子区位点2直接结合。
EMSA结果表明当Hoxd13蛋白质存在时出现阻滞的DNA-蛋白质复合体,当加入Hoxd13抗体时出现超阻滞条带。证明在体外Hoxd13和预测的Hoxd13结合位点2可以直接结合。
8、在孕12.5天大鼠胚胎肢体发育过程中Hoxd13可以和Slim1基因启动子区Hoxd13结合位点2直接结合。
我们应用染色质免疫沉淀技术验证在体内Hoxd13和Slim1基因上游序列的结合作用。沉淀的L6GNR4细胞染色质中有Hoxd13结合位点2的扩增,无对照位点的扩增。为进一步验证在胚胎肢体发育过程中Hoxd13是否和Slim1启动子结合,我们以孕12.5天的大鼠下肢肢芽组织为研究对象进行了染色质免疫沉淀实验,沉淀的肢芽染色质有Hoxd13结合位点2的扩增,无对照位点的扩增,而在对照脑组织中没有Hoxd13结合位点2的扩增。实验结果表明在大鼠胚胎肢体发育过程中Hoxd13蛋白可以和Slim1启动子区的Hoxd13结合位点2结合发挥其转录调节作用。
结论
1、HOXD13基因的编码区突变可能不是CCF发病的原因。
2、HOXD13基因和SLIM1基因的表达下调可能与CCF畸形的发生有关。
3、在大鼠胚胎肢体发育过程中Hoxd13直接结合Hoxd13结合位点2调控Slim1基因的表达。
Introduction
Idiopathic 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.
Methods
Samples: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.
Results
1.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.
Conclusion
1.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.
引文
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