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Molecular mechanism of neural crest cell differentiation, proliferation and death

神经嵴细胞分化、增殖和死亡的分子机制

基本信息

批准号：
16590240
负责人：
HATANO Masahiko
金额：
$ 2.24万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590240/
关键词：
Ncx Neural crest cell Nczf Target genes Neural cell death Transcriptional repressor DNA binding Hirschsprung related diseases Zinc finger protein DNA結合配列腸管神経節遺伝子欠損マウス転写因子クロマチン免疫沈降ノックアウトマウス

项目摘要

The murine Ncx (Tlx2,Enx,Hox11L1) gene is expressed in neural crest derived tissues and regulates neuronal cell death in enteric neurons. We have used chromatin immunoprecipitation method to screen for target genes for Ncx. This screen led to the identification of novel gene termed Nczf. Nczf contains an N-terminal KRAB box domain and 11 Kruppel C2H2 type zinc finger motifs at C terminus. Promoter region of the Nczf gene contains 5 consensus sequences for Ncx binding motifs. Transient transfection assays of the 5'-flanking region fused to the luciferase reporter gene with various amount of Ncx expression vector showed dose dependent increase of the Nczf promoteractivity. Expression of Nczf mRNA was detected in enteric neurons like that of Ncx. The amount of Nczf mRNA roughly correlated with that of the Ncx in enteric ganglia during embryonic development. Nczf localized in the nucleus and functioned as a transcriptional repressor. The consensus binding sequence of core nucleotides contains (A/T/C)CTTT(A/G)TTNT. In a gel mobility shift assay, the probe containing these sequences bound to the fusion protein. In silico analysis, these consensus sequences were found on regulatory regions of the endothelin receptor B and the microphthalmia-associated transcription factor genes, which are involved in neural crest development. Furthermore, overexpression of Nczf incultured neuroblastoma and fibroblast cell lines caused apoptotic cell death. These results suggest that Nczf functions as a sequence specific transcription repressor to regulate neural crest cell development.

小鼠NCX(Tlx2，ENX，Hox11L1)基因在神经脊来源的组织中表达，并调节肠神经细胞的死亡。我们用染色质免疫沉淀法筛选NCX的靶基因。这一筛选导致了命名为Nczf的新基因的鉴定。Nczf含有一个N端的KRAB盒结构域和C端的11个Kruppel C2H2型锌指基序。Nczf基因启动子区域包含5个Ncx结合基序的共同序列。将荧光素酶报告基因的5‘侧翼区与不同数量的NCX表达载体进行瞬时转染，结果显示NCZF启动子的活性呈剂量依赖性增加。与NCX类似，Nczf基因在肠神经细胞中也有表达。胚胎发育过程中肠神经节中Nczf和Ncx的表达大致呈正相关。Nczf定位于胞核，作为转录抑制因子发挥作用。核心核苷酸的共同结合序列包括(A/T/C)CTTT(A/G)TTNT。在凝胶迁移率改变分析中，包含这些序列的探针与融合蛋白结合。在电子计算机分析中，这些共同的序列被发现在内皮素受体B和小眼球相关转录因子基因的调节区，这两个基因与神经脊的发育有关。此外，在培养的神经母细胞瘤和成纤维细胞系中过表达Nczf可导致细胞凋亡。这些结果表明，Nczf作为一种序列特异性转录抑制因子，调节神经脊细胞的发育。