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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) 基因在神经嵴衍生组织中表达，并调节肠神经元中的神经细胞死亡。我们采用染色质免疫沉淀法筛选Ncx的靶基因。该筛选导致了名为 Nczf 的新基因的鉴定。 Nczf 包含 N 端 KRAB 盒结构域和 C 端 11 个 Kruppel C2H2 型锌指基序。 Nczf 基因的启动子区域包含 5 个 Ncx 结合基序的共有序列。用不同量的 Ncx 表达载体对与荧光素酶报告基因融合的 5'-侧翼区域进行瞬时转染测定，结果显示 Nczf 启动子活性呈剂量依赖性增加。在肠神经元中检测到 Nczf mRNA 的表达，如 Ncx 的表达。 Nczf mRNA 的量与胚胎发育期间肠神经节中 Ncx 的量大致相关。 Nczf 定位于细胞核并充当转录抑制因子。核心核苷酸的共有结合序列包含(A/T/C)CTTT(A/G)TTNT。在凝胶迁移率变动测定中，含有这些序列的探针与融合蛋白结合。在计算机分析中，这些共有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域，这些基因参与神经嵴发育。此外，在培养的神经母细胞瘤和成纤维细胞系中过度表达 Nczf 会导致细胞凋亡。这些结果表明 Nczf 作为序列特异性转录抑制因子来调节神经嵴细胞的发育。