三个含SCAN结构域的锌指蛋白基因ZNF191、ZNF396和ZNF397研究

【作者】 吴益民；

【导师】 赵寿元；

【作者基本信息】 复旦大学 ， 遗传学， 2002， 博士

【摘要】 一些编码SCAN结构域的锌指基因在人类基因组中常成簇分布，例如17p12-13的ZNF18, ZNF29, ZNF62和zfp3四个基因。ZNF191是一个早先克隆的含SCAN结构域的锌指基因，并定位于18q12。在此，我们识别了人类两个新型含SCAN结构域基因ZNF396和ZNF397，它们与ZNF191和 ZNF271基因一起成簇地分布于18q12的32850-33000k区域。我们鉴定了SCAN-(C2H2)X基因会在其羧基端截除锌指结构域。这种独特的变异剪接形式使ZNF191、ZNF396 和ZNF397基因分别产生两种不同的蛋白质。编码有锌指结构域的三种异构型分别命名为ZNF191-fu, ZNF396-fu和ZNF397-fu (full zinc finger)，同时，不编码锌指结构域的三种异构型被命名ZNF191-nf, ZNF396-nf and ZNF397-nf (no zinc finger)。含成年人16种组织的northern印迹膜分别用同位素标记的ZNF191-nf, ZNF396-nf或ZNF397-nf cDNA进行杂交，发现ZNF191的 2.0-kb, 3.7-kb和7.5-kb三种剪接本在睾丸组织中同时表达，ZNF396的1.5-kb, 2.5-kb和3.9-kb三种剪接本在肝脏组织中同时高表达，ZNF397的1.7-kb, 2.5-kb, 7.0-kb和9.0-kb四种剪接本在睾丸组织中同时高表达。它们的组织表达谱差异很大。ZNF191-fu在氨基端第46-141位为SCAN结构域，而羧基端有四个串联重复的锌指结构。编码335个氨基酸的ZNF396-fu，在氨基端第46-141位为SCAN结构域，而羧基端在aa251-273, aa279-301和aa307-329位为三个串联重复的锌指结构。编码534个氨基酸的ZNF397-fu，在氨基端第44-139位为SCAN结构域，而羧基端在aa285-307, aa313-335, aa341-363, aa369-391, aa397-419, aa425-447, aa453-475, aa481-503和aa509-531位为九个串联重复的锌指结构。ZNF191-nf，ZNF396-nf和ZNF397-nf分别编码193, 210和198个氨基酸，仅含有SCAN结构域。ZNF191-fu, ZNF191-nf, ZNF396-fu, ZNF396-nf, ZNF397-fu和ZNF397-nf六种异构型蛋白质分别可自身形成二聚化，ZNF191-fu可与ZNF191-nf异构型蛋白质异源性结合，ZNF396-fu可与ZNF396-nf异构型蛋白质异源性结合，ZNF397-fu可与ZNF397-nf异构型蛋白质异源性结合。进而，ZNF397-fu能与含SCAN结构域的ZNF397-fu第aa3-250位片段结合，ZNF397-fu不能与无SCAN结构域的ZNF397-fu第aa224-534位片段结合。ZNF191-nf, ZNF396-nf和 ZNF397-nf异构型蛋白质在瞬时表达的细胞中，定位为整个细胞之中。但是ZNF191-fu, ZNF396-fu和ZNF397-fu异构型蛋白质特异地分布在细胞核之中，与DAPI染色显示的细胞核一致。若ZNF191-fu和ZNF191-nf两质粒同时转染细胞，ZNF191-fu仍是特异地分布于细胞核中，而ZNF191-nf不再散布于整个细胞，在细胞核中分布大大增多。通过缺失实验，含SCAN结构域的ZNF397-fu第aa3-250<WP=5>位肽段弥散分布于整个细胞，与ZNF397-nf的定位相似；无SCAN结构域的ZNF397-fu第aa224-534位片段定位在细胞核，与ZNF397-fu的定位相似。因此推导ZNF397-fu 的核定位信号序列在其锌指结构区域，即氨基酸第251至534位范围。这三个基因的六种异构型蛋白质，除ZNF397-fu之外均能抑制报告基因的转录。然而，ZNF191-fu和ZNF397-nf两种异构型的转录抑制作用更强。若被截除九个锌指重复结构，ZNF397-fu成为一个转录激活因子。通过系列缺失实验，表明ZNF191-fu的各个亚结构域对于其抑制转录所贡献的作用不尽相同。最后，将ZNF191-fu的中间第139-247位片段与GST表位融合，所表达的融合蛋白免疫兔子，该抗体经亲和层析纯化后可灵敏地检测转染表达的和某些组织内源的ZNF191蛋白质分子。更多还原

【Abstract】 Many zinc finger genes encoding the SCAN domains are clustered in human genome, e.g. 17p12-13 (ZNF18, ZNF29, ZNF62 and zfp3). One of the SCAN domain genes, ZNF191 has been cloned and mapped to 18q12 . We have identified two novel human SCAN domain genes ZNF396 and ZNF397, which are clustered in the chromosome 18q12: 32850-33000k regions with ZNF191 and ZNF271 genes. It is firstly characterized for the SCAN-(C2H2)X genes to truncate the zinc finger domains at the carboxyl terminus. The unique alternative splicing makes ZNF191, ZNF396 and ZNF397 genes encode two distinct proteins respectively. Three isoforms encoding zinc finger regions are designated as ZNF191-fu, ZNF396-fu and ZNF397-fu (full zinc finger) while the other isoforms truncating zinc finger regions are designated as ZNF191-nf, ZNF396-nf and ZNF397-nf (no zinc finger). Tissue Northern blots (MTN I and MTN II) from 16 adult human tissues are respectively hybridized with isotope-labeled ZNF191-nf, ZNF396-nf or ZNF397-nf cDNA. Three isoforms of ZNF191 transcript, 2.0-kb, 3.7-kb and 7.5-kb, are expressed in testis. Three isoforms of ZNF396 transcript, 1.5-kb, 2.5-kb and 3.9-kb, are highly expressed in liver. Four isoforms of ZNF397 transcript, 1.7-kb, 2.5-kb, 7.0-kb and 9.0-kb, are expressed in a variety of tissues, but expression levels vary dramatically. ZNF191-fu consists of a SCAN domain at N-terminus and four tandem C2H2 zinc finger repeats at C-terminus. ZNF396-fu encoding 335 amino acids consists of a SCAN domain (aa46-141) at N-terminus and three consecutive C2H2 zinc finger repeats at C-terminus (aa251-273, aa279-301, aa307-329). ZNF397-fu encoding 534 amino acids consists of a SCAN domain (aa44-139) at N-terminus and nine consecutive C2H2 zinc finger repeats at C-terminus (aa285-307, aa313-335, aa341-363, aa369-391, aa397-419, aa425-447, aa453-475, aa481-503, aa509-531). ZNF191-nf, ZNF396-nf and ZNF397-nf encode 193, 210 and 198 amino acids with a SCAN domain only respectively. ZNF191-fu, ZNF191-nf, ZNF396-fu, ZNF396-nf, ZNF397-fu or ZNF397-nf can homo-associate, ZNF191-fu can hetero-associates with ZNF191-nf，so does ZNF396-fu with ZNF396-nf, or ZNF397-fu with ZNF397-nf. Moreover, ZNF397-fu can associate with SCAN-domain-containing polypeptide of<WP=7>ZNF397-fu (aa3-250) but not with non-SCAN-domain polypeptide of ZNF397-fu (aa224-534). ZNF191-nf, ZNF396-nf and ZNF397-nf polypeptides diffuse in the whole cell, but ZNF191-fu, ZNF396-fu and ZNF397-fu polypeptides target specifically to the nuclei, consistent with the DAPI-staining nuclei. If ZNF191-fu and ZNF191-nf are co-transfected into the cells, ZNF191-nf tends to localize to the nuclei while ZNF191-fu still targets to the nuclei. In the deletion experiment, the SCAN-domain-containing polypeptide of ZNF397-fu (aa3-250) has the same sub-cellular localization pattern in cells as that of ZNF397-nf, and non-SCAN-domain polypeptide of ZNF397 (aa224-534) targets to the nuclei just like that of ZNF397-fu. It is inferred that the nuclear localization signal of ZNF397-fu might be within zinc finger regions (aa251-534). All six isoforms except for ZNF397-fu are shown to repress the transcription of reporter gene, but ZNF191-fu and ZNF397-nf have the stronger repression activity. Without its nine zinc finger repeats, ZNF397-fu is a transcriptional activation factor. Through the series deletion, the different sub-domains of ZNF191-fu contribute to the distinct effects on the transcription repression. At last, the central fragment of ZNF191-fu (aa139-247) fused with the GST tag is used to immunize rabbit, and the purified polyclonal antibody can detect both transfected and endogeneous ZNF191 proteins.更多还原