The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191(+/-) mice are normal and fertile. Homozygous Zfp191(-/-) embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191(-/-) and Zfp191(+/-) embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191(-/-) cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191(+/-) intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation.     

Post-genomic era and gene discovery for psychiatric diseases: there is a new art of the trade? The example of the HUMTH01 microsatellite in the Tyrosine Hydroxylase gene

The microsatellite HUMTH01, located in the first intron of the Tyrosine Hydroxylase (TH) gene (encoding the rate-limiting enzyme in the synthesis of catecholamines), is characterized by a TCAT repeated motif and has been used in genetic studies of neuropsychiatric and other complex diseases, in which catecholaminergic neurotransmission is implicated. After reporting a positive association between HUMTH01 and bipolar disorder as well as schizophrenia, the authors established that HUMTH01 alleles display the features of regulatory elements. Thereafter, they cloned two proteins (ZNF191 and HBP1), specifically binding to HUMTH01, and demonstrated that allelic variations of HUMTH01 have a quantitative silencing effect on TH gene expression in vitro, and correlate with quantitative and qualitative changes in the binding by ZNF191. The authors aim to characterize the transduction pathway impinging on the HUMTH01 microsatellite and establish its relevance for TH gene regulation in vivo. Since the TCAT repeated sequence is widespread throughout the genome, their approach may lead to the dissection of the mechanisms underlying the quantitative expression of several genes implicated in complex genetic traits, both normal and pathological. Thus, these investigations on the possible contribution and potential role of the HUMTH01 microsatellite in neuro-pathological conditions may represent an example of the different approaches needed to validate genetic targets in the "post-genomic era."     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.     

Differential expression of the zinc finger gene Zfp105 during spermatogenesis

We report the isolation of Zfp105, the mouse homolog of the human ZNF35 zinc finger gene. Zfp105 and ZNF35 are highly conserved at the protein and nucleotide level, and Zfp105 maps to a region of mouse Chromosome (Chr) 9 that is homologous to the human region containing ZNF35. Zpf105 is highly expressed in the testis, especially in pachytene spermatocytes and round spermatids. The possible role of this gene product in maintaining an ordered germ cell differentiation process is discussed. Received: 11 February 1998 / Accepted: 1 May 1998     

人类锌指结构新基因ZNF18的克隆和表达谱分析

在很多转录因子中发现的锌指结构,被认为在人类心脏的发育和相关疾病的发生过程中发挥重要的作用。本文报道了克隆和表达分析人类新的锌指蛋白基因ZNF18。该基因cDNA长2 767 bp,编码一个有549个氨基酸的蛋白,这一蛋白含有一个SCAN结构域,一个KRAB结构域和5个连续的C2H2型锌指结构域。ZNF18蛋白与小鼠Zfp535有77％的同源性。ZNF18基因定位于人染色体17p12~p13,包含9个外显子和8个内含子。以ZNF18全长编码区为探针进行Northern杂交,结果显示ZNF18在成体小鼠各组织中广泛表达,但在心脏中低丰度表达。整体原位杂交结果显示,ZNF18基因在小鼠胚胎的表达有很高的动态性。ZNF18主要在E7.5小鼠胚胎的胚外组织表达,E8.5出现了胚胎躯干前端表达。ZNF18从E9.0开始在胚胎的心脏和尾部表达,尤其在E10.5胚胎的心脏高丰度表达。这提示ZNF18基因与心脏发育过程可能有密切的关系。     

Chromosomal localization of two human zinc finger protein genes, ZNF24 (KOX17) and ZNF29 (KOX26), to 18q12 and 17p13-p12, respectively

Two members of the zinc finger Krüppel family, ZNF24 (KOX17) and ZNF29 (KOX26), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization to human chromosomes 18q12 and 17p13-p12, respectively. The mapping of ZNF29 together with the previously reported localization of ZFP3 suggests that a zinc finger gene complex is located on human chromosome 17p. ZNF29 maps centromeric to the human p53 tumor antigen gene (TP53). In the analogous murine position, the two mouse zinc finger genes Zfp2 and Zfp3 have recently been assigned to the distal region of mouse chromosome 11, the murine homolog of human chromosome 17. Both human zinc finger genes ZNF24 and ZNF29 are in chromosomal regions that have been noted to be deleted in neoplasms of the lung and of the central nervous system at chromosome 17p and in colorectal neoplasia at chromosomes 17p and 18q.     

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.     

一个与小鼠锌指蛋白基因ZF-12相关的假基因的克隆和鉴定

小鼠类Krüppel锌指蛋白基因ZF-12为人的类Krüppel锌指蛋白基因ZNF191的同源基因,它们都编码368个氨基酸残基,N端存在SCAN结构域,C端有4个连续的锌指模体,最近的研究表明ZNF191是肝癌发生的相关基因。我们以人锌指蛋白基因ZNF191的cDNA为探针,筛选小鼠λ噬菌体基因组文库,意外地获得了1个与小鼠锌指蛋白基因ZF-12相类似的基因,多种组织的RT-PCR和启动子序列分析,暗示该基因不表达,且该基因无内含子,与ZF-12高度相似,存在突变,暗示其为与ZF-12相关的假基因序列,经查新证实它为新的序列后,以ZF12p(ZF-12 pseudogene)命名在GenBank登录(AY040222)。查寻GenBank的人类基因组库以及Southern结果显示人类基因组中无ZNF191假基因序列。ZF12p与ZF-12高度相似,暗示ZF12p在进化过程中产生的时间较晚,这对研究锌指蛋白基因ZF-12的突变与进化具有重要的意义。