论文题目：造血组织表达的krüppel样锌指基因结构和功能研究

众多证据表明，锌指基因家族是最大的人类基因家族之一，其蛋白产物主要通过与DNA相互作用而起转录因子作用，调节靶基因的表达以适应生物体发育、分化、成熟过程的需要。锌指蛋白不仅与造血密切相关，影响着红细胞系、巨核细胞系和淋巴细胞系（如GATA家族和EKLF）、粒细胞系（MZF1）、单核细胞系（Egr-1）等发育、分化过程，而且还与一些造血系统恶性肿瘤有关，如PLZF在急性早幼粒细胞性白血病发病中起重要作用。因此，本研究试图利用Cys₂/His₂型（krüppel样）锌指基因顺序上的保守特征，用一系列分子生物学、细胞生物学方法获得造血组织（骨髓）和急性早幼粒细胞性白血病细胞株NB4表达的新的锌指基因，并初步揭示其功能。

首先根据krüppel样锌指基因特有的保守顺序设计一对引物，通过逆转录-多聚酶链反应（RT-PCR）方法扩增骨髓和NB4细胞株表达的锌指基因片段，经亚克隆和荧光自动测序后查询GenBank，判断是新的锌指基因片段后，即进行克隆全长cDNA或完整开放阅读框架（ORF）研究。克隆全长cDNA工作主要通过筛选骨髓、脑、NB4细胞cDNA文库方法，若仍不能获得全长cDNA，则利用EST（表达顺序标签）数据库进行电脑拼接及RACE（快速cDNA末端扩增）等方法。目前，本研究已获得6个krüppel样锌指基因RSG-A(ZNF191)、BMZF-1(ZNF253), BMZF-2(ZNF255), BMZF-3(ZNF256), BMZF-4(ZNF257),和BMZF-5(ZNF254），经查询GenBank，除1个基因RSG-A已知外，其余5个均为新的锌指基因。

6个锌指基因中，RSG-A全长1.5kb，3’端含poly（A）尾，推导出的氨基酸顺序全长368个氨基酸，C端有4个锌指结构，N端含SCAN盒（81个氨基酸）， 该功能域富含酸性氨基酸，氨基酸顺序与zf 38（鼠）、 zf165（人）等同源 。BMZF-1基因全长1.2kb，3’端虽未见poly（A）尾，但含有完整ORF，编码275个氨基酸，体外翻译为30KD，C端有3个锌指结构，N端含KRAB（kruppel-associated box）顺序，与zn90、zn93、zn43、zn85、zn91、 z117等许多人类基因同源。BMZF-2是经过筛选骨髓、脑cDNA文库仍未获得全长cDNA后，利用EST数据库进行电脑拼接而获得的，BMZF-2全长3kb，3’端有poly（A）尾及加尾信号AATAAA，编码623个氨基酸，C端含18个锌指结构，而N端非锌指区仅有81个氨基酸，未见明确功能域，仅与q14588、FDZF2人类基因同源, 或许是个新的功能域。为获得BMZF-3, 除筛选骨髓、脑cDNA文库外，还设计多个引物，进行5’和3’端RACE, 最后获得全长cDNA，BMZF-3全长2114bp，尾端见加尾信号和poly（A）尾，编码474个氨基酸，C端有14个锌指结构，N端含KRAB顺序, 与鼠zf35和人类的许多锌指基因同源。BMZF-4经筛选骨髓、脑cDNA文库后，仅获得3’端cDNA，设计多个引物进行5’端RACE, 最后获得全长为2181bp 的cDNA，3’端有poly（A）尾，编码535个氨基酸，C端含13个锌指结构，N端含KRAB顺序，与人类锌指基因zn43、zn85、zn91等同源。BMZF-5来自骨髓CD34+细胞，全长1.6kb，3’端可见加尾信号及poly（A）尾，编码353个氨基酸，C端含4个不典型锌指结构，N端有KRAB顺序，与人类锌指基因zn91、zn85、zn43、z117等同源。

用荧光素原位杂交法（FISH）及幅射杂种系方法（Radiation hybrid, RH）进行染色体定位，将RSG-A定位于18q12.1，BMZF-1定位于19p13.1-13.2，BMZF-2定位于19q13.2，BMZF-3定位于19q13.42，BMZF-4定位于19p13.11-13.13 , BMZF-5定位于19p13.11-13.12。半定量RT-PCR显示，RSG-A几乎在所有组织中表达，而BMZF-1在NB4、HL60、胃、肺、心、肝等细胞和组织中表达量低或不表达，在骨髓、睾丸、肾、脑、肝、胰、U937、K562等组织和细胞中表达量较高。

为阐明RSG-A、BMZF-1和BMZF-2的功能，本研究将RSG-A、BMZF-1和BMZF-2非锌指区域构建至pGBT9和pM质粒中Gal4(1-147aa)下游，以产生Gal4-RSG-A和Gal4-BMZF-1融合基因，这里Gal4(1-147aa)替代RSG-A和BMZF-1的锌指功能域，而起与DNA结合作用。将杂合的pGBT9转染至酵母细胞株Y187（酵母单杂交系统，Yeast one-hybrid system）后，检测β-半乳糖苷酶(galactosidase)结果显示，RSG-A和BMZF-2在酵母中可能起转录活化作用，BMZF-1有转录抑制作用。为了进一步了解这三个基因在哺乳类细胞中的功能，将杂合的pM质粒与含有报道基因荧光素酶(Luciferase)的质粒共转染至NIH3T3和CHO细胞，检测Luciferase结果显示，RSG-A和BMZF-1均起转录抑制作用，以后者更明显， 而BMZF-2未见明显的转录抑制作用。

上述结果表明，本研究所采用的寻找新的锌指基因的技术路线是较为有效的，可能适用于含有保守序列特征基因的鉴定。所获得的锌指基因可能是转录因子，RSG-A、BMZF-1在哺乳细胞中起转录抑制作用，而RSG-A在酵母细胞中起转录活化作用。BMZF-2 N端可能存在新的转录调节区。染色体定位提示19号染色体存在krüppel样锌指基因聚集区，尤其19p13和19q13，这种格局的形成可能与进化过程中的基因扩增事件有关。进一步的研究将有可能进一步完善这套技术路线以获得更多新基因。另外，对新基因的结构包括基因组结构及其调控元件也需要加以阐明。尤其重要的是，对这些新基因的功能如转录活性，所结合的DNA元件和所调控的靶基因谱以及在调控基因网络中的位置，均有待于探讨。

Transcriptional regulation is controlled through interaction between DNA and protein complex, the latter containing transcription factors with highly conserved protein motifs. The most well-known motifs are the helix-turn-helix, helix-loop-helix, and zinc finger. During cell differentiation and development, each of these domains is involved in the binding of transcription factors to their cognate DNA recognition site, resulting in the specific activation or repression of gene expression.

Hematopoiesis is a complex physiological process, which requires fine regulation of gene expression during embryogenesis, fetal life and after birth. Disturbance of this regulation, therefore, may be the cause of hematopoietic disorders. It is interesting to note that some zinc finger proteins may regulate hematopoietic differentiation toward erythroid (GATA-1and EKLF), megakaryocytic (GATA-1 and 2), lymphoid (GATA-3 and Ikaros), granulocytic (MZF-1), as well as monocytic (EGR-1) lineages. Moreover, many zinc finger genes have been shown to be involved in the pathogenesis of hematological malignancies. For example, Cys2/His2 type zinc fingers such as HRX (also named as ALL-1 or MLL), PLZF (promyelocytic leukemia zinc finger gene) and Bcl-6, glucocorticoid receptor family member such as RAR? (retinoic acid receptor ?), ring finger such as PML (promyelocytic leukemia), and LIM family member such as Ttg-1 and Ttg-2, have been implicated in a variety of chromosomal translocations which play a primordial role in the pathogenesis of hematological malignancies.

DNA Fragments of Zinc Finger Genes Obtained from NB4 Cell Line and Normal Bone Marrow—Totally, 179 DNA fragments of Cys2/His2 type zinc finger genes were obtained from NB4 cell line and normal bone marrow by RT-PCR using primers corresponding to the consensus sequences of the krüppel-like zinc finger genes. Sequence analysis showed that there were 51 different DNA sequence species including novel ones after searching against GenBank. 3 DNA fragments of novel genes expressed in NB4 and 4 fragments of novel genes expressed in bone marrow were used as probe pool to screen cDNA libraries of NB4 cells or bone marrow, respectively, for obtaining full-length cDNA.

Cloning and Characterization of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 full-length cDNA—5 pure positive clones were isolated after screening cDNA library of NB4 cells using mixed probes. These fragments contained overlapping sequences and could thus be assembled into a contig. Bioinformatic analysis of this cDNA suggested that it contained an ORF (136-1243 nt.) encoding 368 amino acid residues (Fig.1A). This gene was initially named as RSG-A (for retinoic acid suppressed gene-A) because its mRNA could be amplified by homologous RT-PCR only in retinoic acid (RA)-untreated but not in RA-treated NB4 cells. However, a cDNA for the same gene, with longer 3’ UTR and thus 2.5 Kb in length was also cloned by a different approach, and obtained the international nomenclature ZNF191.

12 positive clones were isolated through screening cDNA library of bone marrow. 8 clones with insert size longer than 500bp were chosen for further study. Sequencing and database search of these 8 clones revealed that 4 corresponded to known genes and the 4 others were cDNA fragments of novel zinc finger genes, nominated as ZNF253, ZNF255, ZNF256, and ZNF257. Although in silico cloning and RACE were used to extend the 5’ end of ZNF253 cDNA, no stop codon could be found in frame upstream of an 825 bp ORF encoding 275 amino acid residues. The in vitro transcription and translation of ZNF253 gene revealed a protein product of 30 KD in good agreement with the predicted amino acid sequence derived from its cDNA.

ZNF255 gene full-length cDNA was obtained through the following steps: first, cDNA library screening allowed two overlapping clones to be isolated, which, however, only contained the 3’ part of an ORF. Then the in silico cloning method was utilized which identified two ESTs (GB-EST5: N99348 and GB-EST7: AA011208) and extended largely the 5’ end from the existing sequence. An assembled sequence of 3006 bp was thus acquired which may contain an entire ORF. Finally, a cDNA containing full-length ORF of ZNF255 was amplified from total RNA of bone marrow by RT-PCR using primers based on the assembled sequence. This clone was sequenced again to get the final sequence. As a result, an 1869 bp ORF of ZNF255 gene may encode a protein of 623 amino acids (Fig. 1C).

Two overlapping clones obtained from bone marrow and brain cDNA libraries constitute the initial fragment of ZNF256 gene. No EST was found to overlap with this sequence. Thus, primers were synthesized to get both 5’ and 3’-end of ZNF256 by RACE. The sequences derived from products of RACE PCR were partly overlapping with the initial fragment and these clones collectively defined a 2114 bp cDNA sequence of ZNF256, containing an 1422 bp ORF for a protein of 474 amino acids.

A clone from cDNA library of bone marrow was a partial 3’ cDNA of ZNF257. Primers were synthesized for getting full-length cDNA of ZNF257 by RACE because neither cDNA library screening nor database search found clones overlapping with the initial sequence. The sequences from product of RACE PCR could form a contig with that of the original clone. The collective 2181bp nucleotides sequence thus obtained included an 1605 bp ORF encoding 535 amino acid residues.

Among the six genes, all have a consensus polyadenylation signal AATAAA in the 3’-end. However, only four (ZNF191, ZNF255, ZNF256 and ZNF257) have “in-frame” stop codon upstream of the first ATG, while two genes (ZNF253 and ZNF254) with ORFs, which could continue to the 5’ end. Although so, the two genes have several ESTs matched, which, nevertheless, don’t extend to the 5’ end. The amino acid sequences of the two genes were compared with known genes by search against swissprot database. Both genes share KRAB domain in N-terminal. However, the sequences upstream of the first ATG in KRAB are not homologous with known ones. Thus, we would believe that the two genes might contain whole ORF, though further evidence is needed.

All Six Genes Belong to Krüppel-like Zinc Finger Gene Family? Bioinformatics analyses revealed that ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 were novel genes belonging to Krüppel-like zinc finger gene family. The deduced amino acid sequences of these genes contain 3-18 tandemly repeated zinc finger motifs related to Drosophila Krüppel gene family at the C-terminal and possible transcriptional regulatory elements such as KRAB and SCAN box at their N-terminal. The amino acid “knuckle” between zinc finger motifs, typified by the amino acid sequence TGE(R/K) P (F/Y) X, was also highly conserved in all six deduced amino acid sequences. From these features it was reasonable to predict that all six genes could encode DNA-binding proteins with transcriptional regulatory properties.

A possible Novel Trans-regulatory Domain KRNB Analysis of Non ZF Regions? Deduced 368 amino acid sequence of ZNF 191 had 4 continuous typical krüppel-like zinc finger motifs in C-terminal and contains rich acidic amino acids (15 D, 36 E) in non-zinc finger region. The non-zinc finger region shared high homology with those in some other Cys2/His2 type zinc finger genes such as human ZNF174, ZNF165, Q15776 and mouse ZNF38. An 81 amino acid stretch at the N-terminal of these genes was highly conserved and has been designated as the SCAN box (17). The SCAN box, enriched in hydrophobic and negatively charged residues with the L (X6) L motif at its core commonly found in transcription factors was separated from the zinc finger domains by amino acid regions varying in length and sharing no extensive sequence homology.

In addition to 3, 14, 13 and 4 tandemly arranged typical Cys2/His2 zinc finger motifs respectively, ZNF253, ZNF254, ZNF256, and ZNF257 genes contained Krüppel-associated box (KRAB) in their non-zinc finger regions. For example, the primary structure of KRAB domain of ZNF253 shares high homology with those in a large number of Krüppel-like zinc finger genes such as human ZNF43, ZNF85, ZNF90, ZNF91, ZNF93 and ZNF117. These domains consisting of approximately 75 amino acids are all located at the N-terminal moiety of the genes and enriched in hydrophobic and negatively charged residues with the L (X6) L at its core. This core is flanked by certain residues (e.g. E, L, V, and C) that are frequently found in ?-helices. There is also a high proportion of acidic amino acid residues (e.g. E, D).

Expression Pattern of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 ? Northern blot were performed to explore tissue expression pattern of these genes. However, except for ZNF191, the analysis was not always successful due to relatively low expression level of other genes. Therefore, semi-quantitative RT-PCR was used to determine the expression patterns of these genes in various tissues and leukemia cell lines. All genes were expressed in bone marrow in good agreement with their origin. ZNF191 gene was expressed in almost all tissues and cell lines except for heart and in fact its expression level was stable in NB4 cells during the course of ATRA treatment. ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 genes were selectively expressed in different tissues. Within hematopoietic system, ZNF191 and ZNF255 were expressed in all lineages, whereas ZNF253 expression was restricted to monocytic (U937) and immature erythroid (K562) lines. ZNF256 and ZNF257 tended to be expressed in myelomonocytic lineages (HL-60 and U937), although a low expression level could be detected in T lymphocytes (MOLT-4) and early erythroid cells (K562). ZNF253 expression was observed in all lineages except for K562 cells.

Functional Analysis of KRNB in Comparison with SCAN and KRAB Transcriptional Regulatory Domain? To further address the function of the six genes isolated in the present work, ZNF-191, ZNF253 and ZNF255 were chosen to study their transcriptional regulatory activities, since these genes contain SCAN, KRAB and KRNB, respectively. In order to assess whether ZNF-191 can affect the transcriptional expression of other genes, the non-zinc finger region (1-251aa) including SCAN box was amplified by PCR and subcloned into the expression vectors pGBT9 and pM. The recombinant pGBT9-ZNF191 containing GAL4-ZNF191 chimera and control plasmids pGBT9, pGBT9-HA, and pCL1 were then used to transform Yeast strain Y187. The qualitative and quantitative analyses of ?-galactosidase didn’t show obvious transregulatory activity of ZNF-191 in Y187. However, when a recombinant pM containing GAL4-ZNF191 chimera was cotransfected with a luciferase reporter plasmid into mammalian cells CHO and NIH3T3, it show a significant transrepression activity (P<0.05 to 0.01).

Analysis using yeast one-hybrid system and mammalian cell transfection for defining the functions of KRAB domain from ZNF253 generated quite coherent results. After Y187 was transformed with pGBT9-ZNF253 containing GAL4 BD-ZNF253 (1-174aa) chimera, both qualitative and quantitative assays of ?-galactosidase displayed a suppressive effect of ZNF253 non-zinc finger region on the transcription of reporter gene lacZ, making the galactosidase activity lower than that from pGBT9 with minimal basal stimulation (P<0.01). A similar transcriptional repressor effect was also observed in mammalian cells in that recombinant pM containing GAL4-ZNF253 fusion gene inhibited significantly the expression of reporter plasmid pGAL4₅tkLUC in CHO, NIH3T3 and COS-7 cell lines (P<0.05 to 0.01).

Chromosome Localization of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257? Using FISH, ZNF191 was mapped on chromosome 18q12.1. Interestingly, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 were all mapped on chromosome 19, ZNF253, ZNF254, ZNF257 being located at 19p13 and ZNF255, ZNF256 at 19q13 by RH technique.

Cys2/His2 type (kruppel-like) zinc finger gene family is one of the largest human gene families and plays an important role as transcriptional regulators by mediating interaction between DNA and protein. Some zinc finger proteins not only regulate hematopoiesis, especially the differentiation toward erythoid, megakaryocytic and lymphoid (GATA family and EKLF), granulocytic (MZF1), as well as monocytic (Egr-1) lineages, but also are involved in the pathogenesis of hematological malignancies such as acute leukemia. This study tries to isolate novel kruppel-like zinc finger genes expressed in bone marrow and acute promyelocytic leukemia cell line NB4 and characterize the function of these genes.

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