Proteins of theXenopus laevis zinc finger multigene family as targets for CK II phosphorylation

Zn finger proteins (ZFPs) of the C₂/H₂ type inXenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XIcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites forin vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activityin vivo is discussed.     

Rice <Emphasis Type="BoldItalic">ZFP15</Emphasis> Gene Encoding for a Novel C2H2-type Zinc Finger Protein Lacking DLN box,is Regulated by Spike Development but not by Abiotic Stresses

A novel C2H2-type zinc finger protein gene, ZFP15, was cloned from rice by RT-PCR approach. The ZFP15 gene encodes a protein of 144 amino acid residues with a predicted molecular mass of 15 kDa. The ZFP15 protein comprises two C2H2-type zinc finger domains, a putative nuclear localization signal (NLS) at its N-terminus but the DLN-box identified in all reported plant C2H2-type zinc finger proteins was not found. A homology search revealed that ZFP15 gene was localized within a cluster of C2H2-type zinc finger genes in BAC clone OJ1754_E06 mapped on chromosome 3. All three members in the cluster encoded proteins showed high identities in amino acids and might contribute to a co-regulation. The RT-PCR assay revealed that ZFP15 mRNA was not regulated by cold, salt, drought and ABA stresses, though CRT/DRE and ABRE elements were found in the promoter region of ZFP15 gene. The expression profiling also showed that ZFP15 mRNA was expressed with a lower level in leaves and roots, but not detected in stems. Besides, ZFP15 was shown to accumulate much more in flowering spike than in immature spike. Thus, ZFP15, as the first characterized C2H2-type zinc finger protein in rice, might play a regulatory role on rice spike development.     

ZFP5 encodes a functionally equivalent GIS protein to control trichome initiation

The Arabidopsis thaliana trichome development is a model system for understanding various aspects of plant cell development and differentiation. The C2H2 zinc finger proteins GIS, GIS2, and ZFP8 play important roles in controlling trichome initiation. In our recent study, we reported that a new C2H2 zinc finger protein, ZINC FINGER PROTEIN 5 (ZFP5), controls trichome cell development through GA signaling. ZFP5 acts upstream of GIS gene family and key trichome initiation regulators, and ZFP8 is the direct target gene of ZFP5. Here we show that ZFP5 encodes a protein functionally equivalent to GIS and GIS2 in controlling trichome initiation. Furthermore, similar to GIS2, ZFP5 is not involved in trichome branching.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 °C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.     

Human and mouse ZFP57 proteins are functionally interchangeable in maintaining genomic imprinting at multiple imprinted regions in mouse ES cells

Genomic imprinting is a common epigenetic phenomenon in mammals. Dysregulation of genomic imprinting has been implicated in a variety of human diseases. ZFP57 is a master regulator in genomic imprinting. Loss of ZFP57 causes loss of DNA methylation imprint at multiple imprinted regions in mouse embryos, as well as in embryonic stem (ES) cells. Similarly, mutations in human ZFP57 result in hypomethylation at many imprinted regions and are associated with transient neonatal diabetes and other human diseases. Mouse and human Zfp57 genes are located in the same syntenic block. However, mouse and human ZFP57 proteins only display about 50% sequence identity with different number of zinc fingers. It is not clear if they share similar mechanisms in maintaining genomic imprinting. Here we report that mouse and human ZFP57 proteins are functionally interchangeable. Expression of exogenous wild-type human ZFP57 could maintain DNA methylation imprint at three imprinted regions in mouse ES cells in the absence of endogenous mouse ZFP57. However, mutant human ZFP57 proteins containing the mutations found in human patients could not substitute for endogenous mouse ZFP57 in maintaining genomic imprinting in ES cells. Like mouse ZFP57, human ZFP57 and its mutant proteins could bind to mouse KAP1, the universal cofactor for KRAB zinc finger proteins, in mouse ES cells. Thus, we conclude that mouse and human ZFP57 are orthologs despite relatively low sequence identity and mouse ES cell system that we had established before is a valuable system for functional analyses of wild-type and mutant human ZFP57 proteins.     

水稻TFⅢA型锌指蛋白基因ZFP207的克隆和表达分析

锌指蛋白是一类重要的转录因子, 广泛参与植物的生长发育和胁迫应答过程。文章使用生物信息学方法从水稻中预测并克隆了一个TFⅢA型锌指蛋白基因ZFP207(GenBank 登陆号: AK063147.1), 该基因包含一个567 bp的开放阅读框, 编码一条188个氨基酸组成的多肽,其编码产物的预测分子量为20.72 kDa, 等电点为9.67。锌指蛋白ZFP207含有一个典型的TFⅢA型锌指结构, 并且在C端含有一个可能具有转录抑制功能的EAR-motif, 但不具有任何已知的核定位信号。系统发生分析结果表明, ZFP207与植物中已经发现的单锌指蛋白亲缘关系较近。通过RT-PCR方法分析了ZFP207基因在成株期水稻不同组织中的表达, 发现ZFP207在茎和叶中表达量较高, 而在穗和根中表达量较低。在酵母中的转录激活实验结果显示, ZFP207在酵母中不具有转录激活功能。     

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

Integration of HIV‐1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3′ end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV‐1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2‐LTR‐circle junctions of HIV‐1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV‐1 LTR. A six‐contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (K_d) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen‐bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 degrees C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

A multiscale approach to simulating the conformational properties of unbound multi‐C2H2 zinc finger proteins

The conformational properties of unbound multi‐Cys₂His₂ (mC₂H₂) zinc finger proteins, in which zinc finger domains are connected by flexible linkers, are studied by a multiscale approach. Three methods on different length scales are utilized. First, atomic detail molecular dynamics simulations of one zinc finger and its adjacent flexible linker confirmed that the zinc finger is more rigid than the flexible linker. Second, the end‐to‐end distance distributions of mC₂H₂ zinc finger proteins are computed using an efficient atomistic pivoting algorithm, which only takes excluded volume interactions into consideration. The end‐to‐end distance distribution gradually changes its profile, from left‐tailed to right‐tailed, as the number of zinc fingers increases. This is explained by using a worm‐like chain model. For proteins of a few zinc fingers, an effective bending constraint favors an extended conformation. Only for proteins containing more than nine zinc fingers, is a somewhat compacted conformation preferred. Third, a mesoscale model is modified to study both the local and the global conformational properties of multi‐C₂H₂ zinc finger proteins. Simulations of the CCCTC‐binding factor (CTCF), an important mC₂H₂ zinc finger protein for genome spatial organization, are presented. Proteins 2015; 83:1604–1615. © 2015 Wiley Periodicals, Inc.     

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing

The 3′ cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.     

Protein interactions within the TcZFP zinc finger family members of Trypanosoma cruzi: implications for their functions

The small zinc finger proteins tbZFP1 and tbZFP2 have been implicated in the control of Trypanosoma brucei differentiation to the procyclic form. Here, we report that the complete ZFP family in Trypanosoma cruzi is composed by four members, ZFP1A and B, and ZFP2A and B. ZFP1B is a paralog specific gene restricted to T. cruzi, while the ZFP2A and B paralogs diverged prior to the trypanosomatid lineage separation. Moreover, we demonstrate that TcZFP1 and TcZFP2 members interact with each other and that this interaction is mediated by a WW domain in TcZFP2. Also, TcZFP2B strongly homodimerizes by a glycine rich region absent in TcZFP2A. We propose a model to discuss the relevance of these protein-protein interactions in terms of the functions of these proteins.     

A zinc finger protein array for the visual detection of specific DNA sequences for diagnostic applications

The visual detection of specific double-stranded DNA sequences possesses great potential for the development of diagnostics. Zinc finger domains provide a powerful scaffold for creating custom DNA-binding proteins that recognize specific DNA sequences. We previously demonstrated sequence-enabled reassembly of TEM-1 β-lactamase (SEER–LAC), a system consisting of two inactive fragments of β-lactamase each linked to engineered zinc finger proteins (ZFPs). Here the SEER–LAC system was applied to develop ZFP arrays that function as simple devices to identify bacterial double-stranded DNA sequences. The ZFP arrays provided a quantitative assay with a detection limit of 50 fmol of target DNA. The method could distinguish target DNA from non-target DNA within 5 min. The ZFP arrays provided sufficient sensitivity and high specificity to recognize specific DNA sequences. These results suggest that ZFP arrays have the potential to be developed into a simple and rapid point-of-care (POC) diagnostic for the multiplexed detection of pathogens.