Lineage-specific expansion of the zinc finger associated domain ZAD

The zinc finger associated domain (ZAD), present in almost 100 distinct proteins, characterizes the largest subgroup of C2H2 zinc finger proteins in Drosophila melanogaster and was initially found to be encoded by arthropod genomes only. Here, we report that the ZAD was also present in the last common ancestor of arthropods and vertebrates, and that vertebrate genomes contain a single conserved gene that codes for a ZAD-like peptide. Comparison of the ZAD proteomes of several arthropod species revealed an extensive and species-specific expansion of ZAD-coding genes in higher holometabolous insects, and shows that only few ZAD-coding genes with essential functions in Drosophila melanogaster are conserved. Furthermore, at least 50% of the ZAD-coding genes of Drosophila melanogaster are expressed in the female germline, suggesting a function in oocyte development and/or a requirement during early embryogenesis. Since the majority of the essential ZAD coding genes of Drosophila melanogaster were not conserved during arthropod or at least during insect evolution, we propose that the LSE of ZAD-coding genes shown here may provide the raw material for the evolution of new functions that allow organisms to pursue novel evolutionary paths.     

Selectivity of arsenite interaction with zinc finger proteins

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ? C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.     

The C2H2 -type Zinc Finger Protein ZFP182 is Involved in Abscisic Acid-Induced Antioxidant Defense in Rice

C2H2 -type zinc finger proteins (ZFPs) are thought to play important roles in modulating the responses of plants to drought, salinity and oxidative stress. However, direct evidence is lacking for the involvement of these ZFPs in abscisic acid (ABA)-induced antioxidant defense in plants. In this study, the role of the rice (Oryza sativa L. sub. japonica cv. Nipponbare) C 2 H 2 -type ZFP ZFP182 in ABA-induced antioxidant defense and the relationship between ZFP182 and two rice MAPKs, OsMPK1 and OsMPK5 in ABA signaling were investigated. ABA treatment induced the increases in the expression of ZFP182, OsMPK1 and OsMPK5, and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in rice leaves. The transient gene expression analysis and the transient RNA interference (RNAi) analysis in protoplasts showed that ZFP182, OsMPK1 and OsMPK5 are involved in ABA-induced up-regulation in the activities of SOD and APX. Besides, OsMPK1 and OsMPK5 were shown to be required for the up-regulation in the expression of ZFP182 in ABA signaling, but ZFP182 did not mediate the ABA-induced up-regulation in the expression of OsMPK1 and OsMPK5. These results indicate that ZFP182 is required for ABA-induced antioxidant defense and the expression of ZFP182 is regulated by rice MAPKs in ABA signaling.     

Structural basis of diversity and homodimerization specificity of zinc-finger-associated domains in Drosophila

In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein''s ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.     

Functional Characterization of Maize C<Subscript>2</Subscript>H<Subscript>2</Subscript> Zinc-Finger Gene Family

Plant C2H2-type zinc finger proteins (ZFPs) play essential roles in developmental control and stress responses. The whole complement of ZFP genes has been identified in Arabidopsis and rice, while the genome-scale identification and functional analysis of maize ZFPs is not yet reported. Hence, we performed a comprehensive analysis, including gene structure, chromosome location, duplicated event, selective pressure, phylogeny, gene ontology annotation, and expression profiling under developmental stages and abiotic stresses. Phylogenetic analyses suggested that the ZmZFP gene family can be grouped into three classes (A, B, and C). The analysis of differential gene expression in different developmental stages and stress treatments (drought, salt, and cold) was conducted based on microarray and RNA-seq data. A total of 99.05 % (209 genes) of the total ZmZFP genes (211 genes) were detected in 60 different tissues in microarray data. Under drought stress, 13 differentially expressed genes were found in leaf, of which 7 and 6 genes were up-regulated and down-regulated, respectively. For salt stress, crown root (CR), primary root (PR) and seed root (SR) each had one significantly elevated gene, while 2, 1, and 7 genes were obviously down-regulated in CR, PR and SR, respectively. Additionally, 8 and 3 genes were significantly up-regulated and down-regulated, respectively, in the cold-tolerant line ETH-DH7. This study will lay the foundation for understanding the roles of ZFPs in maize growth and stress resistance, contributing to the molecular breeding of maize for food.     

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.     

中华蜜蜂ZFP37基因的生物信息学分析及高温下表达特性的研究

锌指蛋白(Zinc finger proteins, ZFPs)是一类在真核生物体内广泛分布的蛋白质。锌指蛋白作为一类转录因子,它能够调控基因的表达和细胞的分化,最近的研究显示其在动植物抗逆方面也发挥着重要作用。本研究对中华蜜蜂Apis cerana cerana ZFP37的蛋白结构进行了预测分析,并通过qRT-PCR分析了中华蜜蜂在遭受高温胁迫时ZFP37的表达情况,进一步了解锌指蛋白在中华蜜蜂应对热胁迫过程中的作用。结果显示,中华蜜蜂ZFP37可编码123个氨基酸,蛋白分子量为13.7 kDa,无跨膜结构。氨基酸同源序列比对结果表明,中华蜜蜂ZFP37序列与蜜蜂科昆虫的相似性最高,与其他膜翅目昆虫的相似性存在差异。基因的表达模式显示,ZFP37在高温下表达升高,此外,胁迫时间的增加也可导致ZFP37表达的升高。这些结果表明ZFP37对于中华蜜蜂应对热应激有重要的生物学意义。