Assessment of a zinc finger protein gene (MPZC3H10) as potential RNAi target for green peach aphid Myzus persicae control.

BACKGROUND: RNA interference (RNAi) has potential application in pest control, and selection of the specific target gene is one of the key steps in RNAi. As an important effector, the zinc finger protein (ZFP) gene has high similarity among aphid species, and may have potential use in an RNAi-based pest control strategy. This study assessed the control efficiency of an RNAi target, MPZC3H10, a CCCH-type ZFP gene, against green peach aphid. RESULTS: ZC3H10 amino acid sequence similarity is more than 97.71% among the five tested aphid species: Myzus persicae, Aphis citricidus, Acyrthosiphon pisum, Diuraphis noxia and Rhopalosiphum maidis. However, no homologous sequence was found in the transcriptome of their ladybeetle predator, Propylaea japonica. Spatial expression patterns revealed that MPZC3H10 showed high expression in the muscle and fat body of M. persicae. The RNAi bioassay revealed that silencing of MPZC3H10 resulted in high mortality (53.33%) in M. persicae. By contrast, there were no observed negative effects on the growth and development of P. japonica when fed on aphids treated with double-stranded RNA (dsRNA) or injected with a "high dose" of dsRNA. CONCLUSION: Targeting MPZC3H10 showed promising efficiency for green peach aphid control via artificially designed dsRNA, and was safe for the predatory ladybeetle. © 2022 Society of Chemical Industry.

The KRAB Domain-Containing Protein ZFP961 Represses Adipose Thermogenesis and Energy Expenditure through Interaction with PPARα.

Adipose thermogenesis plays a pivotal role in whole-body metabolic homeostasis. Although transcriptional mechanisms that promote thermogenesis are extensively studied, the negative regulatory network is still poorly understood. Here, a Krüppel-associated box (KRAB) domain-containing zinc finger protein, ZFP961, as a potent repressor of the thermogenic program is identified. ZFP961 expression is induced by cold and ß3-adrenergic agonist in adipose tissue. ZFP961 represses brown fat-selective gene expression and mitochondrial respiration without any effect on general adipogenesis in cultured adipocytes. Adipose-specific knockdown and overexpression of ZFP961 produce remarkable and opposite phenotypes of white fat remodeling. ZFP961 knockout mice display robust inguinal white adipose tissue browning, which is abolished by reexpression of full-length ZFP961, but not by KRAB domain-deleted ZFP961 mutant. ZFP961-deficient mice are cold tolerant and resistant to high-fat diet-induced obesity, hyperglycemia, and hepatic steatosis. ZFP961 suppresses thermogenic gene expression by directly interacting with PPARα and blocking its transcriptional activity, which can be completely negated by the PPARα agonist. The findings uncover ZFP961 as a critical physiological brake that limits adipose thermogenesis and provides insights into the regulatory mechanisms that maintain energy balance and tissue homeostasis.

Chicken ZNF764L gene: mRNA expression profile, alternative splicing analysis and association analysis between first exon indel mutation and economic traits.

Zinc finger proteins are a class of transcription factors with finger-like domains and have diverse uses in biological processes, including development, differentiation, and metabolism. In this study, we identified the absence of the 24 bp sequence in the third exon of the zinc finger protein 764-like (ZNF764L) gene that lead to the production of two new transcripts, ZNF764L-SV1 and ZNF764L-SV2, and the sum of the expression levels of the two transcripts is approximately equal the total RNA expression level. Temporal and spatial expression showed that ZNF764L had higher expression during the embryonic stage. Moreover, the research study revealed a 22-bp indel mutation in the first exon region of ZNF764L gene. Statistically significant results (P < 0.05) were encountered for this indel for chicken growth and carcass traits, which include birth weight, chest breadth and body slanting length at 4 weeks of age and subcutaneous fat weight and others. Genetic parameter analysis showed that D is the predominant allele in the commercial chicken population. Gene expression for each genotype showed that birds carrying the II allele had a higher expression level than the other genotypes. These findings enrich the understanding of ZNF764L gene function and enhance reproduction in the chicken industry.

Comparative analysis of the genetic variability within the Q-type C2H2 zinc-finger transcription factors in the economically important cabbage, canola and Chinese cabbage genomes.

BACKGROUND: Brassica oleracea, B. rapa and B. napus encompass many economically important vegetable and oil crops; such as cabbage, broccoli, canola and Chinese cabbage. The genome sequencing of these species allows for gene discovery with an eye towards discerning the natural variability available for future breeding. The Q-type C2H2 zinc-finger protein (ZFP) transcription factors contain zinc finger motifs with a conserved QALGGH as part of the motif and they may play a critical role in the plants response to stress. While they may contain from one to five ZF domains (ZFD) this work focuses on the ZFPs that contain two zinc-fingers, which bind to the promoter of genes, and negatively regulate transcription via the EAR motif. B. oleracea and rapa are diploid and evolved into distinct species about 3.7 million years ago. B. napus is polyploid and formed by fusion of the diploids about 7500 years ago. RESULTS: This work identifies a total of 146 Q-type C2H2-ZFPs with 37 in B. oleracea, 35 in B. rapa and 74 in B. napus. The level of sequence similarity and arrangement of these genes on their chromosomes have mostly remained intact in B. napus, when compared to the chromosomes inherited from either B. rapa or oleracea. In contrast, the difference between the protein sequences of the orthologs of B. rapa and oleracea is greater and their organization on the chromosomes is much more divergent. In general, the 146 proteins are highly conserved especially within the known motifs. Differences within subgroups of ZFPs were identified. Considering that B. napus has twice the number of these proteins in its genome, RNA-Seq data was mined and the expression of 68 of the 74 genes was confirmed. CONCLUSION: Alignment of these proteins gives a snapshot of the variability that may be available naturally in Brassica species. The aim is to study how different ZFPs bind different genes or how dissimilar EAR motifs alter the negative regulation of the genes bound to the ZFP. Results from such studies could be used to enhance tolerance in future Brassica breeding programs.

Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.

Zinc finger proteins are the most common among families of DNA-binding transcription factors. Designer transcription factors generated by the fusion of engineered zinc finger DNA-binding domains (ZF-DBDs) to effector domains have been valuable tools for the modulation of gene expression and for targeted genome editing. However, ZF-DBDs without effector domains have also been shown to effectively modulate gene expression by competing with sequence-specific DNA-binding transcription factors. Here, we describe the methodology and provide a detailed workflow for the cloning, expression, purification, and direct cell delivery of engineered ZF-DBDs. Using this protocol, ZF-DBDs can be generated with high efficiency in less than 2 weeks. We also describe a nonradioactive method for measuring DNA binding affinity of the purified ZF-DBD proteins as well as a method for direct delivery of the purified ZF-DBDs to mammalian cells.

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline.

After induced mutagenesis and transgenesis, genome editing is the next step in the development of breeding techniques. Genome editing using site-directed nucleases - including meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system - is based on the mechanism of double strand breaks. The nuclease is directed to cleave the DNA at a specific place of the genome which is then repaired by natural repair mechanisms. Changes are introduced during the repair that are either accidental or can be targeted if a DNA template with the desirable sequence is provided. These techniques allow making virtually any change to the genome including specific DNA sequence changes, gene insertion, replacements or deletions with unprecedented precision and specificity while being less laborious and more straightforward compared to traditional breeding techniques or transgenesis. Therefore, the research in this field is developing quickly and, apart from model species, multiple studies have focused on economically important species and agronomically important traits that were the key subjects of this review. In plants, studies have been undertaken on disease resistance, herbicide tolerance, nutrient metabolism and nutritional value. In animals, the studies have mainly focused on disease resistance, meat production and allergenicity of milk. However, none of the promising studies has led to commercialization despite several patent applications. The uncertain legal status of genome-editing methods is one of the reasons for poor commercial development, as it is not clear whether the products would fall under the GMO regulation. We believe this issue should be clarified soon in order to allow promising methods to reach their full potential.

Structural analysis of viral infectivity factor of HIV type 1 and its interaction with A3G, EloC and EloB.

BACKGROUND: The virion infectivity factor (Vif) is an accessory protein, which is essential for HIV replication in host cells. Vif neutralizes the antiviral host protein APOBEC3 through recruitment of the E3 ubiquitin ligase complex. METHODOLOGY: Fifty thousand Vif models were generated using the ab initio relax protocol of the Rosetta algorithm from sets of three- and nine-residue fragments using the fragment Monte Carlo insertion-simulated annealing strategy, which favors protein-like features, followed by an all-atom refinement. In the protocol, a constraints archive was used to define the spatial relationship between the side chains from Cys/His residues and zinc ions that formed the zinc-finger motif that is essential for Vif function. We also performed centroids analysis and structural analysis with respect to the formation of the zinc-finger, and the residue disposal in the protein binding domains. Additionally, molecular docking was used to explore details of Vif-A3G and Vif-EloBC interactions. Furthermore, molecular dynamics simulation was used to evaluate the stability of the complexes Vif-EloBC-A3G and Vif-EloC. PRINCIPAL FINDINGS: The zinc in the HCCH domain significantly alters the folding of Vif and changes the structural dynamics of the HCCH region. Ab initio modeling indicated that the Vif zinc-finger possibly displays tetrahedral geometry as suggested by Mehle et al. (2006). Our model also showed that the residues L146 and L149 of the BC-box motif bind to EloC by hydrophobic interactions, and the residue P162 of the PPLP motif is important to EloB binding. CONCLUSIONS/SIGNIFICANCE: The model presented here is the first complete three-dimensional structure of the Vif. The interaction of Vif with the A3G protein and the EloBC complex is in agreement with empirical data that is currently available in the literature and could therefore provide valuable structural information for advances in rational drug design.

A study on the distribution of 37 well conserved families of C2H2 zinc finger genes in eukaryotes.

BACKGROUND: The C2H2 zinc-finger (ZNF) containing gene family is one of the largest and most complex gene families in metazoan genomes. These genes are known to exist in almost all eukaryotes, and they constitute a major subset of eukaryotic transcription factors. The genes of this family usually occur as clusters in genomes and are thought to have undergone a massive expansion in vertebrates by multiple tandem duplication events (BMC Evol Biol 8:176, 2008). RESULTS: In this study, we combined two popular approaches for homolog detection, Reciprocal Best Hit (RBH) (Proc Natl Acad Sci USA 95:6239-6244, 1998) and Hidden-Markov model (HMM) profiles search (Bioinformatics 14:755-763, 1998), on a diverse set of complete genomes of 124 eukaryotic species ranging from excavates to humans to identify all detectable members of 37 C2H2 ZNF gene families. We succeeded in identifying 3,890 genes as distinct members of 37 C2H2 gene families. These 37 families are distributed among the eukaryotes as progressive additions of gene blocks with increasing complexity of the organisms. The first block featuring the protists had 7 families, the second block featuring plants had 2 families, the third block featuring the fungi had 2 families (one of which was also present in plants) and the final block consisted of metazoans with 25 families. Among the metazoans, the simpler unicellular metazoans had just 15 of the 25 families while most of the bilaterians had all 25 families making up a total of 37 families. Multiple potential examples of lineage-specific gene duplications and gene losses were also observed. CONCLUSIONS: Our hybrid approach combines features of the both RBH and HMM methods for homolog detection. This largely automated technique is much faster than manual methods and is able to detect homologs accurately and efficiently among a diverse set of organisms. Our analysis of the 37 evolutionarily conserved C2H2 ZNF gene families revealed a stepwise appearance of ZNF families, agreeing well with the phylogenetic relationship of the organisms compared and their presumed stepwise increase in complexity (Science 300:1694, 2003).

Prediction of DNA-binding specificity in zinc finger proteins.

Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions -1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure-function relationships of the existing zinc finger-DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

An elastic-network-based local molecular field analysis of zinc finger proteins.

We study two designed and one natural zinc finger peptide each with the Cys(2)His(2) (CCHH) type of metal binding motif. In the approach we have developed, we describe the role of the protein and solvent outside the Zn(II)-CCHH metal-residue cluster by a molecular field represented by generalized harmonic restraints. The strength of the field is adjusted to reproduce the binding energy distribution of the metal with the cluster obtained in a reference all-atom simulation with empirical potentials. The quadratic field allows us to investigate analytically the protein restraints on the binding site in terms of its eigenmodes. Examining these eigenmodes suggests, consistent with experimental observations, the importance of the first histidine (H) in the CCHH cluster in metal binding. Further, the eigenvalues corresponding to these modes also indicate that the designed proteins form a tighter complex with the metal. We find that the bulk protein and solvent response tends to destabilize metal binding, emphasizing that the favorable energetics of metal-residue interaction is necessary to drive folding in this system. The representation of the bulk protein and solvent response by a local field allows us to perform Monte Carlo simulations of the metal-residue cluster using quantum-chemical approaches, here using a semiempirical Hamiltonian. For configurations sampled from this simulation, we study the free energy of replacing Zn(2+) with Fe(2+), Co(2+), and Ni(2+) using density functional theory. The calculated selectivities are in fair agreement with experimental results.

Metalloproteins and the pyrite-based origin of life: a critical assessment.

We critically examine the proposal by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that putative transition metal binding sites in protein components of the translation machinery of hyperthermophiles provide evidence of a direct relationship with the FeS clusters of pyrite and thus indicate an autotrophic origin of life in volcanic environments. Analysis of completely sequenced cellular genomes of Bacteria, Archaea and Eucarya does not support the suggestion by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that aminoacyl-tRNA synthetases and ribosomal proteins bear sequence signatures typical of strong covalent metal bonding whose absence in mesophilic species reveals a process of adaptation towards less extreme environments.

In vitro assessment of zinc finger nuclease activity.

The technical advances in developing artificial endonucleases, such as zinc finger nucleases (ZFNs), have opened a wide field of applications in the genome engineering arena, including the therapeutic correction of mutated genes in the human genome. Gene editing frequencies of up to 50% in human cells under non-selective conditions reveal the power of the ZFN technology. Activity and toxicity of ZFNs are determined by a number of parameters, including the specificity of DNA binding, the kinetics of dimerization of the two ZFN subunits, and the catalytic activity. In order to investigate these parameters individually, a cell-free system that models these reactions is essential. Here, we present a simple and fast method for the functional testing of ZFNs in vitro.

Deducing the energetic cost of protein folding in zinc finger proteins using designed metallopeptides.

Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biological activity. The thermodynamic contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorimetry, potentiometry, and calorimetry to determine the thermodynamics of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation constants for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6 x 10(16), 1.5 x 10(15), or 2.5 x 10(13) M(-1), respectively. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, respectively, in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equivalent at physiological pH, DeltaG = -16.8 kcal/mol or a Ka = 2.0 x 10(12) M(-1). Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to determine the free energy required to fold the latter. Several proteins have identical Zn(II) affinities to GGG. That is, little, if any, of their Zn(II) binding energy is required to fold the protein, whereas some have affinities weakened by up to 5.7 kcal/mol; i.e., the Zn(II) binding energy is being used to fold the protein.