Quantitative trait loci identification reveals zinc finger protein CONSTANS-LIKE 4 as the key candidate gene of stigma color in watermelon(Citrullus lanatus)

Stigma color is a critical agronomic trait in watermelon that plays an important role in pollination.However,there are few reports on the regulation of stigma color in watermelon.In this study,a genetic analysis of the F2 population derived from ZXG1553(P1,with orange stigma)and W1-17(P2,with yellow stigma)indicated that stigma color is a quantitative trait and the orange stigma is recessive compared with the yellow stigma.Bulk segregant analysis sequencing(BSA-seq)revealed a 3.75 Mb segment on chromosome 6 that is related to stigma color.Also,a major stable effective QTL Clqsc6.1(QTL stigma color)was detected in two years between cleaved amplified polymorphic sequencing(CAPS)markers Chr06_8338913 and Chr06_9344593 spanning a~1.01 Mb interval that harbors 51 annotated genes.Cla97C06G117020(annotated as zinc finger protein CONSTANS-LIKE 4)was identified as the best candidate gene for the stigma color trait through RNA-seq,quantitative real-time PCR(qRT-PCR),and gene structure alignment analysis among the natural watermelon panel.The expression level of Cla97C06G117020 in the orange stigma accession was lower than in the yellow stigma accessions with a significant difference.A nonsynonymous SNP site of the Cla97C06G117020 coding region that causes amino acid variation was related to the stigma color variation among nine watermelon accessions according to their re-sequencing data.Stigma color formation is often related to carotenoids,and we also found that the expression trend of ClCHYB(annotated asβ-carotene hydroxylase)in the carotenoid metabolic pathway was consistent with Cla97C06G117020,and it was expressed in low amounts in the orange stigma accession.These data indicated that Cla97C06G117020 and ClCHYB may interact to form the stigma color.This study provides a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.

Diagnostic value of methylated branched chain amino acid transaminase 1/IKAROS family zinc finger 1 for colorectal cancer

BACKGROUND The diagnostic value of combined methylated branched chain amino acid transaminase 1(BCAT1)/IKAROS family zinc finger 1(IKZF1)in plasma for colorectal cancer(CRC)has been explored since 2015.Recently,several related studies have published their results and showed its diagnostic efficacy.AIM To analyze the diagnostic value of methylated BCAT1/IKZF1 in plasma for screening and postoperative follow-up of CRC.METHODS The candidate studies were identified by searching the PubMed,Embase,Cochrane Library,CNKI,and Wanfang databases from May 31,2003 to June 1,2023.Sensitivity,specificity,and diagnostic accuracy were calculated by merging ratios or means.RESULTS Twelve eligible studies were included in the analysis,involving 6561 participants.The sensitivity of methylated BCAT1/IKZF1 in plasma for CRC diagnosis was 60%[95%confidence interval(CI)53-67]and specificity was 92%(95%CI:90-94).The positive and negative likelihood ratios were 8.0(95%CI:5.8-11.0)and 0.43(95%CI:0.36-0.52),respectively.Diagnostic odds ratio was 19(95%CI:11-30)and area under the curve was 0.88(95%CI:0.85-0.91).The sensitivity and specificity for CRC screening were 64%(95%CI:59-69)and 92%(95%CI:91-93),respectively.The sensitivity and specificity for recurrence detection during follow-up were 54%CONCLUSION The detection of methylated BCAT1/IKZF1 in plasma,as a non-invasive detection method of circulating tumor DNA,has potential CRC diagnosis,but the clinical application prospect needs to be further explored.

Zinc finger protein A20 protects rats against chronic liver allograft dysfunction

AIM： To investigate the effect of zinc finger protein A20 on chronic liver allograft dysfunction in rats. METHODS： AIIogeneic liver transplantation from DA rats to Lewis rats was performed. Chronic liver allograft dysfunction was induced in the rats by administering low-dose tacrolimus at postoperative day （POD） 5. Hepatic overexpression of A20 was achieved by recom- binant adenovirus （rAd.）-mediated gene transfer ad- ministered intravenously every 10 d starting from POD 10. The recipient rats were injected with physiologi- cal saline, rAdEasy-A20 （1 × 109 pfu/30 g weight） or rAdEasy （1 × 109 pfu/30 g weight） every 10 d through the tail vein for 3 mo starting from POD 10. Liver tissue samples were harvested on POD 30 and POD 60. RESULTS： Liver-transplanted rats treated with only tacrolimus showed chronic allograft dysfunction with severe hepatic fibrosis. A20 overexpression ameliorated the effects on liver function, attenuated liver allograft fibrosis and prolonged the survival of the recipient rats. Treatment with A20 suppressed hepatic protein pro- duction of tumor growth factor （TGF）-β1, interleukin- 113, caspase-8, CD40, CD40L, intercellular adhesion molecule-i, vascular cell adhesion molecule-1 and E-selectin. A20 treatment suppressed liver cell apopto- sis and inhibited nuclear factor-KB activation of Kupffer cells （KCs）, liver sinusoidal endothelial cells （LSECs） and hepatic stellate cells （HSCs）, and it subsequently decreased cytokine mRNA expression in KCs and LSECs and reduced the production of TGF-β1 in HSCs. CONCLUSION： A20 might prevent chronic liver allogra- ft dysfunction by re-establishing functional homeostasis of KCs, LSECs and HSCs.

Myeloid zinc finger 1(MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin （FS） is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from -1 800 to ＋16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs （-302/＋16 bp）-FS and （-180/＋16 bp）-FS （P〈0.01）. Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 （MZF1） binding sequence had significantly decreased luciferase activity （P〈0.05）. Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence ＂TCCCCACC＂ （the recognition site of transcription factor MZF1） was the most important for FS transcription activation in the porcine.

Zinc finger E-box-binding homeobox 1 mediates aerobic glycolysis via suppression of sirtuin 3 in pancreatic cancer

AIM TO uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1 （ZEB1） was silenced using a and the impact of ZEB1 and lentivirus-mediated method, methyI-CpG binding domain protein 1 （MBD1） on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3 （SIRT3） expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatinimmunoprecipitation assays.RESULTS ZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1.CONCLUSION ZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.

Expression Pattern Analysis of Zinc Finger Protein Genes in Wheat(Triticum aestivum L.) Under Phosphorus Deprivation

Zinc finger protein（ZFP） genes comprise a large and diverse gene family, and are involved in biotic and abiotic stress responses in plants. In this study, a total of 126 ZFP genes classified into various types in wheat were characterized and subjected to expression pattern analysis under inorganic phosphate（Pi） deprivation. The wheat ZFP genes and their corresponding GenBank numbers were obtained from the information of a 4×44K wheat gene expression microarray chip. They were confirmed by sequence similarity analysis and named based on their homologs in Brachypodium distachyon or Oriza sativa. Expression analysis based on the microarray chip revealed that these ZFP genes are categorized into 11 classes according to their gene expression patterns in a 24-h of Pi deprivation regime. Among them, ten genes were differentially up-regulated, ten genes differentially downregulated, and two genes both differentially up- and down-regulated by Pi deprivation. The differentially up- or down-regulated genes exhibited significantly more or less transcripts at one, two, or all of the checking time points（1, 6, and 24 h） of Pi stress in comparison with those of normal growth, respectively. The both differentially up- and down-regulated genes exhibited contrasting expression patterns, of these, TaWRKY70;5 showed significantly up-regulated at 1 and 6 h and down-regulated at 24 h whereas TaAN1AN20-8;2 displayed significantly upregulated at 1 h and downregulated at 6 h under deprivation Pi condition. Real time PCR analysis confirmed the expression patterns of the differentially expressed genes obtained by the microarray chip. Our results indicate that numerous ZFP genes in wheat respond to Pi deprivation and have provided further insight into the molecular basis that plants respond to Pi deprivation mediated by the ZFP gene family.

Zinc finger protein 139 expression in gastric cancer and its clinical significance

AIM: To investigate the expression of zinc finger protein 139 (ZNF139) in gastric cancer (GC), and to analyze its clinical significance.

Zinc Finger Protein-activating Transcription Factor Up-regulates Vascular Endothelial Growth Factor-A Expression in Vitro

Objective To construct the zinc finger protein-activating transcription factor (ZFP-ATF) plasmid and evaluate its efficacy in inducing vascular endothelial growth factor (VEGF) expression in EY.HY926 endothelial cells. Methods Firstly, we constructed the ZFP-ATF plasmid, then testified the quantity of VEGF protein in EY.HY926 endothelial cells after transfected with ZFP-ATP plasmid by Western blot, finally, we used the RT-PCR to testify whether the ZFP-ATF can stimulate expression of VEGF splice variants. Results The ZFP-ATF DNA sequences were located the multiclone sites of PVAX1 vector between the site of BamH Ⅰ and Xhol. Western blot result showed VEGF expression in EY.HY926 endothelial cells transfected with ZFP-ATF plasmid was significantly higher than that in cells transfected with VEGF165 (19.95±3.95 vs. 12.15±1.55 μg/μL, P<0.01). RT-PCR result showed VEGF-A mRNA expression level induced by ZFP-ATF was high than that induced by VEGF165. Conclusion ZFP-ATF can up-regulate the VEGF-A expression in comparison with VEGF165, which might have beneficial effects in angiogenesis process.

Genome-wide analysis of the CCCH zinc finger family in longan:Characteristic identification and expression profiles in Dimocarpus longan Lour

CCCH(C3 H) Zinc finger(Znf) transcription factors(TFs), as a novel type of Znf gene, regulate the expression of genes by binding to their mRNAs and play important roles in plant growth and development and abiotic stress resistance.Longan(Dimocarpous longan) is a tropical/subtropical fruit tree of great economic importance in Southeast Asia.However, genomic information on C3 H and their functions in longan are still unknown. In this study, a comprehensive analysis of the longan C3 H(DlC3 H) gene family was carried out. A total of 49 DlC3 H genes in three clades were identified from the longan genome database. Characteristics of the genes were analyzed with respect to gene structure,motif composition, phylogenetic tree and potential functions. The analysis of alternative splicing(AS) events suggested that AS events in DlC3 H genes were related to the transformation from longan non-embryonic to embryonic cultures.Promoter analysis indicated that most of the DlC3 H genes included cis-acting elements associated with hormones and stresses responses. Quantitative real-time PCR(qRT-PCR) analysis indicated that 26 of the 49 DlC3 Hs, which possess methyl jasmonate(MeJA) and abscisic acid(ABA) responsive cis-acting elements, showed differential expression patterns under treatment with ABA, MeJA and their endogenous inhibitors, suggesting that DlC3 Hs might be involved in the ABA and MeJA signaling pathways. The expression profiles of 17 of the 49 DlC3 Hs in non-embryonic callus and three tissues of embryonic cultures showed that only five of the 17 DlC3 Hs had the same expression trends as the FPKM trends in transcriptome data;the expression levels of DlC3 H07/14/16/36/49 in embryogenic callus and DlC3 H04/38 in globular embryos were high, suggesting that they have different functions in embryonic development. Further, we verified that DlC3 H01/03/05/11/19/39 were regulated by sRNAs by a modified 5’ RLM-RACE method. This study provides the first systematic analysis of C3 H genes in longan, and found that C3 H genes may be involved in hormone and stress responses, and somatic embryogenesis. Our preliminary investigation may provide clues to further studies on the characteristics and functions of this family in longan.

STUDIES ON THE SYNTHESIS AND DNA-BINDING ABILITY OF ZINC FINGER MOTIF

Transcription factor SPI is a protcin present in mammalian cells that binds to GC box promoter clements of Gene and selectively activates mRNA synthesis. The gene contains functional recognition sites. It contains three continuous zinc finger motifs, which are believed being mctalloprotein structures that interact with DNA. We synthesized the second zine finger fragment of SP1 (SP1-ZF2) and its mutant (SP1-ZF2 / HT. E20→H. R23→T), we also synthesized the Cys-Cys loop (ZF6) and the His-His loop (ZF5) of SPI and linked the twoloops together using a β-turn structure to obtain a finger mimic analogue (ZF-15) by stepwise solid-phase technique. Atomic absorption studies show that SP 1-ZF2 and SP1-ZF2 / HT bind zinc cquimolarly, but ZF-15 docs not bind Zn anyway. The CD experiments demonstrate a significant change in secondary structure in the prescnce or absence of Zn to SP1-ZF2 and SP1-ZF2/ HT, but there is no change about ZF-15. Gcl-retardation clectrophoresis assays indicate that SP1-ZF2 binds to DNA sequence specifically in the presence of Zn, but SP1-ZF2 / HT docs not bind as SP 1-ZF2 did. We observed that a single zine finger like SP1-ZF2 is able to bind DNA sequence specifically.

Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating

Molecular Characterization and Expression Analysis of TaZFP15, a C_2H_2-Type Zinc Finger Transcription Factor Gene in Wheat (Triticum aestivum L.)

Based on sequencing of part clones in a root subtractive cDNA library, an expressed sequence tag （EST） sharing high similarity to a rice C2H2 zinc finger transcription factor （ZFP15） was obtained in wheat. Through bioinformatics approach, the wheat C2H2-type ZFP gene referred to TaZFP15 has been identified and characterized. As a full-length cDNA of 670 bp, TaZFP15 has an open reading frame of 408 bp and encodes a 135-aa polypeptide. TaZFP15 contains two C2H2 zinc finger domains and each one has a conserved motif QALGGH. The typical L-box, generally identified in the C2H2 type transcription factors, has also been found in TaZFP15. Phylogenetic analysis suggested that TaZFP15 shares high similarities with rice ZFP15 （GenBank accession no. AY286473）, maize ZFP （GenBank accession no. NM_001159094） and a subset of other zinc-finger transcription factor genes in plant species. The expression of TaZFP15 was up-regulated by starved-Pi stress, showing a pattern to be gradually elevated along with the progression of the Pi-stress in a 23-h treatment regime. Similarly, the transcripts of TaZFP15 in roots were also induced by nitrogen deficiency, and abiotic stresses of drought and salinity. No responses of TaZFP15 were detected in roots to nutrition deficiencies of P, Zn, and Ca, and the external treatment of abscisic acid （ABA）. TaZFP15 could be specifically amplified in genome A, B, and D, and without variability in the sequences, suggesting that TaZFP15 has multi-copies in the homologous hexaploid species. Transgenic analysis in tobacco revealed that up-regulation of TaZFP15 could significantly improve plant dry mass accumulation via increasing the plant phosphorus acquisition capacity under Pi-deficiency condition. The results suggested that TaZFP15 is involved in mediation of signal transductions of diverse external stresses.

Zinc finger structure-function in Ikaros

The zinc finger motif was used as a vehicle for the initial discovery of Ikaros in the context of T-cell differentiation and has been central to all subsequent analyses of Ikaros function.The Ikaros gene is alternately spliced to produce several isoforms that confer diversity of function and consequently have complicated analysis of the function of Ikaros in vivo.Key features of Ikaros in vivo function are associated with six C2H2 zinc fingers;four of which are alternately incorporated in the production of the various Ikaros isoforms.Although no complete structures are available for the Ikaros protein or any of its family members,considerable evidence has accumulated about the structure of zinc fingers and the role that this structure plays in the functions of the Ikaros family of proteins.This review summarizes the structural aspects of Ikaros zinc fingers,individually,and in tandem to provide a structural context for Ikaros function and to provide a structural basis to inform the design of future experiments with Ikaros and its family members.

Zinc finger protein 296 promotes hepatocellular carcinoma progression via intervening interaction between macrophages and B cells

Objective:Hepatocellular carcinoma(HCC)is a prevalent malignancy with poor survival.Different cell types in the tumor microenvironment participate in the tumorigenesis and progression of HCC.This study aimed to analyze the immune microenvironment of HCC and its relationship with clinical outcomes.Methods:We analyzed HCC RNA-seq for cell type identification and prognosis by estimating relative subsets of RNA transcripts using CIBERSORTx.The interaction between B cells and macrophages in HCC was analyzed using a Hepa1-6 orthotopic transplantation mouse model and flow cytometry.The effect of Zinc finger protein 296(ZNF296)on the interaction of B cells and macrophages was verified using human HCC tissues analyzed through western blot,quantitative real-time polymerase chain reaction(qPCR),and multiplex immunofluorescence.A comparative analysis of immune cells associated with HCC prognosis was performed using RNA-seq data from The Cancer Genome Atlas(TCGA),bulk multimodal data,and single-cell transcriptomic data from existing HCC single-cell transcriptomic data employing the Single Cell Inferred Site Specific Omics Resource for Tumor Microenvironments(SCISSOR).Results:Liver hepatocellular carcinoma(LIHC)RNA-seq analysis of TCGA showed that high eosinophil infiltration promoted HCC progression.The proportion of B cells correlated with that of macrophages(r=−0.24)and affected the infiltration and programmed death ligand 1(PD-L1)expression of macrophages in HCC.ZNF296 may participate in the interaction between B cells and macrophages to accelerate the HCC progression by regulating PAFAH1B3 and H2AFX.Moreover,ZNF296 expression positively correlated with LAG3(r=0.27)and CTLA4(r=0.31)expression levels.Among the immune cell phenotypes related to survival and death identified by SCISSOR analysis,T cells correlated with an excellent prognosis of HCC.The normal function of liver and dendritic cells was also associated with a good prognosis in HCC.Conclusions:This study analyzed the interaction of the immune microenvironment with HCC prognosis,identifying ZNF296 as a promising diagnostic and therapeutic target for HCC.

In Vivo Modeling of Zebrafish Zinc Finger,MIZ-Type Containing 1 Expression and Its Effect on Pigmentation

Objective:The zinc finger,MIZ-type containing 1(ZMIZ1)gene has been identified as a possible susceptibility gene associated with vitiligo,therefore we conducted this study to investigate the role ofZMIZ1 in pigmentation.Methods:We generate a zebrafish loss-of-function model using morpholino oligonucleotides(MOs),and two orthologs of humanZMIZ1 have been annotated(ZMIZ1a andZMIZ1b).The expression profiles of ZMIZ1a and ZMIZ1b and their effects on the pigmentation in zebrafish were evaluated by using whole-mount in situ hybridization and melanin quantification.Statistical analysis was performed using the unpaired Studentt-test or one-way analysis.Results:Investigation of the temporal and spatial expressions of these two transcripts suggested that the expressions ofZMIZ1a andZMIZ1b in the brain start to emerge in a ubiquitous fashion from 2 days post-fertilization onwards.After the successful design and validation of MOs,we observed thatZMIZ1a andZMIZ1b MOs caused embryonic developmental delays and malformations in zebrafish.Further analysis of the melanin content in the morphants revealed thatZMIZ1a significantly(49.1%for 0.667 mmol/L inZMIZI1a group,P=0.03)reduced the melanin content in a dose-dependent manner,but only the highest concentration of injectedZMIZ1b MOs significantly(50%for 0.667 mmol/L inZMIZ1b group,P=0.02)reduced the melanin content.A tyrosinase inhibition assay indicated no significant difference between the morphants and wild-type zebrafish.Conclusion:This study successfully modeled a susceptibility gene identified by genome-wide association studies in a zebrafish loss-of-function model and provides insights into the biological mechanism of pigmentation.

Phylogenetic Analysis of the Plant-specific Zinc Finger-Homeobox and Mini Zinc Finger Gene Families

Zinc finger-homeodomain proteins （ZHD） are present in many plants; however, the evolutionary history of the ZHD gene family remains largely unknown. We show here that ZHD genes are plant-specific, nearly all intronless, and related to MINI ZINC FINGER （MIF） genes that possess only the zinc finger. Phylogenetic analyses of ZHD genes from representative land plants suggest that non.seed plant ZHD genes occupy basal positions and angiosperm homologs form seven distinct clades. Several clades contain genes from two or more major angiosperm groups, including eudicots, monocots, magnoliids, and other basal angiosperms, indicating that several duplications occurred before the diversification of flowering plants. In addition, specific lineages have experienced more recent duplications. Unlike the ZHD genes, MIFs are found only from seed plants, possibly derived from ZHDs by loss of the homeodomain before the divergence of seed plants. Moreover, the MIF genes have also undergone relatively recent gene duplications. Finally, genome duplication might have contributed substantially to the expansion of family size in angiosperms and caused a high level of functional redundancy/overlap in these genes.

The C_2H_2 -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） C2H2-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 OsMPKS. 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.

A zinc finger protein, interacted with cyclophilin,affects root development via IAA pathway in rice

The plant hormone auxin plays a crucial role in lateral root development. To better understand the rnolecular mechanisms underlying lateral root formation, an auxin-responsive gene OsCYP2 （Oso2g0121300） was characterized from rice. Compared to the wild type, OsCYP2-RNAi （RNA interference） lines exhibited distinctive defects in lateral root development. Yeast two-hybrid and glutathione S-transferase puIl-down results confirmed that OsCYP2 interacted with a C2HC-type zinc finger protein （OsZFP, O501g0252900） which is located in the rice nucleus. T2OsZFP-RNAi lines had significantly fewer lateral roots than did wild-type plants, which suggests a role for OsCYP2 and OsZFP in regulating lateral root development.Quantitative real-time polymerase chain reaction showed that the expression of certain Aux/IAA （auxin/indole-3- acetic acid） genes was altered in OsCYP2- and OsZFP-RNAi lines in response to IAA. These findings imply that OsCYP2 and OsZFP participate in IAA signal pathways controlling lateral root development. More importantly, OslAA11 showed functional redundancy not only in OsCYP2-RNAi lines but also in OsZFP-RNAi lines, which provides important clues for the elucidation of mechanisms controlling lateral root development in response to auxin.

Potential application of FoldX force field based protein modeling in zinc finger nucleases design

Engineered sequence-specific zinc finger nucleases (ZFNs) make the highly efficient modification of eukaryotic genomes possible.However,most current strategies for developing zinc finger nucleases with customized sequence specificities require the construction of numerous tandem arrays of zinc finger proteins (ZFPs),and subsequent largescale in vitro validation of their DNA binding affinities and specificities via bacterial selection.The labor and expertise required in this complex process limits the broad adoption of ZFN technology.An effective computational assisted design strategy will lower the complexity of the production of a pair of functional ZFNs.Here we used the FoldX force field to build 3D models of 420 ZFP-DNA complexes based on zinc finger arrays developed by the Zinc Finger Consortium using OPEN (oligomerized pool engineering).Using nonlinear and linear regression analysis,we found that the calculated protein-DNA binding energy in a modeled ZFP-DNA complex strongly correlates to the failure rate of the zinc finger array to show significant ZFN activity in human cells.In our models,less than 5% of the three-finger arrays with calculated protein-DNA binding energies lower than 13.132 kcal mol 1 fail to form active ZFNs in human cells.By contrast,for arrays with calculated protein-DNA binding energies higher than 5 kcal mol 1,as many as 40% lacked ZFN activity in human cells.Therefore,we suggest that the FoldX force field can be useful in reducing the failure rate and increasing efficiency in the design of ZFNs.

Involvement of C2H2 zinc finger proteins in the regulation of epidermal cell fate determination in Arabidopsis

Cell fate determination is a basic developmental process during the growth of multicellular organisms.Trichomes and root hairs of Arabidopsis are both readily accessible structures originating from the epidermal cells of the aerial tissues and roots respectively, and they serve as excellent models for understanding the molecular mechanisms controlling cell fate determination and cell morphogenesis. The regulation of trichome and root hair formationis a complex program that consists of the integration of hormonal signals with a large number of transcriptional factors, including MYB and b HLH transcriptional factors.Studies during recent years have uncovered an important role of C2H2 type zinc finger proteins in the regulation of epidermal cell fate determination. Here in this minireview we briefly summarize the involvement of C2H2 zinc finger proteins in the control of trichome and root hair formation in Arabidopsis.