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Background: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation. Methods and results: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1's function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells. Conclusions: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity. [ABSTRACT FROM AUTHOR]

Background: LncRNA DLX6-AS1 has been uncovered to exert effects on various cancers. Nevertheless, the impacts of DLX6-AS1 on endometrial cancer (EC) development remained obscure. The study explored the influence of DLX6-AS1 on EC progression via the microRNA (miR)-374a-3p/zinc-finger protein (ZFX) axis. Methods: EC cell lines were collected and DLX6-AS1, miR-374a-3p, and ZFX levels in EC cell lines were detected. The EC cells were transfected with DLX6-AS1, miR-374a-3p, and ZFX constructs to examine the biological functions of EC cells. The xenograft model was established for detecting tumor growth. Rescue experiments were conducted to verify the interaction of DLX6-AS1, miR-374a-3p, and ZFX in EC cells. Results: DLX6-AS1 and ZFX levels were elevated, while miR-374a-3p exhibited a reduced level in EC cells. Silencing DLX6-AS1 and elevated miR-374a-3p expressions repressed the biological activities of EC cells. Reduced DLX6-AS1 repressed tumor development. MiR-374a-3p silencing reversed the impacts of DLX6-AS1 silencing, while ZFX overexpression abrogated the impacts of miR-374a-3p elevation on EC cell growth. Mechanically, DLX6-AS1 was found to bind to miR-374a-3p, and miR-374a-3p targeted ZFX. Conclusion: DLX6-AS1 depletion restricts the malignant phenotype of EC cells. The study might provide novel therapeutic biomarkers for EC treatment. [ABSTRACT FROM AUTHOR]

Drought-induced 19 (Di19) protein is a Cys2/His2 (C2H2) type zinc-finger protein, which plays a crucial role in plant development and in response to abiotic stress. This study systematically investigated the characteristics of the GhDi19 gene family, including the member number, gene structure, chromosomal distribution, promoter cis-elements, and expression profiles. Transcriptomic analysis indicated that some GhDi19s were up-regulated under heat and salt stress. Particularly, two nuclear localized proteins, GhDi19-3 and GhDi19-4, were identified as being in potential salt stress responsive roles. GhDi19-3 and GhDi19-4 decreased sensitivity under salt stress through virus-induced gene silencing (VIGS), and showed significantly lower levels of H2O2, malondialdehyde (MDA), and peroxidase (POD) as well as significantly increased superoxide dismutase (SOD) activity. This suggested that their abilities were improved to effectively reduce the reactive oxygen species (ROS) damage. Furthermore, certain calcium signaling and abscisic acid (ABA)-responsive gene expression levels showed up- and down-regulation changes in target gene-silenced plants, suggesting that GhDi19-3 and GhDi19-4 were involved in calcium signaling and ABA signaling pathways in response to salt stress. In conclusion, GhDi19-3 and GhDi19-4, two negative transcription factors, were found to be responsive to salt stress through calcium signaling and ABA signaling pathways. [ABSTRACT FROM AUTHOR]

Key message: Overexpression of OsiSAP8 driven by Port Ubi2.3 from Porteresia coarctata imparts drought and salinity stress tolerance in transgenic rice. Stress associated proteins (SAPs) possess the zinc-finger domains that are wildly evolving functional and conserved regions/factors in plants to combat abiotic stresses. In this study, the promoter region of OsiSAP8, an intron-less, multiple stress inducible gene, was compared in silico with a strong constitutive promoter, Port Ubi2.3. This resulted in developing rice, resistant to drought and salinity expressing OsiSAP8 promoted by Port Ubi2.3. (Porteresia coarctata), through Agrobacterium-mediated transformation in the popular rice varieties, IR36 and IR64. Southern blot hybridization confirmed the integration of OsiSAP8, and the T0 transgenic lines of IR36 and IR64 were evaluated for their drought and salinity tolerance. The IR36-T1 progenies showed an enhanced tolerance to water withhold stress compared to wild type and IR64-T1 progenies. Physiological parameters, such as the panicle weight, number of panicles, leaf wilting, and TBARS assay, showed the transgenic IR36 to be superior. The transgenic lines performed better with higher 80–95% relative leaf water content when subjected to drought for 14 days. Gene expression analysis of OsiSAP8 in IR36 T1 showed a 1.5-fold upregulation under mannitol stress. However, IR64 T1 showed a two-fold upregulation in NaCl stress. An enhanced drought and salinity stress tolerance in the transgenic IR36 cultivar through overexpression of OsiSAP8 was observed as it had a native copy of OsiSAP8. This is perhaps the first study using a novel ubiquitin promoter (Port Ubi2.3) to generate drought and salinity stress-tolerant transgenic rice. Thus, we report the overexpression of a rice gene (OsiSAP8) by a rice promoter (Port Ubi2.3) in rice (IR36) to resist drought and salinity. [ABSTRACT FROM AUTHOR]

Plant zinc‐finger proteins play a crucial role in biosynthesis and plant development. However, it is not known whether certain zinc‐finger proteins play a role in rice chloroplast development.In this study, a novel rice zinc‐finger protein mutant asl4 (albino seedling lethality4), which exhibits an albino lethal phenotype at the seedling stage, was used. Chlorophyll fluorescence analysis and TEM were used to investigate features of the asl4 mutant. The genetic behaviour and function of ASL4 gene were then analysed thorough map‐based cloning, transgenic complement and subcellular localization.The albino lethal phenotype was caused by a single nucleotide (G*) deletion mutation on the exon of the ASL4 (LOC_Os09g21710) gene. The ASL4 gene encoded a novel zinc‐finger protein containing two ZnF‐AN1 domains, which was localized to the nucleocytoplasm. The ASL4 transcripts were highly expressed in all leaves but relatively less in other tissues, suggesting its tissue‐specific expression. The transcript levels of associated genes for Chl biosynthesis, photosynthesis and chloroplast development were severely suppressed in asl4 mutants.In conclusion, the absence of ASL4 function caused a defect in chloroplast development and seedling lethality. This is the first published report on the importance of the ZnF‐AN1 type zinc‐finger protein gene in chloroplast development in rice. [ABSTRACT FROM AUTHOR]

Trichomes are unicellular or multicellular epidermal structures that play a defensive role against environmental stresses. Although unicellular trichomes have been extensively studied as a mechanistic model, the genes involved in multicellular trichome formation are not well understood. In this study, we first classified the trichome morphology structures in Capsicum species using 280 diverse peppers. We cloned a key gene (Hairiness) on chromosome 10, which mainly controlled the formation of multicellular non-glandular trichomes (types II, III, and V). Hairiness encodes a Cys2-His2 zinc-finger protein, and virus-induced gene silencing of the gene resulted in a hairless phenotype. Differential expression of Hairiness between the hairiness and hairless lines was due to variations in promoter sequences. Transgenic experiments verified the hypothesis that the promoter of Hairiness in the hairless line had extremely low activity causing a hairless phenotype. Hair controlled the formation of type I glandular trichomes in tomatoes, which was due to nucleotide differences. Taken together, our findings suggest that the regulation of multicellular trichome formation might have similar pathways, but the gene could perform slightly different functions in crops. [ABSTRACT FROM AUTHOR]

We report for the first time a microfluidic electrophoretic mobility reversal assay (MEMRA) for double-stranded DNA (dsDNA) detection using zinc-finger proteins (ZFPs) and a polyacrylamide-gel (PAG) sieving matrix. Microfluidic DNA analysis was actively studied because of its importance in biology and medicine. Most microfluidic DNA detection techniques rely on time-consuming denaturation and hybridization processes. To address this limitation, ZFP was employed as a novel affinity probe, which directly binds to a specific sequence of dsDNA without denaturation and renaturation. A mildly alkaline electrophoresis buffer (pH 8.6) was used for our MEMRA, instead of a strongly alkaline buffer (pH 10.75) for separating the ZFP–dsDNA complex from interfering species. At pH 8.6, the mobility of ZFP was reversed upon binding with dsDNA (complex pI = ~ 5.33), and unbound ZFP (pI = ~ 9.3) was excluded from loading. Therefore, the ZFP–dsDNA complex was detected without zone interferences. Furthermore, nonspecific interactions and band dispersion, observed in strongly alkaline buffer, were effectively mitigated in the MEMRA. The ZFP–dsDNA complex was fully separated (separation resolution ≥ 2.0) and detected rapidly (12–15 s at a separation distance of 160–240 μm) using on-chip photopatterned 3–16%T discontinuous PAG. The MEMRA performance was excellent, providing a detection limit of 50 pM and a detection range of 100 pM–500 nM for seb (Staphylococcus enterotoxin B) gene dsDNA oligonucleotides. We expect that our ZFP-based MEMRA will find broad utility in biology and medicine where the rapid, specific, and quantitative detection of dsDNA is of paramount importance. [ABSTRACT FROM AUTHOR]

Trichomes are unicellular or multicellular epidermal structures that play a defensive role against environmental stresses. Although unicellular trichomes have been extensively studied as a mechanistic model, the genes involved in multicellular trichome formation are not well understood. In this study, we first classified the trichome morphology structures in Capsicum species using 280 diverse peppers. We cloned a key gene (Hairiness) on chromosome 10, which mainly controlled the formation of multicellular non-glandular trichomes (types II, III, and V). Hairiness encodes a Cys2-His2 zinc-finger protein, and virus-induced gene silencing of the gene resulted in a hairless phenotype. Differential expression of Hairiness between the hairiness and hairless lines was due to variations in promoter sequences. Transgenic experiments verified the hypothesis that the promoter of Hairiness in the hairless line had extremely low activity causing a hairless phenotype. Hair controlled the formation of type I glandular trichomes in tomatoes, which was due to nucleotide differences. Taken together, our findings suggest that the regulation of multicellular trichome formation might have similar pathways, but the gene could perform slightly different functions in crops.

Background: ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown. Methods: To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways. Results: ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27S82 and eIF2aS51, and triggers autophagosome formation in lung cancer cells. Conclusions: These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy. [ABSTRACT FROM AUTHOR]

DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA‐binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two‐hybrid screen to discover new Claspin‐binding proteins. This system allowed us to identify the zinc‐finger protein OZF (ZNF146) as a new Claspin‐interacting protein. OZF is also present at replication forks and co‐immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin‐binding protein with a specific function in fork progression under replication stress. [ABSTRACT FROM AUTHOR]

A highly efficient lycopene production system was constructed by assembling enzymes fused to zinc-finger motifs on DNA scaffolds in vitro and in vivo. Three key enzymes of the lycopene synthesis pathway, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, were fused with zinc-finger proteins, expressed and purified. Recombinant plasmids of the pS series containing DNA scaffolds that the zinc-finger proteins can specifically bind to were constructed. In the in vitro system, the production efficiency of lycopene was improved greatly after the addition of the scaffold plasmid pS231. Subsequently, the plasmid pET-AEBI was constructed and introduced into recombinant Escherichia coli BL21 (DE3) for expression, together with plasmids of the pS series. The lycopene production rate and content of the recombinant strain pp231 were higher than that of all strains carrying the DNA scaffold and the control. With the addition of cofactors and substrates in the lycopene biosynthesis pathway, the lycopene yield of pp231 reached 632.49 mg/L at 40 h, representing a 4.7-fold increase compared to the original recombinant strain pA1A3. This DNA scaffold system can be used as a platform for the construction and production of many biochemicals synthesized via multi-enzyme cascade reactions. [ABSTRACT FROM AUTHOR]

Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-β/BMP. It plays important roles in epithelial and germ cell development, particularly by repressing c-Myc and Id2 genes and modulating the balance between proliferation and differentiation of progenitor cells. In this study, we show that Ovol1 negatively regulates its own expression by binding to and repressing the activity of its promoter. We further demonstrate that Ovol1 uses both passive and active repression mechanisms to auto-repress: (1) it antagonizes transcriptional activation of c-Myb, a known positive regulator of proliferation, by competing for DNA binding; (2) it recruits histone deacetylase activity to the promoter via an N-terminal SNAG repressor domain. At Ovol1 cognate sites in the endogenous Ovol1 promoter, c-Myb binding correlates with increased histone acetylation, whereas the expression of Ovol1 correlates with a displacement of c-Myb from the DNA and decreased histone acetylation. Collectively, our data suggest that Ovol1 restricts its own expression by counteracting c-Myb activation and histone acetylation of the Ovol1 promoter.

Homoharringtonine (HHT) and imatinib have a synergistic effect in the clinical treatment of chronic myeloid leukemia (CML). The purpose of the present study was to explore the underlying mechanisms by which HHT enhanced imatinib sensitivity. K562 CML cells were treated with HHT and imatinib separately or in combination. Cell viability was detected by Cell Counting Kit-8 assay; apoptotic rates and protein expression levels of phosphorylated-tyrosine (p-Tyr) and p-CRK like proto-oncogene, adaptor protein (p-Crkl) were analyzed by flow cytometry; zinc-finger protein, X-linked (ZFX) overexpression plasmid was transfected to cells using electroporation; western blotting was used to detect the protein expression levels of PI3K, AKT, p-AKT and ZFX; and reverse transcription-quantitative PCR was used to measure ZFX mRNA expression levels. The results demonstrated that HHT and imatinib co-treatment had significant effects of proliferation inhibition and apoptosis induction on K562 CML cells compared with imatinib alone. Co-treatment also significantly downregulated the expression levels of p-Tyr, p-Crkl, PI3K and p-Akt compared with imatinib or HHT treatment. In addition, HHT downregulated ZFX mRNA and protein expression. ZFX overexpression reversed cell sensitivity to imatinib and HHT and also reduced the HHT-induced imatinib sensitization by increasing p-Akt expression. In conclusion, HHT may enhance the effect of imatinib on CML cells by downregulating ZFX. [ABSTRACT FROM AUTHOR]

Two proteins required for the growth of a skin-like structure called the tegument in parasitic flatworms could be new targets for drugs to kill these parasites.

Aluminum (Al) and proton (H+) ions are major acid soil stress factors deleteriously affecting plant root growth and crop yield. In our preliminary studies, cotton (Gossypium hirsutum L.) seedlings displayed very sensitive phenotypes to Al and H+ rhizotoxicities.Based on previous Arabidopsis results, we aimed to characterise the Al stress responsive Sensitive to Proton rhizotoxicity 1 (GhSTOP1) transcription system in cotton using RNAi‐mediated down‐regulation. With the help of seed embryo apex explants, we developed transgenic cotton plants overexpressing a GhSTOP1‐RNAi cassette with NPTII selection. Kanamycin‐tolerant T1 seedlings were further considered for Al and H+ stress tolerance studies.Down‐regulation of the GhSTOP1 increased sensitivity to Al and proton rhizotoxicities, and root growth was significantly reduced in RNAi lines. The expression profile of GhALMT1 (Aluminum‐activated Malate Transporter 1), GhMATE (Multidrug and Toxic Compound Extrusion), GhALS3 (Aluminium Sensitive 3) and key genes involved in the GABA shunt were down‐regulated in the transgenic RNAi lines. Additionally, the lateral root initiation process was delayed and expression of GhNAC1, which is involved in lateral roots, was also suppressed in transgenic lines. Besides, overexpression of GhSTOP1 in Arabidopsis accelerated root growth and AtMATE and AtALMT1 expression under Al stress conditions.These analyses indicate that GhSTOP1 is essential for the expression of several genes which are necessary for acid soil tolerance mechanisms and lateral root initiation. [ABSTRACT FROM AUTHOR]

Summary: Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome‐wide association study (GWAS) on type‐I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map‐based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc‐finger protein. Transgenic experiments showed that hair‐absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type‐I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein–protein interaction between them was further detected through pull‐down and yeast two‐hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species. [ABSTRACT FROM AUTHOR]

Key message: DownregulatingPagSAP1expression increases salt tolerance in poplar and leads to the alterations in the regulation of genes involved in maintaining cellular ionic homeostasis.Abstract: Plants have evolved defense mechanisms to cope with unfavorable climate conditions such as salinity, drought, and extreme temperatures. Such adaptive strategies include fine-tuning of stress-associated proteins (SAPs). Here, we investigated the molecular and physiological characteristics of the novel gene PagSAP1 from hybrid poplar (Populus alba × P. glandulosa) in response to salt stress. PagSAP1 encodes a putative protein containing an A20 and AN1 zinc-finger domain at its N- and C-terminus, respectively. Salt stress significantly downregulated the expression of PagSAP1 in roots, but not leaves, of poplar at the seven-leaf stage. Compared to control plants, knockdown (RNAi) transgenic poplar plants showed strong tolerance to salt stress, while overexpression (OX) of PagSAP1 increased salt sensitivity. Analysis of Ca2+, Na+, and K+ contents in roots and leaves following salt treatment (150 mM NaCl) revealed that RNAi plants accumulated more Ca2+ and K+, and less Na+ than PagSAP1 OX plants. The RNAi lines exhibited increased expression of genes involved in maintaining cellular ionic homeostasis across the plasma membrane, such as salt overly sensitive 3 (SOS3), SOS1, high-affinity K+ transporter 1 (HKT1), H+-ATPase, AAA-type ATPase, and Arabidopsis K+ channel 2 (AKT2). Taken together, these results indicate that PagSAP1 represents a promising candidate gene for engineering trees with improved salt tolerance, which would increase their suitability for planting in marginal lands, such as reclaimed areas. [ABSTRACT FROM AUTHOR]

During early vertebrate embryogenesis, bone morphogenetic proteins (BMPs) belonging to the transforming growth factor‐β (TGF‐β) family of growth factors play a central role in dorsal–ventral (DV) patterning of embryos, while other growth factors such as Wnt and fibroblast growth factor (FGF) family members regulate formation of the anterior–posterior (AP) axis. Although the establishment of body plan is thought to require coordinated formation of the DV and AP axes, the mechanistic details underlying this coordination are not well understood. Here, we show that a Xenopus homologue of zbtb14 plays an essential role in the regulation of both DV and AP patterning during early Xenopus development. We show that overexpression of Zbtb14 promotes neural induction and inhibits epidermal differentiation, thereby regulating DV patterning. In addition, Zbtb14 promotes the formation of posterior neural tissue and suppresses anterior neural development. Consistent with this, knock‐down experiments show that Zbtb14 is required for neural development, especially for the formation of posterior neural tissues. Mechanistically, Zbtb14 reduces the levels of phosphorylated Smad1/5/8 to suppress BMP signaling and induces an accumulation of β‐Catenin to promote Wnt signaling. Collectively, these results suggest that Zbtb14 plays a crucial role in the formation of DV and AP axes by regulating both the BMP and Wnt signaling pathways during early Xenopus embryogenesis. [ABSTRACT FROM AUTHOR]

The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc‐finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well‐known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat‐stable protein. Overall, the results of this study suggest that MucR might resemble a H‐NS protein. [ABSTRACT FROM AUTHOR]

Aspergillus flavus aswA (AFLA_085170) is a gene encoding a Zn(II) 2 Cys 6 DNA-binding domain and a transcriptional activation domain, DUF3468. Disruption of aswA yielded strains that made a truncated gene transcript and generated a fungus that produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (white) and different from oval and pigmented (dark brown to black) mature sclerotia. Transcriptomic analysis of the Δ aswA strain grown on potato dextrose agar plates and Wickerham agar plates showed that expression of clustering genes involved in the biosynthesis of three sclerotium-associated secondary metabolites was down-regulated. These included gene clusters of asparasone, aflatrem, and aflavarin. In contrast, those of aflatoxin, cyclopiazonic acid and kojic acid were not affected. Metabolite analyses confirmed that the non-pigmented sclerotia contained aflatoxin and cyclopiazonic acid but not other aforementioned metabolites, three asparasone analogs and dihydroxyaflavinine commonly present in mature sclerotia. Impairment in aswA gene function stalls normal sclerotial development, which in turn prevents biosynthesis and accumulation of sclerotium-specific metabolites. [ABSTRACT FROM AUTHOR]

Current studies using knockout mice have revealed that some Cys-Cys-Cys-His (CCCH)-type zinc-finger proteins, namely tristetraprolin (TTP), Roquin and Regnase-1, play important roles in the immune system. These proteins are closely associated with the fate of their target RNAs in normal immune responses. However, the functions of many RNA-binding proteins have not been characterized precisely. To understand the molecular mechanisms of RNA metabolism in the immune system, investigation of TTP/Roquin/Regnase-1 might provide new knowledge. In this review, we will discuss the current understanding of these proteins in immune regulation and homeostasis and discuss RNA metabolism in the immune system. [ABSTRACT FROM AUTHOR]

Key Message: OsiSAP1, an A20/AN1 zinc-finger protein, confers water-deficit stress tolerance at different stages of growth by affecting expression of several endogenous genes in transgenic rice. Abstract: Transgenic lines have been generated from rice constitutively expressing OsiSAP1, an A20/AN1 zinc-finger containing stress-associated protein gene from rice, driven by maize UBIQUITIN gene promoter and evaluated for water-deficit stress tolerance at different stages of growth. Their seeds show early germination and seedlings grow better under water-deficit stress compared to non-transgenic (NT) rice. Leaves from transgenic seedlings showed lesser membrane damage and lipid peroxidation under water-deficit stress. Relatively lower rate of leaf water loss has been observed in detached intact leaves from transgenic plants during late vegetative stage. Delayed leaf rolling and higher relative water content were also observed in transgenic plants under progressive water-deficit stress during reproductive developmental stage. Although reduction in grain yield is observed under unstressed condition, the relative water-deficit stress-induced yield losses are lower in transgenic rice vis-à-vis NT plants thereby resulting in yield loss protection. Transcriptome analysis suggests that overexpression of OsiSAP1 in transgenic rice results in altered expression of several endogenous genes including those coding for transcription factors, membrane transporters, signaling components and genes involved in metabolism, growth and development. A total of 150 genes were found to be more than twofold up-regulated in transgenic rice of which 43 genes are known to be involved in stress response. Our results suggest that OsiSAP1 is a positive regulator of water-deficit stress tolerance in rice. [ABSTRACT FROM AUTHOR]

Intestinal stemcells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair.We used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators.A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division defects, including loss of EdU incorporation, phosphorylated histone 3 staining and expression of the mitotic protein Cdc2. Loss of Charlatan also led to a much reduced histone acetylation staining in precursor cells. Both the histone acetylation and ISC division defects could be rescued by the simultaneous decrease of the Histone Deacetylase 2. The overexpression ofCharlatan blocked differentiation reversibly, but loss ofCharlatan did not lead to automatic differentiation. The results together suggest that Charlatan does not simply act as an anti-differentiation factor but instead functions to maintain a chromatin structure that is compatible with stem cell properties, including proliferation. [ABSTRACT FROM AUTHOR]

Thousands of genes in Drosophila have Pol II paused in the promoter proximal region. Almost half of these genes are associated with either GAGA factor (GAF) or a newly discovered factor we call M1BP. Although both factors dictate the association of Pol II at their target promoters, they are nearly mutually exclusive on the genome and mediate different mechanisms of regulation. High-resolution mapping of Pol II using permanganate-ChIP-seq indicates that pausing on M1BP genes is transient and could involve the +1 nucleosome. In contrast, pausing on GAF genes is much stronger and largely independent of nucleosomes. Distinct regulatory mechanisms are reflected by transcriptional plasticity: M1BP genes are constitutively expressed throughout development while GAF genes exhibit much greater developmental specificity. M1BP binds a core promoter element called Motif 1. Motif 1 potentially directs a distinct transcriptional mechanism from the canonical TATA box, which does not correlate with paused Pol II on the genomic scale. In contrast to M1BP and GAF genes, a significant portion of TATA box genes appear to be controlled at preinitiation complex formation. [ABSTRACT FROM AUTHOR]

Abstract: Fumonisins, toxic secondary metabolites produced by some Fusarium spp. and Aspergillus niger, have strong agro-economic and health impacts. The genes needed for their biosynthesis, named FUM, are clustered and co-expressed in fumonisin producers. In eukaryotes, coordination of transcription can be attained through shared transcription factors, whose specificity relies on the recognition of cis-regulatory elements on target promoters. A bioinformatic analysis on FUM promoters in the maize pathogens Fusarium verticillioides and Aspergillus niger identified a degenerated, over-represented motif potentially involved in the cis-regulation of FUM genes, and of fumonisin biosynthesis. The same motif was not found in various FUM homologues of fungi that do not produce fumonisins. Comparison of the transcriptional strength of the intact FUM1 promoter with a synthetic version, where the motif had been mutated, was carried out in vivo and in planta for F. verticillioides. The results showed that the motif is important for efficient transcription of the FUM1 gene. [Copyright &y& Elsevier]

Background: Soybean is an important oilseed crop that its development and production are affected by environmental stresses (such as saline-alkaline and water deficit)., Objectives: This experiment was performed with the aim of identifying candidate genes in saline-alkaline stress and water-deficit stress conditions using transcriptome analysis and to investigate the expression of these genes under water deficit stress conditions using RTqPCR., Materials and Methods: In this experiment, soybean transcriptome data under saline-alkaline and water-deficit stress were downloaded from the NCBI website, and then the co-expression modules were determined for them and the gene network was plotted for each module, and finally, the hub genes were identified. To compare the expression of genes in saline-alkaline and water deficit conditions, soybean plants were subjected to water deficit stress and their gene expression was determined using RTqPCR., Results: The filtered (Log FC above +2 and below -2) genes of soybean were grouped under saline-alkaline stress in 15 modules and under water-deficit stress in 2 different modules. Within each module, the interaction of genes was identified using the gene network, then three genes of ann11 , cyp450 and zfp selected as hub genes. These hub genes are highly co-expression with other network genes, which not only display differential expression but also differential co-expression. The results of RT-PCR indicated that cyp450 gene expression was not significantly different from the control, while ann11 gene expression significantly increased under water deficit stress, but zfp gene expression decreased significantly under water deficit stress., Conclusions: We identified three genes, ann11 , cyp450 and zfp , as hub genes. According to our results, ann11 gene had a significant increase in expression under water deficit stress, which can indicate the importance of this gene under drought conditions. Therefore, according to the results of this experiment as well as other researchers, we introduce this gene as a key gene in water deficit tolerance and recommend its use in genetic engineering to increase the tolerance of other plants., (Copyright: © 2021 The Author(s); Published by Iranian Journal of Biotechnology.)

Soybean ( Glycine max L.) is a typical short-day crop, and its flowering is strictly restricted by specific photoperiod conditions. CONSTANS ( CO) plays a pivotal role in the photoperiod pathway of flowering regulation. CO-like genes are present in many plant species. Here we describe the isolation of the CO homologue GmCOL11 ( Glycine max CO-like 11) from the soybean cv. Kennong 18. Sequence comparisons show that GmCOL11 is a group II CO-like gene with some similarity to AtCOL6 and AtCOL16. Its sequence includes a conserved B box and a CCT domain. The study of GmCOL11 expression using quantitative real time RT-PCR demonstrated that this gene was regulated in a diurnal rather than in a circadian manner. The gene was expressed throughout the plant, but mainly in adult leaves and maturing seeds; its expression was enhanced following flowering. Apparently GmCOL11 is involved in several aspects of soybean development. [ABSTRACT FROM AUTHOR]

Abstract: The interactions of a zinc-finger (ZF) protein with DNA containing the specific recognition site of the ZF and with a non-specific DNA were studied with the semi-empirical quantum chemical method of PM6/Mozyme. The ZF protein (1MEY)-DNA complex structures were generated by docking calculations. The complex structures were reoptimized with the PM6/Mozyme method with implicit solvation in water. The structures were also calculated in the gas phase. The interaction enthalpies between the protein and DNA within the complexes obtained in the PM6/Mozyme with solvation optimized structures were calculated with the single-point PM6-DH2/Mozyme method (PM6 with dispersion, H-bond correction and Mozyme) with solvation. The results supported the specific and non-specific interactions in the complexes obtained from the docking experiments. The binding enthalpies of the specific and non-specific DNA binding to the protein differed significantly. The interactions between the nucleic acid strands in duplexes were also evaluated; these interactions between the base pairs were different because of the different “G…C:A…T” ratios in the DNA molecules studied. The stacking interactions between the nucleic bases were also characterized in the DNA duplexes. [Copyright &y& Elsevier]

Repression of vascular endothelial growth factor A (VEGF-A) is an attractive approach to cancer therapy. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF- A and constitutive expressions of ATFs were previously shown to downregulate the endogenous VEGF-A gene expression, repression of VEGF-A specifically in hypoxic tumors is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-driven expression of the ATF for hypoxia-specific repression of human VEGF-A gene. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding region. The resulting hypoxia-driven expression plasmids induced the expression of ATFs specifically in hypoxia, and the hypoxia-specific expression of ATFs effectively downregulated the VEGF-A expression in hypoxia, but not in normoxia. Thus, the engineered expression system of ATFs may enable repression of VEGF-A expression specifically in hypoxic tumors without affecting normal, healthy tissues. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 112, 1035–1044. To identify a novel regulatory factor involved in brain development or synaptic plasticity, we applied the differential display PCR method to mRNA samples from NMDA-stimulated and un-stimulated neocortical cultures. Among 64 cDNA clones isolated, eight clones were novel genes and one of them encodes a novel zinc-finger protein, HIT-4, which is 317 amino acid residues (36–38 kDa) in length and contains seven C2H2 zinc-finger motifs. Rat HIT-4 cDNA exhibits strong homology to human ZNF597 (57% amino acid identity and 72% homology) and identity to rat ZNF597 at the carboxyl region. Furthermore, genomic alignment of HIT-4 cDNA indicates that the alternative use of distinct promoters and exons produces HIT-4 and ZNF597 mRNAs. Northern blotting revealed that HIT-4 mRNA (∼6 kb) is expressed in various tissues such as the lung, heart, and liver, but enriched in the brain, while ZNF597 mRNA (∼1.5 kb) is found only in the testis. To evaluate biological roles of HIT-4/ZNF597, targeted mutagenesis of this gene was performed in mice. Homozygous (−/−) mutation was embryonic lethal, ceasing embryonic organization before cardiogenesis at embryonic day 7.5. Heterozygous (+/−) mice were able to survive but showing cell degeneration and vacuolization of the striatum, cingulate cortex, and their surrounding white matter. These results reveal novel biological and pathological roles of HIT-4 in brain development and/or maintenance. [ABSTRACT FROM AUTHOR]

Abstract: Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding regions. The resulting hypoxia-driven expression plasmid induced the ATF expression in hypoxia but not in normoxia, and the hypoxia-specific expression of the ATF activated the VEGF-A expression specifically in hypoxia. Thus, the engineered expression system of ATFs may enable activation of VEGF-A expression specifically in ischemic tissues without affecting normal, healthy tissues in vivo. [Copyright &y& Elsevier]

Abstract: ZFAT (also known as ZNF406), originally identified as a candidate gene for autoimmune thyroid disease, encodes a zinc-finger protein, however, its function has not been elucidated. Here, we report that human ZFAT protein is expressed in peripheral B and T lymphocytes and a human acute T lymphoblastic leukaemia cell line, MOLT-4 cells. Intriguing is that mouse ZFAT expression in CD4+ lymphocytes is increased during blast formation. Furthermore, ZFAT-knockdown in MOLT-4 induces apoptosis via activation of caspases. These results suggested that ZFAT protein is a critical regulator involved in apoptosis and cell survival for immune-related cells. [Copyright &y& Elsevier]

Abstract: LORIEN (encoding a protein that contains a SP-RING/Miz zinc-finger motif present in a group of proteins involved in the Small Ubiquitin-related Modifier -SUMO- conjugation pathway) and MAT2 (encoding the methionine adenosyltransferase -MAT-) genes are arranged as two alternating copies in a head-to-tail configuration, with the LORIEN gene as the first copy of the cluster. The 5880bp preceding the first LORIEN gene copy were compared to the same region of L. major, showing a 93% identity between them. Bioinformatic analysis of this region predicted the presence of a 747-bp ORF encoding a hypothetical protein of 248 amino acids. Transcription of this ORF was confirmed by run-on assays and RT-PCR. Expression of the LORIEN gene was tested in both the promastigote and amastigote stages. Transcription arrest evidenced that LORIEN mRNA stability was very similar in both stages of the parasite life cycle. Protein synthesis inhibition by cycloheximide led to an increase in the steady-state levels of LORIEN transcripts only during the promastigote stage, pointing out to the existence of different stage-dependent mechanisms operating on the post-transcriptional regulation of this gene. The role of the LORIEN untranslated regions (5′UTR and 3′UTR) in post-transcriptional regulation was analysed using the luciferase (luc) reporter gene. Results evidenced that the 5′UTR was responsible for a low reporter gene expression, whereas the intergenic region (IR) between LORIEN and MAT2 genes provided high luc levels. However, the 3′UTR seemed to lack regulatory elements. Basing on these results, a model of regulation for the LORIEN gene is proposed. [Copyright &y& Elsevier]

Abstract: The CCCH class of zinc fingers occurs in a large number of Arabidopsis proteins. Previous studies revealed that one such protein is a nuclease, the activity of which is attributable to one of the CCCH motifs. To examine whether nuclease activity is a more general characteristic of CCCH zinc finger containing proteins, five other such Arabidopsis proteins were assayed for a similar activity. The results indicate that all of these proteins possess nuclease activity. Thus, nuclease activity may be a common characteristic of Arabidopsis CCCH-containing proteins. [Copyright &y& Elsevier]

The Populus euphratica stress responsive zinc-finger protein gene PSTZ, which encodes a protein including typical Cys2/His2 zinc finger structure, was isolated by reverse transcription-polymerase chain reaction from P. euphratica. Northern hybridization revealed that its expression was induced under drought and salt stress conditions. To examine its function, cDNA of the PSTZ gene, driven by the cauliflower mosaic virus 35S promoter, was cloned into a plant expression vector pBin438 and introduced into tobacco plants. Transgenic tobacco showed an enhanced salt tolerance, suggesting that PSTZ may play a role in plant responsiveness to salt stress. [ABSTRACT FROM AUTHOR]

Abstract: WT1 was first described in 1990 as a tumour suppressor gene associated with Wilms tumour (nephroblastoma). It encodes a typical transcription factor with four C2–H2 zinc fingers in the C-terminus. However WT1 is surprisingly complex at multiple levels: it is involved in the development of several organ systems; and is both a tumour suppressor and oncogene. Here we review evidence that has accumulated over the past decade to suggest that as well as binding DNA, WT1 also binds mRNA targets via its zinc fingers and interacts with several splice factors. WT1''s first reported post-transcriptional function is also reviewed. WT1''s complex roles in development and disease now need to be understood in terms of both DNA and mRNA targets. [Copyright &y& Elsevier]