Zinc Finger-like Motif Research Articles

Genome-wide characterization and expression analysis of WRKY transcription factors reveals biotic stress response potential mechanisms in Panax notoginseng

WRKY transcription factors (TFs), among the largest families of transcriptional regulators, are essential players in defense responses in plants. Panax notoginseng, a traditional and precious herbal medicine in China, is widely cultivated due to its excellent medicinal and edible value. However, P. notoginseng is frequently affected by various diseases (like Black blot disease, Root rot disease and Viral diseases). Therefore, the present study aimed to identify and characterize PnWRKYs in the P. notoginseng genome, reveal their structural attributes, regulatory elements, and potential functions in defense responses. 68 PnWRKY TFs that have conserved domains and different physicochemical characteristics were identified in the genome. They can be divided into four groups according to phylogenetic relationships and conserved domain structures. Furthermore, Group II WRKY TFs can be classified into five subgroups (IIa, IIb, IIc, IId, and IIe) according to their zinc finger-like motif. The PnWRKY family expands mainly via segmental duplication. Additionally, the promoter regions of PnWRKYs contain many hormone-responsive and stress-responsive elements. Expression patterns under different hormones (salicylic acid, methyl jasmonate, and melatonin) and disease (root rot disease, black blot disease, and virus disease) treatment analysis highlighted their wide involvement in plant hormone-induced disease defense responses. These findings affirmed the potential functions of PnWRKY TFs in disease-mediated defense responses and provided valuable insights for future agricultural applications.

Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

Genome-wide analysis of LATERAL ORGAN BOUNDARIES DOMAIN-in Physcomitrella patens and stress responses.

LBDs, as the plant-specific gene family, play essential roles in lateral organ development, plant regeneration, as well as abiotic stress and pathogen response. However, the number and characteristic of LBD genes in Pyscomitrella patens were still obscure. This study was performed to identify the LBD family gene in moss and to determine the expression profiles of LBDs under the abiotic and pathogen stress. Complete genome sequences and transcriptomes of P. patens were downloaded from the Ensembl plant database. The hidden Markov model-based profile of the conserved LOB domain was submitted as a query to identify all potential LOB domain sequences with HMMER software. Expression profiles of PpLBDs were obtained based on the GEO public database and qRT-PCR analysis. In this study, a total of 31 LBDs were identified in the P. patens genome, divided into two classes based on the presence of the leucine zipper-like coiled-coil motif. A phylogenetic relationship was obtained between 31 proteins from P. patens and 43 proteins from the Arabidopsis thaliana genome, providing insights into their conserved and potential functions. Furthermore, the exon-intron organization of each PpLBD were analyzed. All PpLBD contain the conserved DNA binding motif (CX2CX6CX3C zinc finger-like motif), and were predicted to be located in cell nuclear. The 31 PpLBD genes were unevenly assigned to 18 out of 27 chromosomes based on the physical positions. Among these genes, PpLBD27 was not only remarkably highest expressed in desiccation, but also a susceptible gene to pathogens through jasmonic acid-mediated signaling pathway. Most of PpLBDs were up-regulated with the treatment of mannitol. These results showed they were differentially induced and their potential functions in the environmental stimulus of the early terrestrial colonizers. Despite significant differences in the life cycle in P. patens and flowering plants, their functions involved in abiotic and biotic stress-regulated by LBDs have been identified and appear to be conserved in the two lineages. These results provided a comprehensive analysis of PpLBDs and paved insights into studies aimed at a better understanding of PpLBDs.

The SrWRKY71 transcription factor negatively regulates SrUGT76G1 expression in Stevia rebaudiana

SrUGT76G1 is vital for the biosynthesis of rebaudioside A, D and M in Stevia rebaudiana Bertoni; however, its transcriptional regulatory mechanism remains unknown. In this study, the 2050-bp promoter region of SrUGT76G1 was isolated by the TAIL-PCR method, and sequence analysis revealed the presence of several W-box cis-elements, which are the recognition motifs of WRKY transcription factors. Furthermore, SrWRKY71, characterized by a typical WRKY domain and a C2H2 zinc finger-like motif, was identified as a putative transcriptional regulator of SrUGT76G1. The transcript of SrWRKY71 predominantly accumulated in leaves and was present at a lower level in stems, roots and flowers. The SrWRKY71-GFP fusion protein was specifically localized to the nucleus in tobacco epidermal cells. In addition, the N and C terminal regions of SrWRKY71 contributed to its transactivation activity. Y1H and EMSA assays validated that SrWRKY71 binds directly to W-box1 and W-box2 in the proximal promoter region of SrUGT76G1. Moreover, SrWRKY71 represses the expression level of SrUGT76G1 in both tobacco leaves and stevia callus. Taken together, the data in this study represent the first identification of an essential upstream transcription factor of SrUGT76G1 and provides new insight into the regulatory network of steviol glycoside biosynthesis in Stevia rebaudiana.

Expression analysis and characterization of dmrt2 in Chinese tongue sole (Cynoglossus semilaevis)

Dmrt2 is a member of the dmrt gene family with a conserved zinc finger-like DNA-binding motif (DM domain). In the present study, CS-dmrt2 was cloned from the gonads of Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of CS-dmrt2 is 2834 bp in length, with a 251 bp 5′-untranslated region (UTR), a 1086 bp 3′-UTR and a 1503 bp open reading frame (ORF) that encodes a 501-amino-acid peptide. qPCR revealed that CS-dmrt2 was mainly expressed in C. semilaevis testes. In situ hybridization (ISH) showed CS-dmrt2 expression throughout early gonadal development (36 days after hatching (dah) and 86 dah), but the expression was higher in male gonads than in female gonads. CS-dmrt2 mRNA was highly expressed in male germ cells. Comparison of methylation levels between females and males demonstrated hypo-methylated levels of the CS-dmrt2 promoter in the male gonads, which is consistent with the high mRNA expression. These results suggest that the CS-dmrt2 gene may play a functional role in gonadal differentiation/development and germ cell maturation in the testis.

Male-specific Dmrt1 is a candidate sex determination gene in spotted scat (Scatophagus argus)

The Scatophagus argus is a cultured marine teleost fish in which females grow faster and larger than males, therefore cloning the master sex-determination (SD) gene will be helpful for its sex control in aquaculture. A well-conserved Dmrt1 (doublesex- and mab-3-related transcription factor-1) gene, encoding zinc finger-like DNA binding motif (DM-domain) plays a critical role in sex determination and differentiation in vertebrates. Here, two pairs of sex-specific primers, Dmrt3-Marker-F/R and Dmrt1-Marker-F/R were developed from Dmrt3 and Dmrt1 transcripts in S. argus respectively. The single-nucleotide polymorphisms (SNP) at position −151 and 166 bp on the DNA amplified by Dmrt3-Marker-F/R are all homozygous CC GG genotype in females and heterozygous CG GA genotype in the males. The Dmrt1-Marker-F/R could only amplify a 3.2 kb fragment in males. Both Markers were confirmed in 246 adult S. argus (113 males and 133 females) from five populations in China. These results indicate that the sexes of S. argus are controlled by sex chromosomes (XX females, XY males). The nucleotide at position 166 bp of the DNA amplified by Dmrt3-Marker-F/R is found to be a donor splice site mutation (c.400 + 1G > A) in Dmrt3 which generates a truncated isoform. The truncated Dmrt3 is linked to male sex and thereafter named as Dmrt3△-Y, and its allele is the normal Dmrt3 on the X chromosome. The DNA amplified by Dmrt1-Marker-F/R is a fragment between exon 2 and exon 3 of a normal Dmrt1 implying that Dmrt1 is male-specific and located on the Y chromosome. A truncated Dmrt1 (Dmrt1b) was found to exist in both males and females. The Dmrt1b is with high similarity with Dmrt1 indicating Dmrt1b is the allele of Dmrt1 located on the X chromosome. RT-PCR revealed that Dmrt1 is highly expressed in the testes, while it is not expressed in the ovaries. Dmrt3△-Y is only expressed weakly in some of the testes, while it is not expressed in the ovaries. The testicular specifically expressed Dmrt1 is the candidate SD gene on the Y chromosome in S. argus.

A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation.IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA.

The adaptor protein ARA55 and the nuclear kinase HIPK1 assist c-Myb in recruiting p300 to chromatin

LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin.

X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR1-105 ) and 8-105 (NTR8-105 ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1-105 packs as a dimer and NTR8-105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.

Expression Analysis of WRKY Transcription Factor Genes in Response to Abiotic Stresses in Horsegram (<i>Macrotyloma uniflorum</i> (Lam.) Verdc.)

Drought and salt stress are two major environmental constraints that limit the productivity of agriculture crops worldwide. WRKY transcription factors are the plant-specific transcription factors that regulate several developmental events and stress responses in plants. The WRKY domain is defined by a 60-amino acid conserved sequence named WRKYGQK at N-terminal and a Zinc Finger-like motif at the C-terminal. WRKY genes are known to respond several stresses which may act as negative or positive regulators. The function of most of the WRKY transcription factors from non-model plants remains poorly understood. This investigation shows the expression levels of eight WRKY transcription factor genes from horsegram plant under drought and salt stress conditions. The increase in mRNA transcript levels of WRKY transcription factor genes was found to be high in drought stressed plants compared to salt-stressed plants. The levels of MDA which indicates the lipid peroxidation were less in drought stress. More ROS is produced in salt stress conditions compared to drought. The results show that the expression of WRKY transcription factors in drought stress conditions is reducing the adverse effect of stress on plants. These results also suggest that, during abiotic stress conditions such as drought and salt stress, WRKY transcription factors are regulated at the transcription level.

Bean metal-responsive element-binding transcription factor confers cadmium resistance in tobacco.

Cadmium (Cd) is highly toxic to plants. Modulation of Cd-responsive transcription is an important way for Cd detoxification in plants. Metal-responsive element (MRE) is originally described in animal metallothionein genes. Although functional MREs also exist in Cd-regulated plant genes, specific transcription factors that bind MRE to regulate Cd tolerance have not been identified. Previously, we showed that Cd-inducible bean (Phaseolus vulgaris) stress-related gene2 (PvSR2) produces a short (S) PvSR2 transcript (S-PvSR2) driven by an intronic promoter. Here, we demonstrate that S-PvSR2 encodes a bean MRE-binding transcription factor1 (PvMTF-1) that confers Cd tolerance in tobacco (Nicotiana tabacum). PvMTF-1 expression was up-regulated by Cd at the levels of RNA and protein. Importantly, expression of PvMTF-1 in tobacco enhanced Cd tolerance, indicating its role in regulating Cd resistance in planta. This was achieved through direct regulation of a feedback-insensitive Anthranilate Synthase α-2 chain gene (ASA2), which catalyzes the first step for tryptophan biosynthesis. In vitro and in vivo DNA-protein interaction studies further revealed that PvMTF-1 directly binds to the MRE in the ASA2 promoter, and this binding depends on the zinc finger-like motif of PvMTF-1. Through modulating ASA2 up-regulation by Cd, PvMTF-1 increased free tryptophan level and subsequently reduced Cd accumulation, thereby enhancing Cd tolerance of transgenic tobacco plants. Consistent with this observation, tobacco transiently overexpressing ASA2 also exhibited increased tolerance to Cd. We conclude that PvMTF-1 is a zinc finger-like transcription factor that links MRE to Cd resistance in transgenic tobacco through activation of tryptophan biosynthesis.

An UBR-box from a non-N-recognin: Crystal Structure of the UBR domain of UBR6

The degradation of many short-lived proteins in eukaryotic cells is carried out by the Ubiquitin Proteasome System. The N-end rule pathway links the half-life of proteins to the identity of its N-terminal residue, also called N-degron. Destabilizing N-degrons, are recognized by E3 ubiquitin ligases termed N-recognins. N-degrons are grouped into type 1, composed of basic residues, and type 2, composed of bulky hydrophobic residues. In mammals, four N-recognins mediate the N-end rule pathway: UBR1, UBR2, UBR4 and UBR5. These proteins share a ~70-residue zinc finger-like motif termed the Ubiquitin Recognin (UBR) box, responsible for their specificity. The mammalian genome encodes at least three more UBR-box proteins: UBR3, UBR6/FBXO11 and UBR7. However, these UBRs cannot recognize any type of N-degrons. Our lab reported the crystal structures of the UBR boxes from the human UBR1 and UBR2, rationalizing the empirical rules for the classification of type 1 N-degrons. Despite the valuable information obtained from those structures there is not a clear explanation for the no recognition of N-degrons by other UBR-box proteins. Here we report the crystal structure of the UBR-box domain from UBR6 also known as FBXO11. UBR6 is a F-box protein of the SKP1-Cullin1-F-box (SCF) ubiquitin ligase complex and does not recognize any type of N-degrons. We crystallized a 77-residue fragment of the UBR-box of UBR6 and determined its structure at 1.7 Å resolution. Unexpectedly, this domain adopts an open conformation compared to UBR1-box, without any N-degron binding pockets. Its zinc-binding residues are conserved as in the N-recognins, but they are arranged in different zinc-binding motifs. Molecules form dimmers stabilized by zinc ions. The crystal had 4 molecules per asymmetric unit and space group P212121. For phasing we used Zn-SAD. With this structure we hope to obtain clues that explain the absence of N-degron recognition in some members of the UBR family.

Molecular identification of dmrt2 and dmrt5 and effect of sex steroids on their expressions in Chlamys nobilis

The dmrt (double-sex and mab-3-related transcription factor) gene family, characterized by a highly conserved zinc finger-like DNA-binding motif known as the DM domain, plays important roles in sex differentiation and sexual development. To better understand the role of dmrt on the sexual development in Chlamys nobilis, 1183 bp encoding cDNAs for dmrt2 and 2362 bp encoding cDNAs for dmrt5 have been cloned and functionally characterized. Dmrt2 and dmrt5 display significant homologies to DMRT2 cluster and DMRT5 cluster, respectively, and exhibit all the typical features of dmrt family. Real-time PCR analyses show that dmrt2 was expressed exclusively in gonads and dmrt5 distributed widely in tissues. Both of them were expressed at all developmental stages examined and peaked at blastula stage. Dmrt2 expression peaked at resting stage in the female gametogenetic cycle, and mature stage in male gonadal development. Dmrt5 expression peaked at resting stage in the female gametogenetic cycle and no significant differences were observed in male gonadal development. Expression of dmrt2 and dmrt5 of C. nobilis at spawning stage was upregulated by different concentration of MT (17 alpha-methyltestosterone) injected into the male and E-2 (17 beta-estradiol) injected into the female, at different time after treatment, and even different duration of function. These results implied that dmrt2 is more likely to be involved in playing a functional role in male gonadal development or maintenance of gonadal function than dmrt5 and dmrt5 may be involved in other biological processes besides gonadal development in C. nobilis. (C) 2014 Elsevier B.V. All rights reserved.

Nuclear RNase P of Trypanosoma brucei: A Single Protein in Place of the Multicomponent RNA-Protein Complex

SummaryRNase P is the endonuclease that removes 5′ extensions from tRNA precursors. In its best-known form, the enzyme is composed of a catalytic RNA and a protein moiety variable in number and mass. This ribonucleoprotein enzyme is widely considered ubiquitous and apparently reached its highest complexity in the eukaryal nucleus, where it is typically composed of at least ten subunits. Here, we show that in the protist Trypanosoma brucei, two proteins are the sole forms of RNase P. They localize to the nucleus and the mitochondrion, respectively, and have RNase P activity each on their own. The protein-RNase P is, moreover, capable of replacing nuclear RNase P in yeast cells. This shows that complex ribonucleoprotein structures and RNA catalysis are not necessarily required to support tRNA 5′ end formation in eukaryal cells.

Role of Intestinal Epithelial Cell Responses to Transforming Growth Factor (TGF)-β in Trichinella Spiralis Infection

Background: Protein kinase D1 (PKD1) is the founding member of a new protein kinase family within the CAMK group and separate from the previously identified PKCs. In most unstimulated cells, PKD1 is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing cysteine-rich zinc finger-like motifs and a pleckstrin homology domain. PKD1 can be activated within intact cells by multiple stimuli via phosphorylation of Ser744 and Ser748 in the PKD1 activation loop. Recent studies demonstrated rapid PKC-dependent and sustained PKC-independent PKD1 activation in response to G protein-coupled receptor (GPCR) agonists. Here, we tested the hypothesis that sustained PKD1 activation plays a key role in intestinal epithelial cell proliferation. Results: In order to determine the kinetics of PKD family activation by agonists that stimulate endogenous Gq-coupled receptors in intestinal epithelial cells (IEC-18 and IEC-6), these cells were stimulated for various times with either angiotensin II (ANG II) or vasopressin. The kinetics of autophosphorylation of PKD1 and PKD2 (indicative of their activation) were strikingly different. The autophosphorylation of PKD2 declined rapidly toward base-line levels whereas that of PKD1 remained elevated for up to 24h, indicating that PKD1 activation is sustained whereas that of PKD2 is transient in the same intracellular environment. Assays of catalytic activity, multi-site phosphorylation and sub-cellular localization demonstrated that GPCR agonists stimulated sustained PKD1 activation via a sequential mechanism consisting of an early PKC-dependent phase and a late PKC-independent phase in both IEC-18 and IEC-6 cells. The sustained activation of PKD1 coincided with its nuclear localization, raising the possibility that this PKD isoform was preferentially associated to long-term cellular responses, including stimulation of DNA synthesis and cell proliferation. To determine the role of endogenous PKD1 in GPCR-induced mitogenesis in intestinal epithelial cells, we depleted its expression using siRNAs that target specifically this PKD isoform. PKD1 knockdown (90%) prevented c-Fos accumulation, increase in DNA synthesis and cell number induced by the GPCR agonists. Conclusion: Our results support the hypothesis that PKD1 plays a key role in mediating GPCR-induced cell proliferation in cultured intestinal epithelial cells. In an accompanying Abstract, we show that PKD1 also plays a role in promoting crypt cell proliferation In Vivo.

Zinc is essential for high-affinity DNA binding and recombinase activity of φC31 integrase

The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of ϕC31 integrase (Int) and its relatives indicate the presence of a conserved motif containing four cysteines resembling a zinc finger. Inductively coupled plasma–mass spectrometry (ICP–MS) confirmed that an Int monomer contains one atom of zinc. Pre-incubation of Int with ethylenediaminetetraacetic acid (EDTA) was detrimental for both recombination activity and DNA binding affinities but full activity could be restored by adding back Zn2+. Mutations in the cysteines and other highly conserved residues yielded proteins that were hypersensitive to proteases, suggesting that without zinc the domain is unfolded. Substitutions in the highly charged region between the conserved cysteines led to lowered DNA binding affinities while circular dichroism revealed that these variant Ints were not greatly affected in overall folding. Int was protected from inhibition by EDTA when DNA containing an attachment site was present suggesting that the zinc finger and the DNA are in close proximity. A truncated mutant of Int, hInt V371SUGA, lacking the putative zinc finger could bind DNA with low affinity. The data are consistent with there being at least two DNA binding motifs in Int one of which is the zinc finger-like motif.

EOR-2 is an obligate binding partner of the BTB-zinc finger protein EOR-1 in Caenorhabditis elegans.

BTB-zinc finger transcription factors play many important roles in metazoan development. In these proteins, the BTB domain is critical for dimerization and for recruiting cofactors to target genes. Identification of these cofactors is important for understanding how BTB-zinc finger proteins influence transcription. Here we show that the novel but conserved protein EOR-2 is an obligate binding partner of the BTB-zinc finger protein EOR-1 in Caenorhabditis elegans. EOR-1 and EOR-2 function together to promote multiple Ras/ERK-dependent cell fates during development, and we show that EOR-1 is a robust substrate of ERK in vitro. A point mutation (L81F) in the EOR-1 BTB domain reduces both ERK phosphorylation and EOR-2 binding and eliminates all detectable biological function without affecting EOR-1 expression levels, localization, or dimerization. This point mutation lies near the predicted charged pocket region of the EOR-1 BTB dimer, a region that, in other BTB-zinc finger proteins, has been proposed to interact with corepressors or coactivators. We also show that a conserved zinc finger-like motif in EOR-2 is required for binding to EOR-1, that the interaction between EOR-1 and EOR-2 is direct, and that EOR-2 can bind to the human BTB-zinc finger protein PLZF. We propose that EOR-2 defines a new family of cofactors for BTB-zinc finger transcription factors that may have conserved roles in other organisms.

The Carboxy-Terminal Domain of Dictyostelium C-Module-Binding Factor Is an Independent Gene Regulatory Entity

The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of jumonji-type (JmjC) transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF) motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD). An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b fromCucumber Vein Yellowing Ipomovirus

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.

Functional Modulation of the Geminivirus AL2 Transcription Factor and Silencing Suppressor by Self-Interaction

The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.