SlHair2 Regulates the Initiation and Elongation of Type I Trichomes on Tomato Leaves and Stems.

Trichomes are hair-like structures that are essential for abiotic and biotic stress responses. Tomato Hair (H), encoding a C2H2 zinc finger protein, was found to regulate the multicellular trichomes on stems. Here, we characterized Solyc10g078990 (hereafter Hair2, H2), its closest homolog, to examine whether it was involved in trichome development. The H2 gene was highly expressed in the leaves, and its protein contained a single C2H2 domain and was localized to the nucleus. The number and length of type I trichomes on the leaves and stems of knock-out h2 plants were reduced when compared to the wild-type, while overexpression increased their number and length. An auto-activation test with various truncated forms of H2 using yeast two-hybrid (Y2H) suggested that H2 acts as a transcriptional regulator or co-activator and that its N-terminal region is important for auto-activation. Y2H and pull-down analyses showed that H2 interacts with Woolly (Wo), which regulates the development of type I trichomes in tomato. Luciferase complementation imaging assays confirmed that they had direct interactions, implying that H2 and Wo function together to regulate the development of trichomes. These results suggest that H2 has a role in the initiation and elongation of type I trichomes in tomato.

Genome-Wide Identification and Expression Patterns of the C2H2-Zinc Finger Gene Family Related to Stress Responses and Catechins Accumulation in Camellia sinensis [L.] O. Kuntze.

The C2H2-zinc finger protein (C2H2-ZFP) is essential for the regulation of plant development and widely responsive to diverse stresses including drought, cold and salt stress, further affecting the late flavonoid accumulation in higher plants. Tea is known as a popular beverage worldwide and its quality is greatly dependent on the physiological status and growing environment of the tea plant. To date, the understanding of C2H2-ZFP gene family in Camellia sinensis [L.] O. Kuntze is not yet available. In the present study, 134 CsC2H2-ZFP genes were identified and randomly distributed on 15 chromosomes. The CsC2H2-ZFP gene family was classified into four clades and gene structures and motif compositions of CsC2H2-ZFPs were similar within the same clade. Segmental duplication and negative selection were the main forces driving the expansion of the CsC2H2-ZFP gene family. Expression patterns suggested that CsC2H2-ZFPs were responsive to different stresses including drought, salt, cold and methyl jasmonate (MeJA) treatment. Specially, several C2H2-ZFPs showed a significant correlation with the catechins content and responded to the MeJA treatment, which might contribute to the tea quality and specialized astringent taste. This study will lay the foundations for further research of C2H2-type zinc finger proteins on the stress responses and quality-related metabolites accumulation in C. sinensis.

Molecular characterization and expression analysis reveal the roles of Cys2/His2 zinc-finger transcription factors during flower development of Brassica rapa subsp. chinensis.

KEY MESSAGE: Conserved motif, gene structure, expression and interaction analysis of C2H2-ZFPs in Brassica rapa, and identified types of genes may play essential roles in flower development, and BrZFP38 was proved to function in flower development by affecting pollen formation. Flower development plays a central role in determining the reproduction of higher plants, and Cys2/His2 zinc-finger proteins (C2H2-ZFPs) widely participate in the transcriptional regulation of flower development. C2H2-ZFPs with various structures are the most widespread DNA-binding transcription factors in plants. In this study, conserved protein motif and gene structures were analyzed to investigate systematically the molecular features of Brassica rapa C2H2-ZFP genes. Expression of B. rapa C2H2-ZFPs in multiple tissues showed that more than half of the family members with different types ZFs were expressed in flowers. The specific expression profiles of these C2H2-ZFPs in different B. rapa floral bud stages were further evaluated to identify their potential roles in flower development. Interaction networks were constructed in B. rapa based on the orthology of flower-related C2H2-ZFP genes in Arabidopsis. The putative cis-regulatory elements in the promoter regions of these C2H2-ZFP genes were thoroughly analyzed to elucidate their transcriptional regulation. Results showed that the orthologs of known-function flower-related C2H2-ZFP genes were conserved and differentiated in B. rapa. A C2H2-ZFP was proved to function in B. rapa flower development. Our study provides a systematic investigation of the molecular characteristics and expression profiles of C2H2-ZFPs in B. rapa and promotes further work in function and transcriptional regulation of flower development.

The role of C2H2 zinc finger proteins in plant responses to abiotic stresses.

Abiotic stresses are important factors affecting plant growth and development and limiting agricultural production worldwide. Plants have evolved complex regulatory mechanisms to respond and adapt to constantly changing environmental conditions. C2H2 zinc finger proteins form a relatively large family of transcriptional regulators in plants. Recent studies have revealed that C2H2 zinc finger proteins function as key transcriptional regulators in plant responses to a wide spectrum of stress conditions, including extreme temperatures, salinity, drought, oxidative stress, excessive light and silique shattering. Here, we summarize recent functional analysis on C2H2 zinc finger proteins in plant responses to abiotic stresses and discuss their roles as part of a large regulatory network in the perception and responses by plants to different environmental stimuli.

Hair, encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato.

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.

Two critical positions in zinc finger domains are heavily mutated in three human cancer types.

A major goal of cancer genomics is to identify somatic mutations that play a role in tumor initiation or progression. Somatic mutations within transcription factors are of particular interest, as gene expression dysregulation is widespread in cancers. The substantial gene expression variation evident across tumors suggests that numerous regulatory factors are likely to be involved and that somatic mutations within them may not occur at high frequencies across patient cohorts, thereby complicating efforts to uncover which ones are cancer-relevant. Here we analyze somatic mutations within the largest family of human transcription factors, namely those that bind DNA via Cys2His2 zinc finger domains. Specifically, to hone in on important mutations within these genes, we aggregated somatic mutations across all of them by their positions within Cys2His2 zinc finger domains. Remarkably, we found that for three classes of cancers profiled by The Cancer Genome Atlas (TCGA)-Uterine Corpus Endometrial Carcinoma, Colon and Rectal Adenocarcinomas, and Skin Cutaneous Melanoma-two specific, functionally important positions within zinc finger domains are mutated significantly more often than expected by chance, with alterations in 18%, 10% and 43% of tumors, respectively. Numerous zinc finger genes are affected, with those containing Krüppel-associated box (KRAB) repressor domains preferentially targeted by these mutations. Further, the genes with these mutations also have high overall missense mutation rates, are expressed at levels comparable to those of known cancer genes, and together have biological process annotations that are consistent with roles in cancers. Altogether, we introduce evidence broadly implicating mutations within a diverse set of zinc finger proteins as relevant for cancer, and propose that they contribute to the widespread transcriptional dysregulation observed in cancer cells.

Regulation of trichome development in tobacco by JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L.

Trichomes are epidermal outgrowths of plant tissues that can secrete or store large quantities of secondary metabolites, which contribute to plant defense responses against stress. The use of bioengineering methods for regulating the development of trichomes and metabolism is a widely researched topic. In the present study, we demonstrate that JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L., can regulate trichome development in transgenic tobacco. To understand the underlying mechanisms, we performed transcriptome profiling of overexpression JcZFP8 transgenic plants and wild-type tobacco. Based on the analysis of differentially expressed genes, we determined that genes of the plant hormone signal transduction pathway was significantly enriched, suggesting that these pathways were modulated in the transgenic plants. In addition, the transcript levels of the known trichome-related genes in Arabidopsis were not significantly changed, whereas CycB2 and MYB genes were differentially expressed in the transgenic plants. Despite tobacco and Arabidopsis have different types of trichomes, all the pathways were associated with C2H2 zinc finger protein genes. Our findings help us to understand the regulation of multicellular trichome formation and suggest a new metabolic engineering method for the improvement of plants.

Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response.

In sexually reproducing organisms, meiosis is an essential step responsible for generation of haploid gametes from diploid somatic cells. The quest for understanding regulatory mechanisms of meiotic recombination in Plasmodium led to identification of a gene encoding a protein that contains 11 copies of C2H2 zinc fingers (ZnF). Reverse genetic approaches were used to create Plasmodium berghei parasites either lacking expression of full-length Plasmodium berghei zinc finger protein (PbZfp) (knockout [KO]) or expressing PbZfp lacking C-terminal zinc finger region (truncated [Trunc]). Mice infected with KO parasites survived two times longer (P < 0.0001) than mice infected with wild-type (WT) parasites. In mosquito transmission experiments, the infectivity of KO and Trunc parasites was severely compromised (>95% oocyst reduction). KO parasites revealed a total lack of trimethylation of histone 3 at several lysine residues (K4, K27, and K36) without any effect on acetylation patterns (H3K9, H3K14, and H4K16). Reduced DNA damage and reduced expression of topoisomerase-like Spo11 in the KO parasites with normal Rad51 expression further suggest a functional role for PbZfp during genetic recombination that involves DNA double-strand break (DSB) formation followed by DNA repair. These finding raise the possibility of some convergent similarities of PbZfp functions to functions of mammalian PRDM9, also a C2H2 ZnF protein with histone 3 lysine 4 (H3K4) methyltransferase activity. These functions include the major role played by the latter in binding recombination hotspots in the genome during meiosis and trimethylation of the associated histones and subsequent chromatin recruitment of topoisomerase-like Spo11 to catalyze DNA DSB formation and DMC1/Rad51-mediated DNA repair and homologous recombination.IMPORTANCE Malaria parasites are haploid throughout their life cycle except for a brief time period when zygotes are produced as a result of fertilization between male and female gametes during transmission through the mosquito vector. The reciprocal recombination events that follow zygote formation ensure orderly segregation of homologous chromosomes during meiosis, creating genetic diversity among offspring. Studies presented in the current manuscript identify a novel C2H2 ZnF-containing protein exhibiting multifunctional roles in parasite virulence, mosquito transmission, and homologous recombination during meiosis. Understanding the transmission biology of malaria will result in the identification of novel targets for transmission-blocking intervention approaches.

The Arabidopsis Cys2/His2 zinc finger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress.

Environmental stress poses a global threat to plant growth and reproduction, especially drought stress. Zinc finger proteins comprise a family of transcription factors that play essential roles in response to various abiotic stresses. Here, we found that ZAT18 (At3g53600), a nuclear C2H2 zinc finger protein, was transcriptionally induced by dehydration stress. Overexpression (OE) of ZAT18 in Arabidopsis improved drought tolerance while mutation of ZAT18 resulted in decreased plant tolerance to drought stress. ZAT18 was preferentially expressed in stems, siliques, and vegetative rosette leaves. Subcellular location results revealed that ZAT18 protein was predominantly localized in the nucleus. ZAT18 OE plants exhibited less leaf water loss, lower content of reactive oxygen species (ROS), higher leaf water content, and higher antioxidant enzyme activities after drought treatment when compared with the wild type (WT). RNA sequencing analysis showed that 423 and 561 genes were transcriptionally modulated by the ZAT18 transgene before and after drought treatment, respectively. Pathway enrichment analysis indicated that hormone metabolism, stress, and signaling were over-represented in ZAT18 OE lines. Several stress-responsive genes including COR47, ERD7, LEA6, and RAS1, and hormone signaling transduction-related genes including JAZ7 and PYL5 were identified as putative target genes of ZAT18. Taken together, ZAT18 functions as a positive regulator and plays a crucial role in the plant response to drought stress.

SMiLE-seq identifies binding motifs of single and dimeric transcription factors.

Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes. All tested TFs yielded DNA-binding models with predictive power comparable to or greater than that of other in vitro assays. De novo motif discovery on all JUN-FOS heterodimers and several nuclear receptor-TF complexes provided novel insights into partner-specific heterodimer DNA-binding preferences. We also successfully analyzed the DNA-binding properties of uncharacterized human C2H2 zinc-finger proteins and validated several using ChIP-exo.

The transcriptional regulator c2h2 accelerates mushroom formation in Agaricus bisporus.

The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation. Its inactivation results in a strain that is arrested at the stage of aggregate formation. In this study, the c2h2 orthologue of Agaricus bisporus was over-expressed in this white button mushroom forming basidiomycete using Agrobacterium-mediated transformation. Morphology, cap expansion rate, and total number and biomass of mushrooms were not affected by over-expression of c2h2. However, yield per day of the c2h2 over-expression strains peaked 1 day earlier. These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development, making its encoding gene a target for breeding of commercial mushroom strains.

TaZAT8, a C2H2-ZFP type transcription factor gene in wheat, plays critical roles in mediating tolerance to Pi deprivation through regulating P acquisition, ROS homeostasis and root system establishment.

Transcription factors (TFs) play critical roles in mediating defense of plants to abiotic stresses through regulating downstream defensive genes. In this study, a wheat C2H2-ZFP (zinc finger protein) type TF gene designated as TaZAT8 was functionally characterized in mediating tolerance to the inorganic phosphate (Pi)-starvation stress. TaZAT8 bears conserved motifs harboring in the C2H2-ZFP type counterparts across vascular plant species. The expression of TaZAT8 was shown to be induced in roots upon Pi deprivation, with a Pi concentration- and temporal-dependent manner. Overexpression of TaZAT8 in tobacco conferred plants improved tolerance to Pi deprivation; the transgenic lines exhibited enlarged phenotype and elevated biomass and phosphorus (P) accumulation relative to wild-type (WT) after Pi-starvation treatment. NtPT1 and NtPT2, the tobacco phosphate transporter (PT) genes, showed increased transcripts in the Pi-deprived transgenic lines, indicative of their transcriptional regulation by TaZAT8. Overexpression analysis of these PT genes validated their function in mediating Pi acquisition under the Pi deprivation conditions. Additionally, the TaZAT8-overexpressing lines also behaved enhanced antioxidant enzyme (AE) activities and enlarged root system architecture (RSA) with respect to WT. Evaluation of the transcript abundance of tobacco genes encoding AE and PIN proteins, including NtMnSOD1, NtSOD1, NtPOD1;2, NtPOD1;5, NtPOD1;6, and NtPOD1;9, and NtPIN1 and NtPIN4 are upregulated in the TaZAT8-overexpressing lines. Overexpression of NtPIN1 and NtPIN4 conferred plants to enlarged RSA and elevated biomass under the Pi-starvation stress conditions. Our investigation provides insights into plant adaptation to the Pi-starvation stress mediated by distinct ZFP TFs through modulation of Pi acquisition and cellular ROS detoxicity.