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Trichomes are specialized glandular or non-glandular structures that provide physical or chemical protection against insect and pathogen attack. Trichomes in Arabidopsis have been extensively studied as typical non-glandular structures. By contrast, the molecular mechanism underlying glandular trichome formation and elongation remains largely unknown. We previously demonstrated that Hair is essential for the formation of type I and type VI trichomes. Here, we found that overexpression of Hair increased the density and length of tomato trichomes. Biochemical assays revealed that Hair physically interacts with its close homolog SlZFP8-like (SlZFP8L), and SlZFP8L also directly interacts with Woolly. SlZFP8L-overexpressing plants showed increased trichome density and length. We further found that the expression of SlZFP6, which encodes a C2H2 zinc finger protein, is positively regulated by Hair. Using chromatin immunoprecipitation, yeast one-hybrid, and dual-luciferase assays we identified that SlZFP6 is a direct target of Hair. Similar to Hair and SlZFP8L, the overexpression of SlZFP6 also increased the density and length of tomato trichomes. Taken together, our results suggest that Hair interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato.

Introduction WT1 missense mutation in exon 8 or 9 causes infantile nephrotic syndrome with early progression to end-stage kidney disease (ESKD), Wilms tumor, and 46,XY female. However, some patients with missense mutations in exon 8 or 9 progress to ESKD in their teens or later. Therefore, we conducted a systematic review and functional analysis of WT1 transcriptional activity. Methods We conducted a systematic review of 174 cases with WT1 exon 8 or 9 missense variants from our cohort (n=13) and previous reports (n=161). Of these cases, mild and severe genotypes were selected for further in vitro functional analysis using luciferase assay. Results The median age of developing ESKD was 1.17 years. A comparative study was conducted among three WT1 genotype classes: mutations of the DNA-binding site (DBS group), mutations outside the DNA-binding site but at sites important for zinc finger structure formation by 2 cysteines and 2 histidines (C2H2 group), and mutations leading to other amino acid changes (Others group). The DBS group showed the severest phenotype and the C2H2 group was intermediate, whereas the Others group showed the mildest phenotype (developing ESKD at 0.90, 2.00, and 3.92 years, respectively, with significant differences). In vitro functional analysis showed dominant-negative effects for all variants; in addition, the DBS and C2H2 mutations were associated with significantly lower WT1 transcriptional activity than the other mutations. Conclusion Not only the DNA-binding site but also C2H2 zinc finger structure sites are important for maintaining WT1 transcriptional activity, and their mutation causes severe clinical symptoms., Graphical abstract

The zinc finger-associated domain (ZAD) is present in over 90 C2H2 zinc finger (ZNF) proteins. Despite their abundance, only a few ZAD-ZNF genes have been characterized to date. Here, we systematically analyze the function of 68 ZAD-ZNF genes in Drosophila female germ cells by performing an in vivo RNA-interference screen. We identified eight ZAD-ZNF genes required for oogenesis, and based on further characterization of the knockdown phenotypes, we uncovered defects broadly consistent with functions in germ cell specification and/or survival, early differentiation, and egg chamber maturation. These results provide a candidate pool for future studies aimed at functionalization of this large but poorly characterized gene family.

C2H2 zinc finger proteins (ZFPs) play essential roles in leaf morphogenesis and floral development, as well as heat stress response and trichome formation, which activate or inhibit gene transcription mainly through interactions with nucleic acids, such as single-strand DNA, RNA binding or RNA/DNA bidirectional binding, and protein interaction. Single C2H2 ZFPs is the subfamily of ZFPs, but little of single C2H2 ZFP family is known in tomato. In this study, we identified 30 single ZFP genes in tomato using bioinformatics-based methods. Gene structures, phylogeny, conserved motifs, cis-element of promoter, chromosomal localization, gene duplication, and expression patterns of these single C2H2 ZFP genes were analyzed. Sequence analysis showed that most single C2H2 ZFP genes possessed only one exon, except for SlC1-liZFP1 and SlC1-liZFP2. These single C2H2 ZFP genes were asymmetrically distributed on 10 chromosomes, excluding 2 and 12 chromosomes. In addition, 24 of these genes were predicated to have experienced segmental duplication. Cis-element prediction indicated that many important elements were located in the putative promoter regions, like light and gibberellic acid (GA)-responsive elements. The expression profiles of these genes in different tissues and various hormones and stress treatment were further analyzed. Many genes were lowly expressed in all tissues, whereas some were specifically expressed in certain tissues, like SlC1-liZFP2 in young leaves, and SlC1-liZFP15 in fruits. Furthermore, these genes could also be induced by several hormones and stresses, including IAA, ETH, GA, cold, and drought. This study sets a good foundation for further characterizing the biological roles of single C2H2 ZFP genes in tomato.

Human ZNF648 is a novel poly C-terminal C2H2 zinc finger (ZnF) protein identified amongst the most dysregulated proteins in erythroid cells differentiated from induced pluripotent stem cells. Its nuclear localization and structure indicate it is likely a DNA-binding protein. Using a combination of ZNF648 overexpression in an induced pluripotent stem cells line and primary adult erythroid cells, ZNF648 knockdown in primary adult erythroid cells and megakaryocytes, comparative proteomics and transcriptomics we show that ZNF648 is required for both erythroid and megakaryocyte differentiation. Orthologues of ZNF648 were detected across Mammals, Reptilia, Actinopterygii, in some Aves, Amphibia and Coelacanthiformes suggesting the gene originated in the common ancestor of Osteichthyes (Euteleostomi or bony fish). Conservation of the C-terminal ZnF domain is higher, with some variation in ZnF number but a core of at least six ZnF conserved across all groups, with the N-terminus recognisably similar within but not between major lineages. This suggests the N-terminus of ZNF648 evolves faster than the C-terminus, however this is not due to exon-shuffling as the entire coding region of ZNF648 is within a single exon. As for other such transcription factors, the N-terminus likely carries out regulatory functions, but showed no sequence similarity to any known domains. The greater functional constraint on the ZnF domain suggests ZNF648 binds at least some similar regions of DNA in the different organisms. However, divergence of the N-terminal region may enable differential expression, allowing adaptation of function in the different organisms.

Different root zones have distinct capacities for nitrate (NO3−) uptake in Populus species, but the underlying physiological and microRNA (miRNA) regulatory mechanisms remain largely unknown. To address this question, two root zones of Populus × canescens (Ait.) Smith. with contrasting capacities for NO3− uptake were investigated. The region of 0–40 mm (root zone I) to the root apex displayed net influxes, whereas the region of 40–80 mm (root zone II) exhibited net effluxes. Concentrations of NO3− and ammonium (NH4+) as well as nitrate reductase activity were lower in zone II than in zone I. Forty one upregulated and twenty three downregulated miRNAs, and 576 targets of these miRNAs were identified in zone II in comparison with zone I. Particularly, growth-regulating factor 4 (GRF4), a target of upregulated ptc-miR396g-5p and ptc-miR396f_L + 1R-1, was downregulated in zone II in comparison with zone I, probably contributing to lower NO3− uptake rates and assimilation in zone II. Furthermore, several miRNAs and their targets, members of C2H2 zinc finger family and APETALA2/ethylene-responsive element binding protein family, were found in root zones, which probably play important roles in regulating NO3− uptake. These results indicate that differentially expressed miRNA–target pairs play key roles in regulation of distinct NO3− uptake rates and assimilation in different root zones of poplars.

Rice (Oryza sativa) spikelets are a unique inflorescence structure and their development directly determines grain size and yield. Although many genes related to spikelet development have been reported, the molecular mechanisms underlying this process have not been fully elucidated. In this study, we identified a new recessive rice mutant, lacking rudimentary glume 1 (lrg1). The lrg1 spikelets only formed one rudimentary glume, which, along with the sterile lemmas, was homeotically transformed into lemma-like organs and acquired lemma identity. The transition from the spikelet to the floral meristem was delayed in the lrg1 mutant, resulting in the formation of an ectopic lemma-like organ between the sterile lemma and the terminal floret. In addition, we found that the abnormal lrg1 grain phenotype resulted from the alteration of cell numbers and the hull size. LRG1 encodes a ZOS4-06-C2H2 zinc-finger protein with the typical EAR motifs, and is expressed in all organs and tissues. LRG1 localizes to the nucleus and can interact with the TOPLESS-RELATED PROTEINs (TPRs) to repress the expressions of their downstream target genes. Taken together, our results reveal that LRG1 plays an important role in the regulation of spikelet organ identity and grain size.

KLF7, one of candidate genes in neurotherapy and metabolic syndrome, has been studied in adipogenesis of mammalian species and birds. However, the effect of the third C2H2 zinc finger of KLF7 for its transcriptional regulation in adipogenesis has not been well understood. Here, the wild-type chicken KLF7 (KLF7) overexpression plasmid, pCMV-myc-KLF7, and two plasmids of chicken KLF7 mutants, i.e. pCMV-myc-KLF7m1 with half of the third zinc finger (KLF7m1) and pCMV-myc-KLF7m2 without the third zinc finger (KLF7m2), were constructed. Luciferase reporter assay in DF1 cells showed that the effect of chicken KLF7 overexpression on the promoter activity of LPL was greater than those of KLF7m1 and KLF7m2 (P 0.05). Additionally, the effects of KLF7, KLF7m1 and KLF7m2 overexpression on the promoter activity of PPARγ were different. KLF7 overexpression had no significant effect on the PPARγ promoter activity (P > 0.05), KLF7m1 overexpression suppressed PPARγ promoter activity (P < 0.05), while KLF7m2 overexpression facilitated the promoter activity of PPARγ (P < 0.05), consistent with the results of western blot analysis. Our results suggested that the third zinc finger of chicken KLF7 may play a role in its transcriptional regulation of LPL and PPARγ but has no effect on its regulation of C/EBPα, FASN and FABP4. The third zinc finger of KLF7 might be a target for the treatment of metabolic disorder in chicken.

Objective Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA–RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA–RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the most common class of transcription factors. These factors have great potential for the management of developmental progression by regulating the specific spatiotemporal expression of genes. In this study, we cloned one C2H2-ZF protein gene of Bombyx mori, BGIBMGA000319, that is orthologous to B-lymphocyte-induced maturation protein-1 (Blimp-1); we thus named it as Bombyx mori Blimp-1 (BmBlimp-1). In the silkworm, the BmBlimp-1 gene is specifically upregulated during day 2 of the pupal to adult stage and is highly expressed in wing discs on day 3 of the pupa. Knockdown of its expression level in the pupal stage results in a crumpled-winged silkworm moth. Using the predicted DNA-binding sequences of BmBlimp-1 to search the silkworm genome to screen target genes of BmBlimp-1, 7049 genes were identified to have at least one binding site of BmBlimp-1 on their 1 kb upstream and downstream genome regions. Comparisons of those genes with a reported pupal wing disc transcriptome data resulted in 4065 overlapping genes being retrieved. GO enrichment analysis of the overlapping genes showed that most of the genes were enriched in the binding term. Combining functional annotation and real-time quantitative PCR, 15 genes were identified as the candidate target genes of BmBlimp-1, including several wing cuticular protein genes, chitin synthase A, and wing disc development genes, such as Wnt1, cubitus interruptus (ci) and engrailed (en). Moreover, the amino acid sequence of the zinc finger motif of Blimp-1 gene was highly conserved among the 15 insect species. We propose that BmBlimp-1 is an important regulatory factor in silkworm wing development.

The C2H2-zinc finger proteins (ZFP) comprise a large family of transcription factors with various functions in biological processes. In maize, the function regulation of C2H2- zine finger (ZF) genes are poorly understood. We conducted an evolution analysis and functional prediction of the maize C2H2-ZF gene family. Furthermore, the ZmZFP126 gene has been cloned and sequenced for further favorable allelic variation discovery. The phylogenetic analysis of the C2H2-ZF domain indicated that the position and sequence of the C2H2-ZF domain of the poly-zinc finger gene are relatively conserved during evolution, and the C2H2-ZF domain with the same position is highly conserved. The expression analysis of the C2H2-ZF gene family in 11 tissues at different growth stages of B73 inbred lines showed that genes with multiple transcripts were endowed with more functions. The expression analysis of the C2H2-ZF gene in P1 and P2 inbred lines under drought conditions showed that the C2H2-ZF genes were mainly subjected to negative regulation under drought stress. Functional prediction indicated that the maize C2H2-ZF gene is mainly involved in reproduction and development, especially concerning the formation of important agronomic traits in maize yield. Furthermore, sequencing and correlation analysis of the ZmZFP126 gene indicated that this gene was significantly associated with the SDW-NAP and TDW-NAP. The analysis of the relationship between maize C2H2-ZF genes and C2H2-ZF genes with known functions indicated that the functions of some C2H2-ZF genes are relatively conservative, and the functions of homologous genes in different species are similar.

The PRDM9 protein determines sites of meiotic recombination in humans by directing meiotic DNA double-strand breaks to specific loci. Targeting specificity is encoded by a long array of C2H2 zinc fingers that bind to DNA. This zinc finger array is hypervariable, and the resulting alleles each have a potentially different DNA binding preference. The assessment of PRDM9 diversity is important for understanding the complexity of human population genetics, inheritance linkage patterns, and predisposition to genetic disease. Due to the repetitive nature of the PRDM9 zinc finger array, the large-scale sequencing of human PRDM9 is challenging. We, therefore, developed a long-read sequencing strategy to infer the diploid PRDM9 zinc finger array genotype in a high-throughput manner. From an unbiased study of PRDM9 allelic diversity in 720 individuals from seven human populations, we detected 69 PRDM9 alleles. Several alleles differ in frequency among human populations, and 32 alleles had not been identified by previous studies, which were heavily biased to European populations. PRDM9 alleles are distinguished by their DNA binding site preferences and fall into two major categories related to the most common PRDM9-A and PRDM9-C alleles. We also found that it is likely that inter-conversion between allele types is rare. By mapping meiotic double-strand breaks (DSBs) in the testis, we found that small variations in PRDM9 can substantially alter the meiotic recombination landscape, demonstrating that minor PRDM9 variants may play an under-appreciated role in shaping patterns of human recombination. In summary, our data greatly expands knowledge of PRDM9 diversity in humans.

C2H2 zinc finger proteins (ZFPs) play important roles in plant development and response to abiotic stresses, and have been studied extensively. However, there are few studies on ZFPs in mangroves and mangrove associates, which represent a unique plant community with robust stress tolerance. MpZFP1, which is highly induced by salt stress in the mangrove associate Millettia pinnata, was cloned and functionally characterized in this study. MpZFP1 protein contains two zinc finger domains with conserved QALGGH motifs and targets to the nucleus. The heterologous expression of MpZFP1 in Arabidopsis increased the seeds’ germination rate, seedling survival rate, and biomass accumulation under salt stress. The transgenic plants also increased the expression of stress-responsive genes, including RD22 and RD29A, and reduced the accumulation of reactive oxygen species (ROS). These results indicate that MpZFP1 is a positive regulator of plant responses to salt stress due to its activation of gene expression and efficient scavenging of ROS.

The presence of awns on the ear is associated with a number of important plant properties, such as drought resistance, quality of the grain mass during processing, etc. The main manifestations of this trait are controlled by the B1 gene, which has recently been identified and encodes the C2H2 zinc finger transcription factor. Based on the previously identified SNPs in the promoter region of this gene, we constructed markers for dominant and recessive alleles which determine awnless and awned phenotypes, respectively. The markers were successful for use in targeting the respective alleles of the B1 gene in 176 varieties of common wheat, accessions of T. spelta L., as well as on F2/F3 hybrids from crosses between awned and awnless forms of T. aestivum. We first identified a new allele, b1mite, which has both an insert of a miniature Stowaway-like transposon, 261 bp in length, and 33 novel SNPs in the promoter region. Despite these changes, this allele had no effect on the awned phenotype. The possible mechanisms of the influence of the analyzed gene on phenotype are discussed.

Plant epidermal cells, such as trichomes, root hairs, salt glands, and stomata, play pivotal roles in the growth, development, and environmental adaptation of terrestrial plants. Cell fate determination, differentiation, and the formation of epidermal structures represent basic developmental processes in multicellular organisms. Increasing evidence indicates that C2H2 zinc finger proteins play important roles in regulating the development of epidermal structures in plants and plant adaptation to unfavorable environments. Here, we systematically summarize the molecular mechanism underlying the roles of C2H2 zinc finger proteins in controlling epidermal cell formation in plants, with an emphasis on trichomes, root hairs, and salt glands and their roles in plant adaptation to environmental stress. In addition, we discuss the possible roles of homologous C2H2 zinc finger proteins in trichome development in non-halophytes and salt gland development in halophytes based on bioinformatic analysis. This review provides a foundation for further study of epidermal cell development and abiotic stress responses in plants.

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates andDrosophila, CTCF is involved in the regulation of homeotic genes. Previously, null mutations in thedCTCFgene were found to result in death during the stage of pharate adults, which failed to eclose from their pupal case. Here, we obtained several new nulldCTCFmutations and found that the complete inactivation of dCTCF appears to be limited to phenotypic manifestations of theAbd-Bgene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the nulldCTCFmutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation inDrosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not essential for the functional activity of dCTCFin vivo.HighlightsThe dCTCF 715–735 aa region interacts with the BTB and M domains of CP190Interaction with CP190 is not essential for dCTCF functionin vivoNulldCTCFmutants are viable but show severely reduced fertility

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila , CTCF is involved in the regulation of homeotic genes . Previously, it was found that null mutations in the dCTCF gene died as pharate adults, which failed to eclose from their pupal case, or shortly after hatching of adults. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears is limited mainly to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not important for the functional activity of dCTCF in vivo.

Transcription factors (TFs) are biological regulators of gene function in response to various internal and external stimuli. C2H2 zinc finger proteins (C2H2-ZFPs) are a large family of TFs that play crucial roles in plant growth and development, hormone signalling and response to biotic and abiotic stresses. While C2H2-ZFPs have been well characterized in many model and crop plants, they are yet to be ascertained in the evolutionarily important C3 plant Dichanthelium oligosanthes (Heller's rosette grass). In the present study, we report 32 C2H2-ZF genes (DoZFs) belonging to three different classes-Q type, C-type and Z-type based on structural elucidation and phylogenetic analysis. Sequence comparisons revealed paralogs within the DoZFs and orthologs among with rice ZF genes. Motif assignment showed the presence of the distinctive C2H2-ZF conserved domain "QALGGH" in these proteins. Cis-element analysis indicated that majority of the predicted C2H2-ZFPs are associated with hormone signalling and abiotic stress responses. Further, their role in nucleic acid binding and transcriptional regulation was also observed using predicted functional assignment. Thus, we report an overview of the C2H2-ZF gene family in D. oligosanthes that could serve as the basis for future experimental studies on isolation and functional implication of these genes in different biological mechanism of C3 plants.

The ZNF280D is a member of the C2H2 zinc finger family, which plays important roles in cell and organismal growth, secretion, and other biological processes. This study aimed to identify the alternative splicing of pig ZNF280D, and to predict the structure and function of the encoded proteins, as well as to study the spatio-temporal expression patterns of each transcript. RNA-seq and qPCR technology were used to identify the transcripts of pig ZNF280D. We found two transcripts, ZNF280D1 and ZNF280D2 (GenBank accession number: MF279143). ZNF280D1 is the common transcript, whereas ZNF280D2 lacked a large region at exon 8. In addition, ZNF280D2 had an additional C2H2 zinc finger domain, changing the protein from a tC2H2 to a maC2H2 zinc finger protein. The expression of two transcripts in various tissues of pig was differ significant. ZNF280D2 is the main subtype. The expression of ZNF280D2 in pig muscle was found to increase gradually with age. The ZNF280D1 was first decreased and then increased slightly. We successfully identified two transcripts of pig ZNF280D, and we suggested important functions for ZNF280D in digestive absorption and growth as well as in the development of skeletal muscles, the specific mechanisms for which would require further study.

C2H2 type of zinc finger transcription factors (C2H2-ZFP TFs) play crucial roles in plant developments and stress response. Regarding its importance, genome-wide study of C2H2-ZFs were performed in multiple important plant species, but any such investigation was not fulfilled in Triticum turgidum ssp. Durum (durum wheat) as an important nutritional crop. The present study identified 122 C2H2-ZFs in durum wheat and physically mapped them onto the genome. The phylogenetic analysis classified these TFs into six major groups. Genes structure and conserved motifs assay showed TtC2H2-ZF involvement in the important cellular functions. Comparative phylogeny between durum wheat TtC2H2-ZF genes and the orthologs in rice revealed the evolutionary relationships of C2H2-ZF proteins. The gene ontology and promoter cis-element analysis indicated that most of TtC2H2-ZF genes are involved in multiple molecular functions including metal ion-binding and various stimuli responses. Further, the miRNAs targeting TtC2H2-ZF transcripts, homology modeling and proteins interaction network were also demonstrated, suggesting the vital cellular functions of TtC2H2-ZFs during various circumstances. The expression heatmap demonstrated differential and tissue-specific expression patterns of these genes. Expression profiling of this gene family members in response to dehydration and heat stresses showed differential expression pattern of these genes at multiple time points of stresses. This study can prepare a comprehensive overview of the durum wheat C2H2-ZF gene family and may provide a new perspective on the evolution of them, which will form the basis for further investigation of the roles of this family members and future genetic engineering studies in crops.

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.

Miz-1 (ZBTB17) is a poly-Zinc Finger BTB/POZ transcription factor with 12 consecutive C2H2 Zinc Fingers (ZFs). Miz-1 binds transcriptional start sites (TSS), through a consensus sequence, to regulate the expression of genes involved in the cell cycle, autophagy, trafficking as well as T- and B-cell development. Although it is not known how exactly Miz-1 recognises this consensus, evidence and predictions suggest that ZFs 7 to 12 are able to bind it contiguously. As observed in the other members of the ZBTB family, striking divergences in highly conserved positions in the primary structures of the ZFs and linkers of Miz-1 are present. In fact, compared to the conserved TGEKP/Y found in the rest of ZFs 7-12, the linker between ZFs 10 and 11 has the following sequence: DNIRP/H586. We show by solution-state NMR and 15N-relaxation studies that this linker generates two coupled conformational exchange phenomena on the “biological” μs-ms timescale. On the one hand, we demonstrate that the His residue (H586) weakens the βα tertiary structure in ZF11 and promotes the population of an opened state in which the inter-ZF contacts, necessary for canonical DNA binding, is lacking. The existence of this opened state is supported by the local unfolding of the first helical turn of the recognition helix of ZF11. On the other hand, although this local unfolding is quenched by the replacement of the His by the conserved Tyr residue (Y), we found that another conformational exchange is present in the DNIRP linker. Most interestingly, we observe that the DNIRP linker adopts a helical structure that also impedes interZF contacts necessary for canonical and contiguous binding of the consensus. This implies that the recognition of the consensus by ZFs 7-12 is accomplished in a segmented fashion. Supported by bioinformatics predictions and DNA binding assays, our structural and dynamical data are congruent with a model whereby ZFs 7-9 and 11-12 can independently recognize both ends and strands of the consensus. Finally, considering the dynamic nature of DNA and the conformational exchange in ZFs 10-11, we further demonstrate that the simultaneous binding of ZF 7-9 or ZFs 11-12 to their respective subsites is physically possible in a coiled conformation of the consensus.

Arabidopsis trichome is specialized structure that develops from epidermal cells, and is an excellent model system for studying various aspects of plant cell development and cell differentiation. Our previous studies have shown that C2H2 zinc finger protein family genes, including GIS, GIS2, GIS3, ZFP5, ZFP6 and ZFP8, play an important role in controlling trichome initiation in Arabidopsis. Here, our novel results showed a C2H2 zinc finger protein, ZFP1, which also plays an important role in trichome initiation in Arabidopsis. ZFP1 over-expression lines display significantly increased trichome number on cauline leaves, lateral branches and main stems in comparison with wild type plants. ZFP1 RNAi lines and loss-of-function mutants showed the opposite phenotype. Furthermore, our study also found that ZFP1 mediates the regulation of trichome initiation by cytokinin signaling. The molecular and genetic analyses reveal that ZFP1 acts upstream of key trichome initiation factors, GL3 and TRY.

Arrays of repeat domains are critical to the proper function of a significant fraction of protein families. These repeats are easily identified in sequence, and are thought to have arisen primarily through the simultaneous duplication of multiple domains. However, for most repeat domain protein families, very little is typically known about the specific domain duplication events that occurred in their evolutionary histories. Here we extend existing reconciliation formulations that use domain trees and sequence trees to infer domain duplication and loss events to additionally consider simultaneous domain duplications under arbitrary cost models. We develop a novel integer linear programming (ILP) solution to this reconciliation problem, and demonstrate the accuracy and robustness of our approach on simulated datasets. Finally, as proof of principle, we apply our approach to an orthogroup containing the C2H2 zinc finger repeat domain, and identify simultaneous domain duplications that occurred at the onset of the primate lineage. Simulation and ILP code is available at https://github.com/Singh-Lab/treeSim.

Background C2H2 zinc finger proteins (C2H2 ZFPs) play vital roles in shaping many aspects of plant growth and adaptation to the environment. Plant genomes harbor hundreds of C2H2 ZFPs, which compose one of the most important and largest transcription factor families in higher plants. Although the C2H2 ZFP gene family has been reported in several plant species, it has not been described in the model leguminous species Medicago truncatula. Results In this study, we identified 218 C2H2 type ZFPs with 337 individual C2H2 motifs in M. truncatula. We showed that the high rate of local gene duplication has significantly contributed to the expansion of the C2H2 gene family in M. truncatula. The identified ZFPs exhibit high variation in motif arrangement and expression pattern, suggesting that the short C2H2 zinc finger motif has been adopted as a scaffold by numerous transcription factors with different functions to recognize cis-elements. By analyzing the public expression datasets and quantitative RT-PCR (qRT-PCR), we identified several C2H2 ZFPs that are specifically expressed in certain tissues, such as the nodule, seed, and flower. Conclusion Our genome-wide work revealed an expanded C2H2 ZFP gene family in an important legume M. truncatula, and provides new insights into the diversification and expansion of C2H2 ZFPs in higher plants.

Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11–13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.

One striking feature of the large KRAB-containing zinc finger protein (KZFP) family is its rapid expansion and divergence since its formation about 400 million years ago. However, the evolutionary characteristics of KRAB domains, C2H2 zinc fingers and the full protein of KZFPs have not been fully analyzed. As for the drivers of the rapid evolution, it’s partly due to their coevolution with transposable elements (TEs). But their diverse functions besides inhibiting TEs suggest other reasons exist. Here we address these two issues by the systematic analysis of the divergence time and diversification pattern of KZFPs at three aspects and the functional analysis of the potential target genes besides TEs. We found that old-zinc-finger-containing KZFPs tend to have varied and disordered KRAB domains, indicating there are two ways of the evolution of KZFPs, including the variation of KRAB domains and the diversification of zinc fingers. Among them, the divergence of zinc fingers mainly contributes to the rapid evolution of KZFPs. Thus, we mainly focused on the functional requirements of the evolution of zinc fingers. Different from the classical regulation pattern of this family, we found and experimentally confirmed that KZFPs tend to bind to non-TE regions and can positively regulate target genes. Although most young genes tend to be with a low expression level, young-zinc-finger-containing KZFPs tend to be highly expressed in early embryonic development or early mesoderm differentiation, indicating their particular evolutionarily novel functional roles in these two processes. We further validated a young KZFP, ZNF611, can bind to non-TE region of STK38 gene and positively regulates its expression in ESCs. The emergence of new sequence in STK38 promoter may drive the evolution of zinc fingers in ZNF611. Finally, we proposed a ‘two-way evolution model’ of KZFP family.