C2H2 Domain Research Articles

Transcription factors containing both C2H2 and homeobox domains play different roles in Verticillium dahliae.

Verticillium dahliae is a soilborne fungus that causes plant wilt and can infect a variety of economic crops and woody trees. The molecular basis of microsclerotial formation and infection by this fungus remains to be further studied. In this study, we analyzed the functions of seven C2H2-homobox transcription factors. Notably, VdChtf3 and VdChtf4 exhibited the most severe defects, affecting phenotypes associated with critical developmental stages in the V. dahliae disease cycle. Our results indicate that VdChtf3 is a potential specific regulator of microsclerotial formation, modulating the expression of pectinase-encoding genes. This finding could contribute to a better understanding of microsclerotial development in V. dahliae. Moreover, VdChtf4 was associated with cell wall integrity, reactive oxygen species (ROS) stress resistance, and increased virulence. These discoveries shed light on the biological significance of C2H2-homeobox transcription factors in V. dahliae's adaptation to the environment and infection of host plants.

C2H2 proteins: Evolutionary aspects of domain architecture and diversification.

The largest group of transcription factors in higher eukaryotes are C2H2 proteins, which contain C2H2-type zinc finger domains that specifically bind to DNA. Few well-studied C2H2 proteins, however, demonstrate their key role in the control of gene expression and chromosome architecture. Here we review the features of the domain architecture of C2H2 proteins and the likely origin of C2H2 zinc fingers. A comprehensive investigation of proteomes for the presence of proteins with multiple clustered C2H2 domains has revealed a key difference between groups of organisms. Unlike plants, transcription factors in metazoans contain clusters of C2H2 domains typically separated by a linker with the TGEKP consensus sequence. The average size of C2H2 clusters varies substantially, even between genomes of higher metazoans, and with a tendency to increase in combination with SCAN, and especially KRAB domains, reflecting the increasing complexity of gene regulatory networks.

New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity.

In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.

Possible New Candidates Involved to Thalidomide-Related Limbs and Cardiac Defects: A Systems Biology Approach.

Thalidomide is a known teratogen that causes malformations especially in heart and limbs. Its mechanism of teratogenicity is still not fully elucidated. Recently, a new target of thalidomide was described, TBX5, and was observed a new interaction between HAND2 and TBX5 that is disrupted in the presence of thalidomide. Therefore, our study aimed to raise potential candidates for thalidomide teratogenesis, through systems biology, evaluating HAND2 and TBX5 interaction and heart and limbs malformations of thalidomide. Genes and proteins related to TBX5 and HAND2 were selected through TF2DNA, REACTOME, Human Phenotype Ontology, and InterPro databases. Networks were assembled using STRING © database. Network analysis were performed in Cytoscape © and R v3.6.2. Differential gene expression (DGE) analysis was performed through gene expression omnibus. We constructed a network for HAND2 and TBX5 interaction; a network for heart and limbs malformations of TE; and the two joined networks. We observed that EP300 protein seemed to be important in all networks. We also looked for proteins containing C2H2 domain in the assembled networks. ZIC3, GLI1, GLI3, ZNF148, and PRDM16 were the ones present in both heart and limbs malformations of TE networks. Furthermore, in the DGE analysis after treatment with thalidomide, we observed that FANCB, ESCO2, and XRCC2 were downregulated and present both in heart and limbs networks. Through systems biology, we were able to point to different new proteins and genes, and selected specially EP300, which was important in all the analyzed networks, to be further evaluated in the TE teratogenicity.

Functional characterization of plant specific Indeterminate Domain (IDD) transcription factors in tomato (Solanum lycopersicum L.)

Plant-specific transcription factors (TFs) are responsible for regulating the genes involved in the development of plant-specific organs and response systems for adaptation to terrestrial environments. This includes the development of efficient water transport systems, efficient reproductive organs, and the ability to withstand the effects of terrestrial factors, such as UV radiation, temperature fluctuations, and soil-related stress factors, and evolutionary advantages over land predators. In rice and Arabidopsis, INDETERMINATE DOMAIN (IDD) TFs are plant-specific TFs with crucial functions, such as development, reproduction, and stress response. However, in tomatoes, IDD TFs remain uncharacterized. Here, we examined the presence, distribution, structure, characteristics, and expression patterns of SlIDDs. Database searches, multiple alignments, and motif alignments suggested that 24 TFs were related to Arabidopsis IDDs. 18 IDDs had two characteristic C2H2 domains and two C2HC domains in their coding regions. Expression analyses suggest that some IDDs exhibit multi-stress responsive properties and can respond to specific stress conditions, while others can respond to multiple stress conditions in shoots and roots, either in a tissue-specific or universal manner. Moreover, co-expression database analyses suggested potential interaction partners within IDD family and other proteins. This study functionally characterized SlIDDs, which can be studied using molecular and bioinformatics methods for crop improvement.

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality

BackgroundCTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer–promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif.ResultsPreviously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein.ConclusionsOur findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

Roles of stress-associated proteins in plant abiotic stress responses.

As a consequence of irregular and rapid changes in the external environment, plants are constantly exposed to a diverse range of stresses, including desiccation, high salinity, extreme temperatures, and metal toxicity. To contend with these stresses, plants have evolved an elaborate array of defense mechanisms and defense-related molecules, including a range of regulatory proteins. Stress-associated proteins (SAPs), a novel subclass of zinc-finger proteins, have been established to play key roles in plant stress responses. These SAPs, which tend to be structurally conserved among different plant species and are typically characterized by a combination of the A20, AN1, and C2H2 domains, have attracted considerable interest in recent decades on account of their involvement in diverse abiotic stress responses. In this review, we aim to provide a brief overview of the discovery and identification of SAPs in plants, with a particular focus on their roles in response to different abiotic stresses. In this regard, phenotypic analyses using transgenic plants have highlighted the dual regulatory role of SAPs in modulating stress responses, wherein by inducing physiological and morphological alterations at the cellular level, they can contribute to both positive and negative effects. However, despite significant advances in the characterization of these proteins, their precise functions remain incompletely understood. This review consolidates our current knowledge regarding SAPs and delineates potential avenues for future research, which we anticipate will unravel the intricate functional dynamics of SAPs in plant stress responses.

Molecular characterization of the B lymphocyte-induced maturation protein-1 (blimp1) gene of common carp (Cyprinus carpio) and its transcription repression involves recruitment of histone deacetylase HDAC3

Blimp1 is the master regulator of B cell terminal differentiation in mammals, it inhibits expression of many transcription factors including bcl6, which provides the basis for promoting further development of activated B lymphocytes into plasma cells. Blimp-1 is thought to act as a sequence-specific recruitment factor for chromatin-modifying enzymes including histone deacetylases (HDAC) and methyltransferases to repress target genes. The cDNA of Ccblimp1a (Cyprinus carpio) open reading frame is 2337 bp encoding a protein of 777 amino acids. CcBlimp1a contains a SET domain, two Proline Rich domains, and five ZnF_C2H2 domains. Blimp1 are conserved in vertebrate species. Ccblimp1a transcripts were detected in common carp larvae from 1 dpf (day post fertilization)to 31 dpf. Ccblimp1a expression was up-regulated in peripheral blood leukocytes (PBL) and spleen leukocytes (SPL) of common carp stimulated by intraperitoneal lipopolysaccharide (LPS) injection. Ccblimp1a expression in PBL and SPL of common carp was induced by TNP-LPS and TNP-KLH. The results indicated TNP-LPS induced a rapid response in PBL and TNP-KLH induced much stronger response in SPL and PBL. IHC results showed that CcBlimp1 positive cells were distributed in the head kidney, trunk kidney, liver, and gut. Immunofluorescence stain results showed that CcBlimp1 was expressed in IgM + lymphocytes. The subcellular localization of CcBlimp1 in the nuclei indicated CcBlimp1 may be involved in the differentiation of IgM + lymphocytes. Further study focusing on the function of CcBlimp1 transcriptional repression was performed using dual luciferase assay. The results showed that the transcription repression of CcBlimp1 on bcl6aa promoter was affected by the histone deacetylation inhibitor and was synergized with histone deacetylase 3 (HDAC3). The results of Co-IP in HEK293T and immunoprecipitation in SPL indicated that CcBlimp1 recruited HDAC3 and might be involved in the formation of complexes. These results suggest that CcBlimp1 is an important transcription factor in common carp lymphocytes. Histone deacetylation modification mediated by HDAC3 may have important roles in CcBlimp1 transcriptional repression during the differentiation of lymphocytes.

ZNF500 abolishes breast cancer proliferation and sensitizes chemotherapy by stabilizing P53 via competing with MDM2.

Zinc finger protein 500 (ZNF500) has an unknown expression pattern and biological function in human tissues. Our study revealed that the ZNF500 mRNA and protein levels were higher in breast cancer tissues than those in their normal counterparts. However, ZNF500 expression was negatively correlated with advanced TNM stage (p = 0.018), positive lymph node metastasis (p = 0.014), and a poor prognosis (p < 0.001). ZNF500 overexpression abolished in vivo and in vitro breast cancer cell proliferation by activating the p53-p21-E2F4 signaling axis and directly interacting with p53 via its C2H2 domain. This may prevent ubiquitination of p53 in a manner that is competitive to MDM2, thus stabilizing p53. When ZNF500-∆C2H2 was overexpressed, the suppressed proliferation of breast cancer cells was neutralized in vitro and in vivo. In human breast cancer tissues, ZNF500 expression was positively correlated with p53 (p = 0.022) and E2F4 (p = 0.004) expression. ZNF500 expression was significantly lower in patients with Miller/Payne Grade 1-2 than in those with Miller/Payne Grade 3-5 (p = 0.012). ZNF500 suppresses breast cancer cell proliferation and sensitizes cells to chemotherapy.

Promoter hypermethylation and comprehensive regulation of ncRNA lead to the down-regulation of ZNF880, providing a new insight for the therapeutics and research of colorectal cancer

The human genome encodes more than 350 kinds of Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), KRAB-type ZNF transcription factor family (KZNF) plays a vital role in gene regulatory networks. The KZNF family members include a large number of highly homologous genes, gene subtypes and pseudogenes, and their expression has a high degree of tissue specificity and precision. Due to the high complexity of its regulatory network, the KZNF gene family has not been researched in sufficient, and the role of its members in the occurrence of cancer is mostly unexplored. In this study, ZNF880 was significantly associated with overall survival (OS) and disease-free survival (DFS) in colorectal carcinoma (CRC) patients. Low ZNF880 expression resulted in shorter OS and DFS. Combined with Colon adenocarcinoma (COAD) and Rectum adenocarcinoma (READ) data collection in the TCGA database, we found that ZNF880 was significantly down-regulated in CRC. Further analysis of the sequence variation of ZNF880 in CRC showed that ZNF880 accumulated a large number of SNV in the C2H2 domain and KRAB domain, while promoter region of ZNF880 also showed high methylation in COAD and READ. Combined with the Cbioportal and TIMER databases, the expression of mutant ZNF880 was significantly lower in COAD compared to the wild type. Simultaneously, the lncRNA-miRNA-ZNF880 ceRNA regulatory network was constructed through co-expression and miRNAs target gene prediction, demonstrating the precision of the ZNF880 regulatory network. In addition, the decreased expression of ZNF880 caused the significant immune infiltration decreases of CD8 + cells in COAD. In contrast, the immune infiltration of CD4 + cells and macrophages in COAD is positively correlated with ZNF880. Finally, through protein–protein interaction (PPI) network analysis and transcription factor target gene prediction, we screened out the genes most likely to be related to the function of ZNF880. CENPK, IFNGR2, REC8 and ZBTB17 were identified as the most closely functioning genes with ZNF880, which may indicate that ZNF880 has important links with the formation of cell centromere, tumor immunity, cell cycle and other pathways closely related to the occurrence of CRC. These studies show that the down-regulation of ZNF880 gene is closely related to CRC, and the targeted change of the expression of its regulatory molecules (miRNA and lncRNA) may be a new perspective for CRC treatment.

CeGAL: Redefining a Widespread Fungal-Specific Transcription Factor Family Using an In Silico Error-Tracking Approach.

In fungi, the most abundant transcription factor (TF) class contains a fungal-specific 'GAL4-like' Zn2C6 DNA binding domain (DBD), while the second class contains another fungal-specific domain, known as 'fungal_trans' or middle homology domain (MHD), whose function remains largely uncharacterized. Remarkably, almost a third of MHD-containing TFs in public sequence databases apparently lack DNA binding activity, since they are not predicted to contain a DBD. Here, we reassess the domain organization of these 'MHD-only' proteins using an in silico error-tracking approach. In a large-scale analysis of ~17,000 MHD-only TF sequences present in all fungal phyla except Microsporidia and Cryptomycota, we show that the vast majority (>90%) result from genome annotation errors and we are able to predict a new DBD sequence for 14,261 of them. Most of these sequences correspond to a Zn2C6 domain (82%), with a small proportion of C2H2 domains (4%) found only in Dikarya. Our results contradict previous findings that the MHD-only TF are widespread in fungi. In contrast, we show that they are exceptional cases, and that the fungal-specific Zn2C6-MHD domain pair represents the canonical domain signature defining the most predominant fungal TF family. We call this family CeGAL, after the highly characterized members: Cep3, whose 3D structure is determined, and GAL4, a eukaryotic TF archetype. We believe that this will not only improve the annotation and classification of the Zn2C6 TF but will also provide critical guidance for future fungal gene regulatory network analyses.

CgBlimp-1 inhibits granulocytes proliferation and interleukin production in the immune response of oyster Crassostrea gigas

B lymphocyte-inducible maturation protein 1 (Blimp-1) is a SET domain and zinc fingers containing transcriptional repressor, which is necessary for regulating the development of many immune cell lineages and keeping immune homeostasis. In the present study, a Blimp-1 homologue (designated as CgBlimp-1) was identified from oyster Crassostrea gigas, which contained a conserved SET domain and five ZnF_C2H2 domains and shared high homology with Blimp-1 from other species. The mRNA transcripts of CgBlimp-1 were highly expressed in gill and hepatopancreas. CgBlimp-1 protein was detected to be specifically expressed in granulocytes. After V. splendidus stimulation, the mRNA expression level of CgBlimp-1 in haemocytes up-regulated significantly at 24, 48, and 96 h, which was 4.39-fold (p < 0.05), 7.68-fold (p < 0.01) and 2.65-fold (p < 0.05) of that in control group, respectively. When the expression of CgBlimp-1 was knocked-down in vivo by RNAi, the mRNA expressions of downstream transcription factor CgMyc-A (1.63-fold of that in control group, p < 0.05) and cell cycle related gene CgCDK2 (1.70-fold, p < 0.05) increased significantly at 24 h after V. splendidus stimulation. Concomitantly, the ratio of EdU+ haemocytes increased notably (p < 0.01) while the proportion of haemocytes in G0/G1 phase decreased dramatically (p < 0.001), compared to that in control group. More specifically, the proportion of granulocytes in total haemocytes increased apparently (p < 0.05) in CgBlimp-1-RNAi oysters, together with up-regulation (p < 0.05) of the ratio of EdU+ granulocytes and down-regulation (p < 0.001) of the proportion of granulocytes in G0/G1 phase. Furthermore, the mRNA expression levels of CgIL17-1, CgIL17-2 and CgIL17-4 in haemocytes increased significantly in CgBlimp-1-RNAi oysters, which was 1.71-fold (p < 0.05), 144.70-fold (p < 0.01) and 1.93-fold (p < 0.05) of that in control group, respectively. Aforementioned results suggested that CgBlimp-1 could reduce the proliferation of granulocytes by arresting cell cycle in G1/G0 phase and avoid over-expression of interleukin to maintain homeostasis in the immune response of oyster.

Structural Evaluation and Conformational Dynamics of ZNF141T474I Mutation Provoking Postaxial Polydactyly Type A.

This study delineates the pathogenic association, miss-fold aggregation, and conformational paradigm of a missense variant (c.1420C &gt; T; p.T474I) in ZNF141 gene segregating PAP through a molecular dynamics simulations approach. In ZNF141 protein, helices play a crucial role by attaching three specific target DNA base pairs. In ZNF141T474I protein, H1, H3, and H6 helices attain more flexibility by acquiring loop conformation. The outward disposition of the proximal portion of H9-helix in mutant protein occurs due to the loss of prior beta-hairpins at the C terminal region of the C2-H2 domain. The loss of hydrogen bonds and exposure of hydrophobic residues to solvent and helices turning to loops cause dysfunction of ZNF141 protein. These significant changes in the stability and conformation of the mutant protein were validated using essential dynamics and cross-correlation maps, which revealed that upon point mutation, the overall motion of the proteins and the correlation between them were completely different, resulting in Postaxial polydactyly type A. This study provides molecular insights into the structural association of ZNF141 protein with PAP type A. Identification of active site residues and legends offers new therapeutic targets for ZNF141 protein. Further, it reiterates the functional importance of the last residue of a protein.

Variation in the fruit development gene POINTED TIP regulates protuberance of tomato fruit tip

The domestication of tomato has led to striking variations in fruit morphology. Here, we show a genome-wide association study (GWAS) to understand the development of the fruit tip and describe a POINTED TIP (PT) gene that encodes a C2H2-type zinc finger transcription factor. A single nucleotide polymorphism is found to change a histidine (H) to an arginine (R) in the C2H2 domain of PT and the two alleles are referred to as PTH and PTR. Knocking out PTH leads to development of pointed tip fruit. PTH functions to suppress pointed tip formation by downregulating the transcription of FRUTFULL 2 (FUL2), which alters the auxin transport. Our evolutionary analysis and previous studies by others suggest that the PTR allele likely hitch-hiked along with other selected loci during the domestication process. This study uncovers variation in PT and molecular mechanism underlying fruit tip development in tomato.

ZNF276 promotes the malignant phenotype of breast carcinoma by activating the CYP1B1-mediated Wnt/β-catenin pathway

Zinc finger proteins (ZNFs) have been demonstrated to participate extensively in breast cancer progression by functioning as transcription factors, but there are still a variety of ZNFs whose biological mechanisms remain unknown. Here, we show that zinc finger protein 276 (ZNF276) is highly expressed in breast cancer tissues and cell lines. Higher level of ZNF276 correlated with poor prognosis. Gain-of and loss-of function suggested that ZNF276 is essential for the proliferation, migration and invasion of breast cancer cells in vitro and metastasis in vivo. RNA-sequencing and CUT&Tag assay revealed that ZNF276 controlled a variety of growth and metastasis-related genes expression. ZNF276 transcriptionally promoted the expression of CYP1B1 by directly binds to the promoter region of the CYP1B1 through its C2H2 domain. ZNF276 facilitated the translocation of β-catenin from cytoplasm to nucleus through CYP1B1, leading to the upregulation of cyclin D1 and c-Myc, and the activation of the Wnt/β-catenin pathway. Knockdown of CYP1B1 significantly blocked the ZNF276-mediated effects on cell proliferation, migration and invasion. Lastly, ZNF276 interacted with MAGEB2 which enhanced the binding of ZNF276 at the CYP1B1 promoter, promoted CYP1B1 expression and Wnt signaling activation. Collectively, these findings highlight the oncogenic role of ZNF276 on breast cancer cell proliferation and metastasis. Targeting ZNF276/MAGEB2 axis may serve as a potential therapeutic strategy for breast cancer patients.

Genome-Wide Analyses of Tea Plant Stress-Associated Proteins (SAPs) Reveal the Role of CsSAP12 in Increased Drought Tolerance in Transgenic Tomatoes

Plant stress-associated proteins (SAPs) contain A20/AN1 zinc finger domains and are involved in plant response to abiotic stresses. In this study, we aimed to explore the biological function of tea plant CsSAPs. A total of 14 CsSAP genes were identified in the tea plant genome using a reference genome database (Camellia sinensis var. sinensis). The CsSAPs were divided into the following two groups: Group I, containing one AN1 domain and/or one A20 domain; and Group II, containing two AN1 domains and/or two C2H2 domains. The sequence alignments and conserved domains analysis indicated that the CsSAPs were highly structurally conserved in terms of amino acid sequence and protein structure. The CsSAPs showed different transcript levels in spatio-temporal expression and in response to cold and drought stress in tea plants. Furthermore, the expression of CsSAP12 was considerably upregulated under drought stress. The overexpression of CsSAP12 in transgenic tomatoes showed increased tolerance to drought stress compared with the wild type. Altogether, the results showed that CsSAP12 might be involved in drought stress. Thus, CsSAP12 might be a target gene in genetic engineering to improve drought tolerance in tea plants.

A novel Ush transcription factor involving in hematopoiesis of Eriocheir sinensis.

The FOG transcriptional factor is a co-regulator that recognizes and binds to the GATA N-terminal zinc-finger domain and participates in hemocyte production and differentiation. In this study, an FOG-like gene, Ush, was characterized from Eriocheir sinensis, which consists of an 897bp full-length open reading frame, encoding a polypeptide of 298 amino acids with four ZnF_C2H2 domains. The EsUsh mRNA transcripts were mainly expressed in the hematopoietic tissue (HPT) and hemocytes, and were significantly higher in hyalinocytes than semi-granulocytes and granulocytes, which were separated by Percoll gradient centrifugation. The transcription levels of EsUsh were found to be significantly upregulated in HPT, but downregulated in hemocytes after exsanguination. By using flow cytometry to determine the percentage of hemocyte sub-population after exsanguination, the percentage of hyalinocytes was found to significantly downregulated, while the percentage of granulocytes was significantly upregulated. Silencing EsUsh by dsRNA interference significantly decreased the percentage of hyalinocytes and small granulocytes, and increased the percentage of medium granulocytes and large granulocytes. Such findings suggest that EsUsh might be involved in hemocyte production and differentiation, especially in promoting hyalinocyte formation and limiting granulocyte generation and differentiation.

Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

KLF13 induces apoptotic cell clearance in Penaeus vannamei as an essential part of shrimp innate immune response to pathogens

Although, in mammals, the Krüppel-like transcription factor 13 (KLF13) plays an essential role in cell proliferation, survival, differentiation, apoptosis, tumorigenesis, immune regulation, and inflammation, its role in penaeid shrimp is unclear. In the current study, we characterized a KLF13 homolog in Penaeus vannamei (PvKLF13), with full-length cDNA of 1677 bp and 1068 bp open reading frame (ORF) encoding a putative protein of 355 amino acids, which contains three ZnF_C2H2 domains. Sequence and phylogenetic analysis revealed that PvKLF13 shares a close evolutionary relationship with KLF13 from invertebrates. Transcript levels of PvKLF13 were ubiquitously expressed in shrimp and induced in hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, and white spot syndrome virus (WSSV), suggesting the involvement of PvKLF13 in shrimp immune response to pathogens. Besides, knockdown of PvKLF13 decreased hemocytes apoptosis in terms of increased expression of pro-survival PvBcl-2, but decreased expression of pro-apoptotic PvBax and PvCytochrome C, coupled with high PvCaspase3/7 activity, especially upon V. parahaemolyticus challenge. The findings here indicate the involvement of PvKLF13 in apoptotic cell clearance as an essential part of shrimp innate immune response to pathogens.

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.