C2H2-type Zinc Finger Protein Research Articles

ZF protein C2H2-71 regulates soluble solids content by inhibiting LIN5 in tomato

Soluble solids content (SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC has been shown to be transcriptionally regulated via sugar metabolism. Previous studies have been predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanism of C2H2 in the accumulation of soluble solids in tomato fruits are yet to be fully understood. This study involved the selection of eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits. The study found that the levels of SlC2H2-71 expressions were significantly reduced in high-SSC accessions compared to low-SSC accessions. The Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, leading to elevated levels of soluble solids, fructose, glucose, malic, and citric acids in mature red ripe fruits. However, sucrose content in the edited Slc2h2-71 mutant lines generally decreased. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to sugar and acid metabolic pathways, which was further confirmed by quantitative real time PCR. Specifically, there was an observed increase in the expression of SlLIN5 encoding the cell wall invertase (CWIN). The yeast one-hybrid (Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay (EMSA) demonstrated that SlC2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of SlLIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase in the SlC2H2-71 knock-out lines exhibited a significantly higher level compared to that observed in the control lines. In summary, the regulation of tomato fruit SSC by SlC2H2-71 involves the inhibition of SlLIN5 expression.

ZNF775 inhibits MCF-7 breast cancer cell migration by downregulating Wnt5a

C2H2 zinc finger protein is widely involved in the occurrence and development of cancer. However, the function and mechanism of most C2H2 zinc finger proteins in breast caner (BC) remains unclear. Here, we reported the expression prognosis of C2H2 type zinc finger protein ZNF775 in BC patients and its possible biological mechanism. First, multiple public databases showed that ZNF775 was significantly overexpressed in BC tissues. Interestingly, high expression of ZNF775 was significantly associated with a better prognosis. Immunohistochemistry were used for verification, and the expression of ZNF775 was consistent with the databases. Considering the large heterogeneity of different breast cancer cells, we temporarily selected MCF-7 cell line for verification. In vitro overexpression experiments showed that overexpression of ZNF775 significantly inhibited the proliferation and migration of MCF-7 BC cell. We further combined RNA-sequencing (RNA-seq) and CUT & Tag, and found that overexpression of ZNF775 can down-regulate the expression of most genes in the Wnt signaling pathway. The cBioportal database showed that ZNF775 was negatively correlated with the expression of Wnt5a, suggesting that its downstream target was likely Wnt5a. Finally, we discovered that Wnt5a could partially reverse the inhibitory effect of ZNF775 on MCF-7 BC cell migration through transwell migration experiments. In conclusion, our findings will provide new ideas for the diagnosis, treatment and prognosis assessment of BC in the future.

AtZAT10/STZ1 improves drought tolerance and increases fiber yield in cotton.

Drought poses a significant challenge to global crop productivity, necessitating innovative approaches to bolster plant resilience. Leveraging transgenic technology to bolster drought tolerance in crops emerges as a promising strategy for addressing the demands of a rapidly growing global populace. AtZAT10/STZ1, a C2H2-type zinc finger protein transcription factor has shown to significantly improve Arabidopsis' tolerance to various abiotic stresses. In this study, we reports that AtSTZ1 confers notable drought resistance in upland cotton (Gossypium hirsutum), amplifying cotton fiber yield under varying conditions, including irrigated and water-limited environments, in field trials. Notably, AtSTZ1-overexpressing transgenic cotton showcases enhanced drought resilience across critical growth stages, including seed germination, seedling establishment, and reproductive phases. Morphological analysis reveals an expanded root system characterized by an elongated taproot system, increased lateral roots, augmented root biomass, and enlarged cell dimensions from transgenic cotton plants. Additionally, higher contents of proline, chlorophyll, soluble sugars, and enhanced ROS-scavenging enzyme activities are observed in leaves of transgenic plants subjected to drought, underscoring improved physiological adaptations. Furthermore, transgenic lines exhibit heightened photosynthetic rate, increased water use efficiency, and larger stomatal and epidermal cell sizes, coupled with a decline in leaf stomatal conductance and density, as well as diminished transpiration rates compared to the wild type counterparts. Transcriptome profiling unveils 106 differentially expressed genes in transgenic cotton leaves post-drought treatment, including protein kinases, transcription factors, aquaporins, and heat shock proteins, indicative of an orchestrated stress response. Collectively, these findings underscore the capacity of AtSTZ1 to augment the expression of abiotic stress-related genes in cotton following drought conditions, thus presenting a compelling candidate for genetic manipulation aimed at enhancing crop resilience.

Comprehensive Annotation and Expression Profiling of C2H2 Zinc Finger Transcription Factors across Chicken Tissues.

As the most abundant class of transcription factors in eukaryotes, C2H2-type zinc finger proteins (C2H2-ZFPs) play critical roles in various biological processes. Despite being extensively studied in mammals, C2H2-ZFPs remain poorly characterized in birds. Recent accumulation of multi-omics data for chicken enables the genome-wide investigation of C2H2-ZFPs in birds. The purpose of this study is to reveal the genomic occurrence and evolutionary signature of chicken C2H2-ZFPs, and further depict their expression profiles across diverse chicken tissues. Here, we annotated 301 C2H2-ZFPs in chicken genome, which are associated with different effector domains, including KRAB, BTB, HOMEO, PHD, SCAN, and SET. Among them, most KRAB-ZFPs lack orthologues in mammals and tend to form clusters by duplication, supporting their fast evolution in chicken. We also annotated a unique and previously unidentified SCAN-ZFP, which is lineage-specific and highly expressed in ovary and testis. By integrating 101 RNA-seq datasets for 32 tissues, we found that most C2H2-ZFPs have tissue-specific expression. Particularly, 74 C2H2-ZFPs-including 27 KRAB-ZFPs-show blastoderm-enriched expression, indicating their association with early embryo development. Overall, this study performs comprehensive annotation and expression profiling of C2H2 ZFPs in diverse chicken tissues, which gives new insights into the evolution and potential function of C2H2-ZFPs in avian species.

SUPERMAN Targets PHRAGMOPLAST ORIENTING KINESIN to Negatively Regulate Leaf Cell Division in Poplar

ABSTRACTPlant organs achieve their specific size and shape through the coordination of cell division and cell expansion, processes that are profoundly influenced by environmental cues. Cytokinesis during cell division depends on the position of the cytokinetic wall, but how this process responses to environment fluctuations remains underexplored. Here, we investigated a regulatory module involving C2H2‐type zinc finger protein (C2H2‐ZFP) in leaf morphology during drought stress. A total of 123 C2H2‐ZFP members were identified through a comparative genome survey in Populus alba × P. glandulosa ‘84K’. Among them, PagSUPa, an orthologous gene of Arabidopsis SUPERMAN, was selected due to its responsiveness to drought stress and was further confirmed to play a role in leaf development. Phenotypic characterization and cellular analysis revealed that PagSUPa fine‐tunes the duration of cell proliferation in the adaxial epidermis, thereby influencing leaf morphology by modulating leaf adaxial‐abaxial polarity. Additionally, we found that PagSUPa directly suppresses the expression of PHRAGMOPLAST ORIENTING KINESIN1 (PagPOK1) and PagPOK2, genes encoding proteins involved in phragmoplast orientation and position, which results in impaired cytokinesis and cell wall organization. This study provides novel insights into the regulatory network governed by the SUP gene during leaf development, specifically in relation to cell division.

C2H2-type zinc-finger protein BCL11B suppresses avian Leukosis virus subgroup J replication by regulating apoptosis

Apoptosis plays a crucial role in host antiviral defense. The avian leukosis virus subgroup J (ALV-J), an avian oncogenic retrovirus, has been shown to suppress apoptosis while promoting its own replication. ALV-J induces myeloid tumors and hemangiomas in chickens resulting in significant economic losses for commercial layer and meat-type chicken production. B-cell lymphoma/leukemia 11B (Bcl11b) encodes a C2H2-type zinc finger protein—BCL11B, that exerts critical functions in cell proliferation, differentiation, and plays an essential role in the immune system. Previous study has been shown that Bcl11b is associated with ALV-J infection. In this study, we further investigated the pathological changes in ALV-J infected cells and examined the role and expression regulation of chicken Bcl11b. Our results demonstrate that Bcl11b, as an interferon-stimulated gene (ISG), encodes C2H2-type zinc finger protein BCL11B that promotes apoptosis to inhibit ALV-J infection. Additionally, gga-miR-1612 and gga-miR-6701-3p regulate apoptosis and are involved in ALV-J infection by targeting Bcl11b, thus revealing immune response strategies between the host and ALV-J. Although the underlying mechanisms require further validation, Bcl11b and its regulatory miRNAs are the first to demonstrate inhibition of ALV-J replication via apoptosis. BCL11B can a valuable target for treating diseases triggered by ALV-J infection.

SETDB1 deletion causes DNA demethylation and upregulation of multiple zinc-finger genes

BackgroundSETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation.Methods and resultsWe investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1’s function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells.ConclusionsSETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

Hydrogen peroxide positively regulates ABA signaling via oxidative modification of the C2H2-type zinc finger protein ZFP36 in rice

The rice zinc finger protein ZFP36 serves as a pivotal regulator of the hydrogen peroxide (H2O2) signaling pathway in response to abscisic acid (ABA). Its role is crucial for integrating H2O2 signals with the plant defense mechanisms against water deficit and oxidative stress. However, it remains unclear whether ZFP36 directly modulates ABA-induced H2O2 signaling. This study explored the effects of oxidative post-translational modifications (OxiPTMs) on ZFP36 in rice, with an emphasis on the H2O2-induced oxidation through its cysteine (Cys) residues. We found that ZFP36 undergoes oxidative modification as a target of H2O2 in the presence of ABA, specifically at Cys32. Employing quantitative detection and fluorescence assays, we observed that ZFP36 oxidation enhances the expression and activity of genes encoding protective antioxidant enzymes. Moreover, our investigation into the thioredoxin (Trx) and glutaredoxin (Grx) families revealed that OsTrxh1 facilitates the reduction of oxidized ZFP36. Genetic evidence indicates that ZFP36 positively influences rice resilience to oxidative and water stress, while OsTrxh1 exerts an opposing effect. These insights reveal a distinctive pathway for plant cells to perceive ABA-induced H2O2 signaling, advance our comprehension of H2O2 signaling dynamics, and ABA-related plant responses, and lay a vital groundwork for enhancing crop stress tolerance.

SUPPRESSOR OF FRIGIDA 4 cooperates with the histone methylation reader EBS to positively regulate root development.

Maintenance and homeostasis of the quiescent center (QC) in Arabidopsis (Arabidopsis thaliana) root apical meristems are critical for stem cell organization and root development. Despite great progress in relevant research, the molecular mechanisms that determine the root stem cell fate and QC still need further exploration. In Arabidopsis, SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes a C2H2-type zinc finger protein that represses flowering by transcriptional activation of FLOWERING LOCUS C (FLC) through the FRIGIDA (FRI) pathway, and EARLY BOLTING IN SHORT DAYS (EBS) is a bivalent histone reader that prevents premature flowering. Here, we found that SUF4 directly interacts with EBS in vivo and in vitro. Loss of function of SUF4 and/or EBS resulted in disorganization of the QC, aberrant cell division, and stunted root growth. RNA-seq and reverse transcription quantitative real-time polymerase chain reaction analysis revealed that SUF4 and EBS coregulate many root development-related genes. A series of biochemical analyses demonstrated that SUF4 directly binds to the promoter of SCARECROW (SCR), which encodes a key regulator of root development. Chromatin immunoprecipitation assay indicated that both SUF4 and EBS are recruited to the SCR locus in an interdependent manner to promote H3K4me3 levels and suppress H3K27me3 levels, thereby activating the expression of SCR. These findings improve our understanding of the function of SUF4 and EBS and provide insights into the molecular mechanism that couples a transcription factor and a histone methylation reader to modulate QC specification and root development in Arabidopsis.

A novel MYC-ZNF706-SLC7A11 regulatory circuit contributes to cancer progression and redox balance in human hepatocellular carcinoma.

The oncogenic potential of chromosome 8q22 copy number gain in liver cancer remains to be depicted. Here, we report that ZNF706, encoded by a gene mapped to chromosome 8q22, is a C2H2-type zinc finger protein. However, the biological function and mechanism of ZNF706 have been poorly investigated. Clinically, ZNF706 expression was elevated in hepatocellular carcinoma (HCC), and high ZNF706 expression was associated with unfavorable survival in HCC patients. Functional experiments revealed that ZNF706 knockdown inhibited HCC progression both in vitro and in vivo. RNA sequencing (RNA-seq) and chromatin immunoprecipitation-based deep sequencing (ChIP-seq) revealed that mechanistically, ZNF706 is a crucial ferroptosis regulator and that SLC7A11 is a critical target of ZNF706. In addition, ZNF706 knockdown inhibited SLC7A11 expression, increased lipid peroxidation, and promoted ferroptosis. Further analysis revealed that ZNF706 is a novel direct target transcriptionally activated by MYC in HCC cells. Importantly, MYC depletion reduced SLC7A11-mediated redox homeostasis, and this effect was reversed by ZNF706 reexpression. Collectively, our data demonstrate that ZNF706 is a potential oncogene in liver cancer and functions as a ferroptosis regulator by modulating SLC7A11 expression, constituting a potential therapeutic target for HCC.

Genome-wide analysis of C2H2 zinc finger family and their response to abiotic stresses in apple

C2H2-type zinc finger proteins are one of the most widely studied families in plants and play important roles in abiotic stress responses. In the present study, the physicochemical properties, chromosomal locations, evolutionary relationships, and gene structures of 54 C2H2 zinc finger protein (ZFP) family members were analyzed in apple. The MdC2H2-ZFP genes were phylogenetically clustered into seven subfamilies distributed in different densities on 16 chromosomes. The RNA-seq data from various tissues revealed that MdC2H2-ZFPs differentially expressed among root, stem, leaf, flower, and fruits. Quantitative analysis of its expression characteristics showed that the MdC2H2-ZFP genes were rapidly induced as exposure to abiotic stresses such as drought, salt and low temperature etc. Under drought stress, the expression of eight members was significantly up-regulated, and the highest was obtained from MdC2H2-17; as exposure to salt stress, nine MdC2H2-ZFPs was obviously up-regulated, with the highest expression of MdC2H2-13; and under low temperature stress, the expression of seven members was highly up-regulated, and MdC2H2-13 also demonstrated the highest expression which is same as the case under salt stress. Therefore, some members of MdC2H2-ZFP gene family considerably involve in the multiple abiotic stress responses, which may better understand the function of this family and facilitate the breeding of apple for stress tolerance.

Arabidopsis SEC13B Interacts with Suppressor of Frigida 4 to Repress Flowering.

SECRETORY13 (SEC13) is an essential member of the coat protein complex II (COPII), which was reported to mediate vesicular-specific transport from the endoplasmic reticulum (ER) to the Golgi apparatus and plays a crucial role in early secretory pathways. In Arabidopsis, there are two homologous proteins of SEC13: SEC13A and SEC13B. SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes a C2H2-type zinc finger protein that inhibits flowering by transcriptionally activating the FLOWERING LOCUS C (FLC) through the FRIGIDA (FRI) pathway in Arabidopsis. However, it remains unclear whether SEC13 proteins are involved in Arabidopsis flowering. In this study, we first identified that the sec13b mutant exhibited early flowering under both long-day and short-day conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that both SEC13A and SEC13B were expressed in all the checked tissues, and transient expression assays indicated that SEC13A and SEC13B were localized not only in the ER but also in the nucleus. Then, we identified that SEC13A and SEC13B could interact with SUF4 in vitro and in vivo. Interestingly, both sec13b and suf4 single mutants flowered earlier than the wild type (Col-0), whereas the sec13b suf4 double mutant flowered even earlier than all the others. In addition, the expression of flowering inhibitor FLC was down-regulated, and the expressions of flowering activator FLOWERING LOCUS T (FT), CONSTANS (CO), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) were up-regulated in sec13b, suf4, and sec13b suf4 mutants, compared with Col-0. Taken together, our results indicated that SEC13B interacted with SUF4, and they may co-regulate the same genes in flowering-regulation pathways. These results also suggested that the COPII component could function in flowering in Arabidopsis.

Self S-RNase reduces the expression of two pollen-specific COBRA genes to inhibit pollen tube growth in pear

Due to self-incompatibility (SI) prevents self-fertilization, natural or artificial cross-pollination has been conducted in many orchards to stabilize fruit yield. However, it is still puzzled which routes of self S-RNase arresting pollen tube growth. Herein, 17 COBRA genes were isolated from pear genome. Of these genes, the pollen-specifically expressed PbCOB.A.1 and PbCOB.A.2 positively mediates pollen tube growth. The promoters of PbCOB.A.1 and/or PbCOB.A.2 were bound and activated by PbABF.E.2 (an ABRE-binding factor) and PbC2H2.K16.2 (a C2H2-type zinc finger protein). Notably, the expressions of PbCOB.A.1, PbCOB.A.2, and PbC2H2.K16.2 were repressed by self S-RNase, suggesting that self S-RNase reduces the expression of PbCOB.A.1 and PbCOB.A.2 by decreasing the expression of their upstream factors, such as PbC2H2.K16.2, to arrest pollen tube growth. PbCOB.A.1 or PbCOB.A.2 accelerates the growth of pollen tubes treated by self S-RNase, but can hardly affect level of reactive oxygen species and deploymerization of actin cytoskeleton in pollen tubes and cannot physically interact with any reported proteins involved in SI. These results indicate that PbCOB.A.1 and PbCOB.A.2 may not relieve S-RNase toxicity in incompatible pollen tube. The information provides a new route to elucidate the arresting pollen tube growth during SI reaction.Graphical

A C2H2-type zinc finger protein ZAT12 of Poncirus trifoliata acts downstream of CBF1 to regulate cold tolerance.

The Cys2/His2 (C2H2)-type zinc finger family has been reported to regulate multiple aspects of plant development and abiotic stress response. However, the role of C2H2-type zinc finger proteins in cold tolerance remains largely unclear. Through RNA-sequence analysis, a cold-responsive zinc finger protein, named as PtrZAT12, was identified and isolated from trifoliate orange (Poncirus trifoliata L. Raf.), a cold-hardy plant closely related to citrus. Furthermore, we found that PtrZAT12 was markedly induced by various abiotic stresses, especially cold stress. PtrZAT12 is a nuclear protein, and physiological analysis suggests that overexpression of PtrZAT12 conferred enhanced cold tolerance in transgenic tobacco (Nicotiana tabacum) plants, while knockdown of PtrZAT12 by virus-induced gene silencing (VIGS) increased the cold sensitivity of trifoliate orange and repressed expression of genes involved in stress tolerance. The promoter of PtrZAT12 harbors a DRE/CRT cis-acting element, which was verified to be specifically bound by PtrCBF1 (Poncirus trifoliata C-repeat BINDING FACTOR1). VIGS-mediated silencing of PtrCBF1 reduced the relative expression levels of PtrZAT12 and decreased the cold resistance of trifoliate orange. Based on these results, we propose that PtrZAT12 is a direct target of CBF1 and plays a positive role in modulation of cold stress tolerance. The knowledge gains new insight into a regulatory module composed of CBF1-ZAT12 in response to cold stress and advances our understanding of cold stress response in plants.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae

BackgroundMelanin plays important roles in morphological development, survival, host–pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear.ResultsWe identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae.ConclusionsOur results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

FRI285 Evidence For ZIC1 As A Novel Gene For Isolated Hypogonadotropic Hypogonadism With Phenotypic Pleiotropic Intersection With Cerebellar Malformation

Abstract Disclosure: J. Cassin: None. D.L. Keefe: None. K.E. Sung: None. C.C. Bojo: None. G. Santiago: None. L. Plummer: None. K.B. Salnikov: None. S.B. Seminara: None. R. Balasubramania: None. P.L. Mellon: None. Isolated Hypogonadotropic Hypogonadism (IHH) is a condition marked by absent pubertal development and low sex steroids in the setting of low/normal gonadotropins. Mutations in known IHH genes only explain <50% of the genetic etiology suggesting significant missing heritability in IHH. To explore this missing IHH heritability, we examined whole exome sequencing data from approximately 1400 IHH probands for rare genetic variants in novel genes that are enriched in IHH patients. We identified four IHH probands with rare, novel heterozygous variants in ZIC1 (p.H134Rfs*21, p.Y287X, p.E299K, p.S413Y), a gene encoding C2H2-type zinc finger protein (rare variant enrichment p <0.0001 for both truncating and missense alleles). Prior literature review show that heterozygous deletions affecting the ZIC1 gene have been linked to Dandy Walker malformation [DWM], a cerebellar malformation, while gain-of-function ZIC1 single nucleotide variants have been linked to coronal craniosynostosis (premature cranial suture fusion). Notably, two IHH subjects with novel ZIC1 variants in our cohort exhibited posterior fossa abnormalities (DWM [p.H134Rfs*21] and ventriculomegaly [E299K]) but none had any cranial suture defects. Given the above genetic enrichment, phenotypic overlap between DWM and IHH, and ZIC1’s known high expression in the hypothalamus, we explored whether ZIC1 regulates the expression of hypothalamic releasing factors. We determined that ZIC1 overexpression increases GNRH1 luciferase expression in the immortalized GnRH cell line, GT1-7. We next explored whether the rare ZIC1 variants identified in the four IHH probands would affect this phenotype. We discovered that three of the four ZIC1 variants significantly reduced GNRH1-luciferase expression. Of those, one truncating mutation, p.H134Rfs*21, resulted in lower ZIC1 protein content in the cell and failure to localize to the nucleus, suggesting a hypomorphic mechanism of action. These findings implicate ZIC1 variants as putatively causal for IHH. Further studies will define the precise ZIC1 transcriptional targets that relate to ZIC1’s pleotropic effects on GnRH transcription, neuronal function, cerebellar development, and cranial suture fusion. Presentation: Friday, June 16, 2023

Genome-Wide Analysis of ZAT Gene Family in Osmanthus fragrans and the Function Exploration of OfZAT35 in Cold Stress.

Osmanthus fragrans is a popular ornamental and odorant plant with high commercial value, but its cultivation and exploitation are limited by low temperature. The ZAT (zinc finger of Arabidopsis thaliana) genes as a subclass of the C2H2-type zinc finger proteins (C2H2-ZFP) family play essential roles in various abiotic stresses. However, their roles in cold stress response in O. fragrans remain unclear. This study identified 38 OfZATs, which could be divided into 5 subgroups based on the phylogenetic tree, with OfZATs in the same subgroup harboring similar gene structures and motif patterns. In addition, 49 segmental and 5 tandem duplication events were detected among OfZAT genes, while some OfZAT genes exhibited specific expression patterns in different tissues. Furthermore, two OfZATs were induced in salt stress and eight OfZATs responded to cold stress. Interestingly, OfZAT35 showed a continuously increasing expression trend under cold stress, while its protein showed nucleus localization with no transcriptional activation activity. Transiently transformed tobacco overexpressing OfZAT35 exhibited a significantly higher relative electrolyte leakage (REL) level and increased activities of superoxide dismutase (SOD), peroxidase (POD), and Ascorbate peroxidase (APX), while there was significantly decreased activity of catalase (CAT). Moreover, CAT, DREB3, and LEA5, which are associated with cold stress, were dramatically decreased after cold treatment in transiently transformed tobacco, suggesting that overexpression of OfZAT35 negatively regulated cold stress. This study provides a basis for exploring the roles of ZAT genes and contributes to uncovering the mechanism of ZAT-mediated cold stress response in O. fragrans.

PhDREB1-PhZFP1-PhGolS1-1 regulator cascade contribute to cold tolerance by mediating galactinol and raffinose accumulation in petunia

The C-repeat/DRE Binding Factor (CBF/DREB1) gene, which plays a critical role in plant responses to cold stress, could positively regulate the accumulation of galactinol and raffinose, but the underlying molecular mechanisms remain elusive. We have previously confirmed that the C2H2-type zinc finger protein PhZFP1 plays a positive role in cold stress tolerance by modulating galactinol accumulation by directly regulating PhGolS1-1 in Petunia hybrida. However, whether and how CBF/DREB1 regulates PhZFP1 to mediate galactinol accumulation under cold stress remains largely unclear. Here, we report the biological and molecular characterization of PhDREB1F and PhDREB1I in petunia. We demonstrate that PhDREB1F and PhDREB1I function as nuclear transcription factors and activate PhZFP1 expression. Silencing of PhDREB1F or PhDREB1I mediated by virus-induced gene silencing (VIGS) increased the sensitivity to freezing stress in petunia plants. Besides, knocking-down of PhDREB1F or PhDREB1I decreased the expression of PhZFP1 and PhGolS1-1. Similarly, silencing of PhGolS1-1 increased cold sensitivity in petunia. And the contents of galactinol and raffinose in PhGolS1-1-VIGS plants were significantly decreased compared with those in TRV plants under chilling stress. What's more, PhDREB1F/1I, PhZFP1, and PhGolS1-1 all showed cold-induced expression patterns with different peaks. Collectively, our data indicate that PhDREB1F and PhDREB1I positively modulate cold stress tolerance through the PhDREB1s-PhZFP1-PhGolS1–1 pathway in petunia. This study provides new insights into the molecular mechanism of plant responsiveness to cold stress through CBF/DREB1 transcription factors.

Fine mapping and genetic analysis identified a C2H2-type zinc finger as a candidate gene for heading date regulation in wheat.

A minor-effect QTL, Qhd.2AS, that affects heading date in wheat was mapped to a genomic interval of 1.70-Mb on 2AS, and gene analysis indicated that the C2H2-type zinc finger protein gene TraesCS2A02G181200 is the best candidate for Qhd.2AS. Heading date (HD) is a complex quantitative trait that determines the regional adaptability of cereal crops, and identifying the underlying genetic elements with minor effects on HD is important for improving wheat production in diverse environments. In this study, a minor QTL for HD that we named Qhd.2AS was detected on the short arm of chromosome 2A by Bulked Segregant Analysis and validated in a recombinant inbred population. Using a segregating population of 4894 individuals, Qhd.2AS was further delimited to an interval of 0.41cM, corresponding to a genomic region spanning 1.70Mb (from 138.87 to 140.57Mb) that contains 16 high-confidence genes based on IWGSC RefSeq v1.0. Analyses of sequence variations and gene transcription indicated that TraesCS2A02G181200, which encodes a C2H2-type zinc finger protein, is the best candidate gene for Qhd.2AS that influences HD. Screening a TILLING mutant library identified two mutants with premature stop codons in TraesCS2A02G181200, both of which exhibited a delay in HD of 2-4days. Additionally, variations in its putative regulatory sites were widely present in natural accession, and we also identified the allele which was positively selected during wheat breeding. Epistatic analyses indicated that Qhd.2AS-mediated HD variation is independent of VRN-B1 and environmental factors. Phenotypic investigation of homozygous recombinant inbred lines (RILs) and F2:3 families showed that Qhd.2AS has no negative effect on yield-related traits. These results provide important cues for refining HD and therefore improving yield in wheat breeding programs and will deepen our understanding of the genetic regulation of HD in cereal plants.

The C2H2-type zinc-finger regulator AoKap5 is required for the growth and kojic acid synthesis in Aspergillus oryzae

Aspergillus oryzae is an important filamentous fungus widely used for the industrial production of fermented foods and secondary metabolites. The clarifying of the mechanism of the growth and secondary metabolites in A. oryzae is important for its industrial production and utilization. Here, the C2H2-type zinc-finger protein AoKap5 was characterized to be involved in the growth and kojic acid production in A. oryzae. The Aokap5-disrupted mutants were constructed by the CRISPR/Cas9 system, which displayed increased colony growth but decreased conidial formation. Deletion of Aokap5 enhanced the tolerance to cell-wall and oxidative but not osmotic stress. The transcriptional activation assay revealed that AoKap5 itself didn’t have transcriptional activation activity. Disruption of Aokap5 resulted in the reduced production of kojic acid, coupled with the reduced expression of the kojic acid synthesis genes kojA and kojT. Meanwhile, overexpression of kojT could rescue the decreased production of kojic acid in Aokap5-deletion strain, indicating that Aokap5 serves upstream of kojT. Furthermore, the yeast one-hybrid assay demonstrated that AoKap5 could directly bind to the kojT promoter. These findings suggest that AoKap5 regulates kojic acid production through binding to the kojT promoter. This study provides an insight into the role of zinc finger protein in the growth and kojic acid biosynthesis of A. oryzae.