Zinc Finger Research Articles

De novo variants in RYBP are associated with a severe neurodevelopmental disorder and congenital anomalies.

Polycomb group (PcG) proteins are key epigenetic regulators of gene transcription. Multiple neurodevelopmental disorders have been associated with pathogenic variants in genes encoding PcG proteins. RYBP is a core component of the non-canonical Polycomb Repressor Complex 1; however, its role in disease is unclear. Functional consequences of RYBP variants were assessed through in vitro cellular studies and in vivo Drosophila melanogaster studies. We describe seven individuals with heterozygous de novo variants in RYBP and clinical findings including severe developmental delay, dysmorphisms and multiple congenital anomalies. We show that all the single nucleotide variants in RYBP localize to the N-terminal domain of the gene, which encodes the zinc finger domain and ubiquitin binding moiety. In vitro studies demonstrate that the RYBP c.132C>G p.(Cys44Trp) variant causes reduced protein expression but does not affect binding of YY1, RING1B or ubiquitin. In vivo overexpression studies in Drosophila melanogaster show a dramatic functional difference from human RYBP and variant forms affecting the C44 amino acid residue. DNA methylation studies suggest a possible episignature that may be associated with RYBP-related disorder. Heterozygous de novo variants in RYBP are associated with an identifiable syndromic neurodevelopmental disorder with multiple congenital anomalies.

Modified Vaccinia Virus Ankara Selectively Targets Human Cancer Cells With Low Expression of the Zinc-Finger Antiviral Protein.

Oncolytic viruses are emerging as promising cancer therapeutic agents, with several poxviruses, including vaccinia virus (VACV) and myxoma virus, showing significant potential in preclinical and clinical trials. Modified vaccinia virus Ankara (MVA), a laboratory-derived VACV strain approved by the FDA for mpox and smallpox vaccination, has been shown to be incapable of replicating in human cells unless zinc finger antiviral protein (ZAP) is repressed. Notably, ZAP deficiency is prevalent in various cancer types. We hypothesized that MVA could selectively target and replicate in ZAP-deficient cancer cells. Our study examined MVA's replication across multiple cancer cell lines with varying ZAP expression levels, revealing that MVA replicates more efficiently in cells with lower ZAP expression. Additionally, we assessed MVA's oncolytic potential using a xenograft mouse model, where cancer cells were transplanted into immunodeficient mice. The data demonstrated that MVA significantly reduced tumors with lower ZAP expression without causing morbidity in nude mice. These findings suggest that MVA holds promise for further development as a targeted therapy for ZAP-deficient cancers.

Denovo variants in POGZ and YY1 genes: The novel mega players for neurodevelopmental syndromes in two unrelated consanguineous families.

Novel denovo variants of exome sequences are major cause of pathogenic neurodevelopmental disorders with a dominant genetic mechanism that emphasize their heterogeneity and complex phenotypes. White Sutton syndrome and Gabriele-de-Vries syndrome are congenital neuro-impairments with overlap of severe intellectual disability, microcephaly, convulsions, seizures, delayed development, dysmorphism of faces, retinal diseases, movement disorders and autistic traits. POGZ gene codes for pogo transposable element-derived zinc-finger protein and YY1 gene regulates transcription, chromatin, and RNA-binding proteins that have been associated with White Sutton and Gabriele-de-Vries syndromes, in recent data. We present probands of two unrelated consanguineous families with complicated, unexplained neurocognitive syndromic characteristics clinically undiagnosed. Objectives of the study were to identify altered genetics and protein characteristics underlying molecular pathological pathways in both the patients. Whole exome sequencing identifies novel, denovo missense variant NM_015100.4: c.776 C>T (p. Pro259Leu) in exons 19 of POGZ gene and non-frameshift variant NM_003403.5: c.141_143delGGA (p. Glu47del) in exon 1 of YY1 gene for White Sutton syndrome in eight years five-month-old girl and Gabriele-de-Vries syndrome in seven years eight months old boy residing in Rawalpindi and Chakwal districts of Punjab, Pakistan respectively. Protein modelling for identified variants predicts size and conformation modifications in mutated amino acid residues that lead to damaging effects in the conserved domains expressed as neurological pathophysiology. The present study widens the diversely ethnic and highly inbred gene pool of Punjab, Pakistan population for spontaneously originated deleterious mutations and contributes to the continuously expanding phenotypic canvas. Molecular genetic identification and personalized diagnosis for the patients suffering from complicated neurodevelopmental phenotypes, for better care, management of day-to-day activities and prolonged life span are the utmost hopes.

Feedback regulation of m6A modification creates local auxin maxima essential for rice microsporogenesis.

N6-methyladenosine (m6A) RNA modification and its effectors control various plant developmental processes, yet whether and how these effectors are transcriptionally controlled to confer functional specificity so far remain elusive. Herein, we show that a rice C2H2 zinc-finger protein, OsZAF, specifically activates the expression of OsFIP37 encoding a core component of the m6A methyltransferase complex during microsporogenesis in rice anthers. OsFIP37, in turn, facilitates m6A modification and stabilization of an auxin biosynthesis gene OsYUCCA3 to promote auxin biosynthesis in anthers. This elevates auxin levels coinciding with upregulation of an auxin response factor OsARF12 that positively controls OsZAF, thus creating a positive feedback circuit whereby OsFIP37 is continuously activated for local auxin production. Our findings suggest that OsZAF-dependent feedback regulation of m6A modification is integral to local auxin biosynthesis and signaling in anthers, which facilitates the timely generation of auxin maxima required for male meiosis in rice.

Understanding the genetics of sex determination in insects and its relevance to genetic pest management.

Sex determination pathways regulate male and female-specific development and differentiation and offer potential targets for genetic pest management methods. Insect sex determination pathways are comprised of primary signals, relay genes and terminal genes. Primary signals of coleopteran, dipteran, hymenopteran and lepidopteran species are highly diverse and regulate the sex-specific splicing of relay genes based on the primary signal dosage, amino acid composition or the interaction with paternally inherited genes. In coleopterans, hymenopterans and some dipterans, relay genes are Transformer orthologs from the serine-arginine protein family that regulate sex-specific splicing of the terminal genes. Alternative genes regulate the splicing of the terminal genes in dipterans that lack Transformer orthologs and lepidopterans. Doublesex and Fruitless orthologs are the terminal genes. Doublesex and Fruitless orthologs are highly conserved zinc-finger proteins that regulate the expression of downstream proteins influencing physical traits and courtship behaviours in a sex-specific manner. Genetic pest management methods can use different mechanisms to exploit or disrupt female-specific regions of different sex determination genes. Female-specific regions of sex determination genes can be exploited to produce a lethal gene only in females or disrupted to impede female development or fertility. Reducing the number of fertile females in pest populations creates a male-biased sex ratio and eventually leads to the local elimination of the pest population. Knowledge on the genetic basis of sex determination is important to enable these sex determination pathways to be exploited for genetic pest management.

The drosophila zinc finger Protein CG9609 interacts with the Deubiquitinating (DUB) module of the SAGA complex and participates in the regulation of Transcription

In previous studies, we found that the zinc finger proteins Su(Hw) and CG9890 interact with the Drosophila SAGA complex and participate in the formation of active chromatin structure and transcriptional regulation. In this research, we discovered the interaction of the DUB module of the SAGA complex with another zinc finger protein, CG9609. ChIP-Seq analysis was performed and CG9609 binding sites in the Drosophila genome were identified. Analysis of binding sites showed that they are localized predominantly at gene promoters. The CG9609 protein has been shown to be involved in the regulation of gene expression.

The Drosophila Zinc Finger Proteins Aef1 and Cg10543 Are Co-Localized with SAGA, SWI/SNF and ORC Complexes on Gene Promoters and Involved in Transcription Regulation

In previous studies, we purified the DUB-module of the Drosophila SAGA complex and showed that a number of zinc proteins interact with it, including Aef1 and CG10543. In this work, we conducted a genome-wide study of the Aef1 and CG10543 proteins and showed that they are localized predominantly on the promoters of active genes. The binding sites of these proteins colocalize with the SAGA and dSWI/SNF chromatin modification and remodeling complexes, as well as with the ORC replication complex. It has been shown that the Aef1 and CG10543 proteins are involved in the regulation of the expression of some genes on the promoters of which they are located. Thus, the Aef1 and CG10543 proteins are new participants in the cell transcriptional network and colocalize with the main transcription and replication complexes of Drosophila.

Functional anatomy of zinc finger antiviral protein complexes.

ZAP is an antiviral protein that binds to and depletes viral RNA, which is often distinguished from vertebrate host RNA by its elevated CpG content. Two ZAP cofactors, TRIM25 and KHNYN, have activities that are poorly understood. Here, we show that functional interactions between ZAP, TRIM25 and KHNYN involve multiple domains of each protein, and that the ability of TRIM25 to multimerize via its RING domain augments ZAP activity and specificity. We show that KHNYN is an active nuclease that acts in apartly redundant manner with its homolog N4BP1. The ZAP N-terminal RNA binding domain constitutes a minimal core that is essential for antiviral complex activity, and we present a crystal structure of this domain that reveals contacts with the functionally required KHNYN C-terminal domain. These contacts are remote from the ZAP CpG binding site and would not interfere with RNA binding. Based on our dissection of ZAP, TRIM25 and KHNYN functional anatomy, we could design artificial chimeric antiviral proteins that reconstitute the antiviral function of the intact authentic proteins, but in the absence of protein domains that are otherwise required for activity. Together, these results suggest a model for the RNA recognition and action of ZAP-containing antiviral protein complexes.

Genome-wide association mapping in safflower (Carthamus tinctorius L.) for genetic dissection of drought tolerance using DArTseq markers

Understanding the genetic basis of drought tolerance in safflower (Carthamus tinctorius L.) is essential for developing resilient varieties. In this study, we performed a genome-wide association study (GWAS) using DArTseq markers to identify marker-trait associations (MTAs) linked to drought tolerance across 90 globally diverse safflower genotypes. These genotypes were evaluated under both rainfed (drought) and irrigated conditions over three consecutive years (2016–2018). Significant variation in seed yield and morphological traits was observed between genotypes and across environments, with notably reduced performance under rainfed conditions. A total of 66 MTAs were identified for key agronomic traits, including seed yield, 1000-seed weight, number of heads per plant, and days to maturity, with 45 MTAs showing significant associations with the stress-tolerance index (STI). Twelve MTAs were consistently detected across both stress and non-stress conditions, suggesting stable loci for breeding applications. BLAST searches indicated that several identified markers corresponded to genes involved in abiotic stress response, including biotin carboxylase, serine/threonine-protein kinase, and zinc finger domain proteins. The clustering of genotypes based on drought-tolerance coefficient (DC) and STI values grouped genotypes into drought-susceptible and drought-tolerant categories, with no strong correlation to geographic origin. These findings provide valuable insights into the genetic architecture of drought tolerance in safflower and highlight candidate loci for marker-assisted selection, supporting the development of drought-resilient varieties.

LncRNA DNM1P35 sponges hsa-mir-326 to promote ovarian cancer progression

Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in cancer progression. We found lncRNA DNM1P35 is elevated in ovarian tumors compared to normal tissues, and demonstrated that lncRNA DNM1P35 promoted cancer cell proliferation, migration and invasion in SK-OV-3 and OVCAR-3 cell lines. Furthermore, lncRNA DNM1P35 also facilitated the epithelial-mesenchymal transition (EMT) of ovarian cancer cells. Mechanistic studies identified microRNA-326 (miR-326) as a target of lncRNA DNM1P35. Overexpression of miR-326 diminished the tumor-promoting activity of lncRNA DNM1P35, resulting in reduction of Zinc finger E-box-binding homeobox 1 (ZEB1) expression and EMT features. We further revealed that ZEB1, a master transcription factor for EMT that is negatively regulated by miR-326, was essential for lncRNA DNM1P35-mediated cancer cell progression and EMT. Loss of ZEB1 led to compromised pro-tumoral activity of lncRNA DNM1P35. In vivo studies using a xenograft mouse model of ovarian cancer revealed that tumors with higher levels of lncRNA DNM1P35 led to shorter survival, increased tumor burden, as well as elevated expression of proliferative marker Ki67 and EMT marker ZEB1. Our comprehensive study underscored the significance of lncRNA DNM1P35 in ovarian cancer progression, elucidating the underlying mechanism through miR-326/ZEB1 axis to promote ovarian cancer progression.

Human cytomegalovirus RNA2.7 inhibits ferroptosis by upregulating ferritin and GSH via promoting ZNF395 degradation.

Human cytomegalovirus (HCMV) is a herpes virus with a long replication cycle. HCMV encoded long non-coding RNA termed RNA2.7 is the dominant transcript with a length of about 2.5kb, accounting for 25% of total viral transcripts. Studies have shown that HCMV RNA2.7 inhibits apoptosis caused by infection. The effect of RNA2.7 on other forms of cell death is still unclear. In this work, we found that RNA2.7 deletion significantly decreased the viability of HCMV-infected cells, while treatment with ferroptosis inhibitor Fer-1 rescued the infection-induced cell death, demonstrating an anti-ferroptosis role of RNA2.7. The results further showed that RNA2.7 inhibited ferroptosis via enhancing Ferritin Heavy Chain 1 (FTH1) and Solute Carrier Family 7 Member 11 (SLC7A11) expression in Erastin treated cells without involving other viral components. Pooled Genome-wide CRISPR screening revealed zinc finger protein 395 (ZNF395) as a new regulator repressing the expression of FTH1 and SLC7A11. HCMV RNA2.7 promoted proteasome-mediated degradation of ZNF395 that resulted in upregulation of FTH1 and SLC7A11 to inhibit ferroptosis, therefore maintain survival in host cells and complete replication of virus.

C2H2 Zinc Finger Protein Family Analysis of Rosa rugosa Identified a Salt-Tolerance Regulator, RrC2H2-8.

Rosa rugosa is a representative aromatic species. Wild roses are known for their strong tolerance to highly salty environments, whereas cultivated varieties of roses exhibit lower salt stress tolerance, limiting their development and industrial expansion. Previous studies have shown that C2H2-type zinc finger proteins play a crucial role in plants' resistance to abiotic stresses. In this study, 102 C2H2-type zinc finger genes (RrC2H2s) were identified in R. rugosa via a comprehensive approach. These genes were categorized into three lineages, and their motif constitutions were grouped into four classes. RrC2H2s were distributed across all seven rose chromosomes, with 15 paralogous gene pairs identified within synteny regions. Additionally, 43 RrC2H2s showed differential expression across various tissues under salt stress, with RrC2H2-8 being the only gene consistently repressed in all tissues. Subcellular localization analysis revealed that the RrC2H2-8 protein was localized in the nucleus. The heterologous expression of RrC2H2-8 in Arabidopsis significantly improved its growth under salt stress compared to the wild-type (WT) plants. Furthermore, the malondialdehyde content in the roots of transgenic Arabidopsis was significantly lower than that in the WT, suggesting that RrC2H2-8 enhanced salt tolerance by reducing cellular damage. This study provides a systematic understanding of the RrC2H2 family and identifies RrC2H2-8 as a regulator of salt tolerance, laying a foundation for future research on the mechanisms of salt stress regulation by RrC2H2.

Genetic Investigation of Regulatory Regions of MKRN3 and DLK1 Genes in Children with Central Precocious Puberty.

Most of the loss-of-function mutations described in children with central precocious puberty (CPP) is located into the coding regions of MKRN3 or DLK1 genes. Notably, potential abnormalities in the regulatory regions of these CPP-genes are rarely explored. To search for pathogenic variants in the regulatory regions of MKRN3 and DLK1 genes in patients with familial or idiopathic CPP. A cohort of 217 individuals with CPP (205 girls and 12 boys; 143 sporadic cases and 74 familial cases) was investigated. Rare and potentially pathogenic variants in the coding regions of both genes were previously excluded. Analyses of the regulatory regions of MKRN3 and DLK1 were performed using polymerase chain reaction and direct automated sequencing (Sanger method). Circulating serum levels of MKRN3 and DLK1 proteins were measured using ELISA assay. We identified a heterozygous allelic variant (c.-265G>A), previously associated with CPP, located in the promoter region of the MKRN3 gene in three girls from two unrelated families. In silico prediction analysis indicated that the c.-265G>A variant was in the ZNF384 binding region. ZNF384 gene encodes a C2H2-type zinc finger protein, which might act as a transcription factor. MKRN3 serum levels varied from 197.5 pg/mL to 1,907 pg/mL and were relatively lower in patients with CPP who carried the c.-265G>A variant. No pathogenic allelic variant was found in the regulatory region of the DLK1 gene. Pathogenic variants in the regulatory region of MKRN3 gene are rare and can be associated with the CPP phenotype.

Genome-Wide Analysis of C2H2 Zinc Finger Gene Family and Exploration of Genes Related to Prickle Development in Zanthoxylum armatum

Zanthoxylum armatum (Z. armatum) is a significant economic tree species known for its medicinal and edible properties. However, the presence of prickles on Z. armatum poses a considerable challenge to the advancement of its industry. Numerous studies have indicated that the C2H2 zinc finger protein (C2H2-ZFPs) families are crucial in the development of plant trichomes or prickles. This study identified 78 ZaC2H2 genes from the Z. armatum genome, categorizing them into three groups and analyzing their protein physicochemical properties, chromosomal locations, conserved domains, and gene structures. The evolutionary analysis indicates that the amplification of ZaC2H2 genes primarily results from whole-genome duplication or segmental duplication, and these genes have undergone strong purifying selection pressure throughout their evolutionary history. The analysis of cis-acting elements revealed that they contain various hormone response elements, such as ABRE, AuxRR, the CGTCA motif, GARE motifs, and TCA elements, which are responsive to ABA, IAA, MeJA, GA, and SA signals. RT-qPCR was employed to assess the expression levels of the candidate genes ZaC2H2-45, ZaC2H2-46, ZaC2H2-49, and ZaC2H2-55 under the treatment of five hormones. The results indicated that the expression levels of the ZaC2H2-46 and ZaC2H2-55 genes were significantly up-regulated under NAA, SA, and MeJA treatments. These results will help to further understand the characteristics of the ZaC2H2 gene family and provide a theoretical basis for studying the development of prickles.

The molecular framework balancing growth and defense in response to PEP-induced signals in Arabidopsis.

Elevated stress signaling compromises plant growth by suppressing proliferative and formative division in the meristem. Plant Elicitor Peptide (PEP), an endogenous danger signal triggered by biotic and abiotic stresses in Arabidopsis (Arabidopsis thaliana), suppresses proliferative division, alters xylem vessel organization, and disrupts cell-to-cell symplastic connections in roots. To gain insight into the dynamic molecular framework that modulates root development under elevated danger signals, we performed a time-course RNA-sequencing analysis of the root meristem after synthetic PEP1 treatment. Our analyses revealed that SALT TOLERANCE ZINC FINGER (STZ) and its homologs are a potential nexus between the stress response and proliferative cell cycle regulation. Through functional, phenotypic, and transcriptomic analyses, we observed that STZ differentially controls the cell cycle, cell differentiation, and stress response genes in various tissue layers of the root meristem. Moreover, we determined the STZ expression level critical for enabling the growth-defense tradeoff. These findings provide valuable information about the dynamic gene expression changes that occur upon perceiving danger signals in the root meristem and potential engineering strategies to generate stress-resilient plants.

The Impact of Bevacizumab and miR200c on EMT and EGFR-TKI Resistance in EGFR-Mutant Lung Cancer Organoids.

Objectives: This research aims to investigate the mechanisms of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC), particularly focusing on the role of the epithelial-mesenchymal transition (EMT) within the tumor microenvironment (TME). Materials and Methods: We employed an in vitro three-dimensional organoid model that mirrors the physiology of human lung cancer. These organoids consist of lung cancer cells harboring specific EGFR mutations, human mesenchymal stem cells, and human umbilical vein endothelial cells. We analyzed EMT and drug resistance markers, and evaluated the effects of the anti-angiogenic agent Bevacizumab and micro-RNA miR200c. Results: The study identified a significant link between EMT and EGFR-TKI resistance. Notable findings included a decrease in E-cadherin and an increase in Zinc Finger E-Box Binding Homeobox 1 (ZEB1), both of which influenced EMT and resistance to treatment. Bevacizumab showed promise in improving drug resistance and mitigating EMT, suggesting an involvement of the Vascular Endothelial Growth Factor (VEGF) cascade. Transfection with miR200c was associated with improved EMT and drug resistance, further highlighting the role of EMT in TKI resistance. Conclusions: Our research provides significant insights into the EMT-driven EGFR-TKI resistance in NSCLC and offers potential strategies to overcome resistance, including the use of Bevacizumab and miR200c. However, due to the limitations in organoid models in replicating precise human cancer TME and the potential influence of specific EGFR mutations, further in vivo studies and clinical trials are necessary for validation.

ZDHHC20 mediated S-palmitoylation of fatty acid synthase (FASN) promotes hepatocarcinogenesis

BackgroundProtein palmitoylation is a reversible fatty acyl modification that undertakes important functions in multiple physiological processes. Dysregulated palmitoylations are frequently associated with the formation of cancer. How palmitoyltransferases for S-palmitoylation are involved in the occurrence and development of hepatocellular carcinoma (HCC) is largely unknown.MethodsChemical carcinogen diethylnitrosamine (DEN)-induced and DEN combined CCl4 HCC models were used in the zinc finger DHHC-type palmitoyltransferase 20 (ZDHHC20) knockout mice to investigate the role of ZDHHC20 in HCC tumourigenesis. Palmitoylation liquid chromatography-mass spectrometry analysis, acyl-biotin exchange assay, co-immunoprecipitation, ubiquitination assays, protein half-life assays and immunofluorescence microscopy were conducted to explore the downstream regulators and corresponding mechanisms of ZDHHC20 in HCC.ResultsKnocking out of ZDHHC20 significantly reduced hepatocarcinogenesis induced by chemical agents in the two HCC mouse models in vivo. 97 proteins with 123 cysteine sites were found to be palmitoylated in a ZDHHC20-dependent manner. Among these, fatty acid synthase (FASN) was palmitoylated at cysteines 1471 and 1881 by ZDHHC20. The genetic knockout or pharmacological inhibition of ZDHHC20, as well as the mutation of the critical cysteine sites of FASN (C1471S/C1881S) accelerated the degradation of FASN. Furthermore, ZDHHC20-mediated FASN palmitoylation competed against the ubiquitin-proteasome pathway via the E3 ubiquitin ligase complex SNX8-TRIM28.ConclusionsOur findings demonstrate the critical role of ZDHHC20 in promoting hepatocarcinogenesis, and a mechanism underlying a mutual restricting mode for protein palmitoylation and ubiquitination modifications.

ZFP36L2 Is a Potential Prognostic Marker of IL1β+ Osteosarcoma.

Background: Osteosarcoma stands as the predominant bone malignancy afflicting children and young adults. Despite strides in treatment, the enduring reality is that the long-term survival rates for patients grappling with recurrences and metastases linger at a mere 30%. This underscores the pressing demand for novel prognostic markers and therapeutic avenues to improve outcomes and offer hope to those battling this formidable disease. ZFP36L2, a member of the tristetraprolin family of CCCH zinc finger proteins, stands out for its pivotal role in posttranscriptional modifications and its ability to modify tumor microenvironments. Methods: We obtained RNA-seq data from TCGA and GTEx cohorts to investigate the expression of ZFP36L2 in tumor and normal tissues. We also utilized GO/KEGG analysis and immune infiltration analysis to verify the relationship between ZFP36L2 and immune cells. A Kaplan-Meier survival curve was used to study the relationship between ZFP36L2 and IL1β in osteosarcoma. Single-cell data analysis and cell-cell communication analysis were used to discover the therapeutic potential of ZFP36L2 in osteosarcoma. Results: This study elucidates the specific expression pattern of ZFP36L2 in tumors. ZFP36L2 is associated with metastasis in sarcoma, but has no statistically significant correlation with survival rate. ZFP36L2 has been shown to be associated with T cells and macrophages in the tumor microenvironment through GO/KEGG analysis and immune infiltration analysis. The survival analysis results show that ZFP36L2 can serve as a biomarker in IL1β+ osteosarcoma. Single-cell sequencing analysis shows that ZFP36L2 is present in IL1β+ macrophages. Cell-cell communication analysis indicates that ZFP36L2 targets TNF in IL1β+ osteosarcoma, thereby improving prognosis. Conclusions: ZFP36L2 has potential as a prognostic marker in IL1β+ osteosarcoma patients.

The Wilms' Tumor Suppressor WT1 in Cardiomyocytes: Implications for Cardiac Homeostasis and Repair.

The Wilms' tumor suppressor WT1 is essential for the development of the heart, among other organs such as the kidneys and gonads. The Wt1 gene encodes a zinc finger transcription factor that regulates proliferation, cellular differentiation processes, and apoptosis. WT1 is also involved in cardiac homeostasis and repair. In adulthood, WT1-expression levels are lower compared to those observed through development, and WT1 expression is restricted to a few cell types. However, its systemic deletion in adult mice is lethal, demonstrating that its presence is also key for organ maintenance. In response to injury, the epicardium re-activates the expression of WT1, but little is known about the roles it plays in cardiomyocytes, which are the main cell type affected after myocardial infarction. The fact that cardiomyocytes exhibit a low proliferation rate in the adult heart in mammals highlights the need to explore new approaches for cardiac regeneration. The aim of this review is to emphasize the functions carried out by WT1 in cardiomyocytes in cardiac homeostasis and heart regeneration.

Zinc and RNA Binding Is Linked to the Conformational Flexibility of ZRANB2: A CCCC-Type Zinc Finger Protein.

Ran-binding domain-containing protein 2 (ZRANB2) is a zinc finger (ZF) protein that plays a key role in alternative splicing. ZRANB2 is composed of two ZF domains that contain four invariant cysteine residues per domain. ZRANB2 binds RNA targets that contain AGGUAA sequence motifs. Three constructs of ZRANB2, ZRANB2-ZF1 (first ZF domain), ZRANB2-ZF2 (second ZF domain), and ZRANB2-2D (both ZF domains), were isolated in the apo form and shown to bind Zn(II) via UV-visible-monitored competitive titrations with Co(II) as a spectroscopic probe. Zn binding to each construct led to the adoption of a limited secondary structure of each domain, as measured by circular dichroism (CD). Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) of the two-domain construct, ZRANB2-2D, revealed that both ZF domains adopt a more rigid structure upon Zn binding. Zn binding to the first ZF domain resulted in a greater decrease in the conformational dynamics than Zn binding to the second ZF domain. RNA binding to TRA2B pre-mRNA, a physiological splicing target, was measured by fluorescence anisotropy (FA), and high-affinity RNA binding was found to require Zn coordination to both domains. HDX-MS of ZRANB2-2D with TRA2B RNA as well as two optimized RNA sequences that contain a single and double AGGUAA hexamer revealed additional protection from H/D exchange for ZRANB2 in the presence of RNA. Here, greater protection was observed for the second ZF of ZRANB2-2D, suggesting a larger effect on conformational dynamics. A model for zinc-mediated RNA binding of ZRANB2 is proposed.