Zinc finger proteins: guardians of genome stability.

Zinc finger proteins (ZNF), a unique yet diverse group of proteins, play pivotal roles in fundamental cellular mechanisms including transcription regulation, chromatin remodeling, protein/RNA homeostasis, and DNA repair. Consequently, the mis regulation of ZNF proteins can result in a variety of human diseases, ranging from neurodevelopmental disorders to several cancers. Considering the promising results of DNA damage repair (DDR) inhibition in the clinic, as a therapeutic strategy for patients with homologous recombination (HR) deficiency, identifying other potential targetable DDR proteins as emerged vulnerabilities in resistant tumor cells is essential, especially when considering the burden of acquired drug resistance. Importantly, there are a growing number of studies identifying new ZNFs and revealing their significance in several DDR pathways, highlighting their great potential as new targets for DDR-inhibition therapy. Although, there are still many uncharacterized ZNF-containing proteins with unknown biological function. In this review, we highlight the major classes and observed biological functions of ZNF proteins in mammalian cells. We briefly introduce well-known and newly discovered ZNFs and describe their molecular roles and contributions to human health and disease, especially cancer. Finally, we discuss the significance of ZNFs in DNA repair mechanisms, their potential in cancer therapy and advances in exploiting ZNF proteins as future therapeutic targets for human disease.

Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.

Identification and expression analysis of CCCH gene family and screening of key low temperature stress response gene CbuC3H24 and CbuC3H58 in Catalpa bungei.

Catalpa bungei, a tree indigenous to China, is renowned for its superior timber quality and as an ornamental in horticulture. To promote the cultivation of C. bungei in cold regions and expand its distribution, enhancing its cold tolerance is essential. The CCCH gene family is widely involved in plant growth, development, and expression under stress conditions, including low-temperature stress. However, a comprehensive identification and analysis of these genes have not yet been conducted. This study aims to identify key cold-tolerance-related genes within the CCCH gene family of C. bungei, providing the necessary theoretical support for its expansion in cold regions. In this study, 61 CCCH genes within C. bungei were identified and characterized. Phylogenetic assessment divided these genes into 9 subfamilies, with 55 members mapped across 16 chromosomes. The analysis of gene structures and protein motifs indicated that members within the same subfamily shared similar exon/intron distribution and motif patterns, supporting the phylogenetic classification. Collinearity analysis suggested that segmental duplications have played a significant role in the expansion of the C. bungei CCCH gene family. Notably, RNA sequencing analysis under 4 °C cold stress conditions identified CbuC3H24 and CbuC3H58 as exhibiting the most significant responses, highlighting their importance within the CCCH zinc finger family in response to cold stress. The findings of this study lay a theoretical foundation for further exploring the mechanisms of cold tolerance in C. bungei, providing crucial insights for its cultivation in cold regions.

ZNF91 is an endogenous repressor of the molecular phenotype associated with X-linked dystonia-parkinsonism (XDP).

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder resulting from an inherited intronic SINE-Alu-VNTR (SVA) retrotransposon in the TAF1 gene that causes dysregulation of TAF1 transcription. The specific mechanism underlying this dysregulation remains unclear, but it is hypothesized to involve the formation of G-quadruplexes (G4) structures within the XDP-SVA that impede transcription. In this study, we show that ZNF91, a critical repressor of SVA retrotransposons, specifically binds to G4-forming DNA sequences. Further, we found that genetic deletion of ZNF91 exacerbates the molecular phenotype associated with the XDP-SVA insertion in patient cells, while no difference was observed when ZNF91 was deleted from isogenic control cells. Additionally, we observed a significant age-related reduction in ZNF91 expression in whole blood and brain, indicating a progressive loss of repression of the XDP-SVA in XDP. These findings indicate that ZNF91 plays a crucial role in controlling the molecular phenotype associated with XDP. Since ZNF91 binds to G4-forming DNA sequences in SVAs, this suggests that interactions between ZNF91 and G4-forming sequences in the XDP-SVA minimize the severity of the molecular phenotype. Our results showing that ZNF91 expression levels significantly decrease with age provide a potential explanation for the age-related progressive neurodegenerative character of XDP. Collectively, our study provides important insights into the protective role of ZNF91 in XDP pathogenesis and suggests that restoring ZNF91 expression, destabilization of G4s, or targeted repression of the XDP-SVA could be future therapeutic strategies to prevent or treat XDP.

Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses.

Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif 'QALGGH' were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.

Drosophila Protein Z4 Possesses ZAD Dimerization Domain.

The transcription factor Z4 (putzig) is one of the key proteins that determine the chromatin structure in Drosophila. Z4 is found at the boundaries of bands on polytene chromosomes, and the bands are currently thought to correlate with chromatin domains. Z4 is a component of a protein complex that additionally includes Chromator and BEAF-32, and a conserved domain is necessary to occur at the N end of Z4 to ensure its interaction with the two proteins. In this study, a zinc finger-associated domain (ZAD) domain was identified in Z4. The capability of dimerization was confirmed for the domain by biochemical methods. A dimer model of the domain was obtained using AlphaFold2, and the model structure was confirmed using small-angle X-ray scattering (SAXS). The dimer structure shows a fold typical of ZAD domains.

Rice A20/AN1 protein, OsSAP10, confers water-deficit stress tolerance via proteasome pathway and positive regulation of ABA signaling in Arabidopsis.

KEY MESSAGE: Overexpression of rice A20/AN1 zinc-finger protein, OsSAP10, improves water-deficit stress tolerance in Arabidopsis via interaction with multiple proteins. Stress-associated proteins (SAPs) constitute a class of A20/AN1 zinc-finger domain containing proteins and their genes are induced in response to multiple abiotic stresses. The role of certain SAP genes in conferring abiotic stress tolerance is well established, but their mechanism of action is poorly understood. To improve our understanding of SAP gene functions, OsSAP10, a stress-inducible rice gene, was chosen for the functional and molecular characterization. To elucidate its role in water-deficit stress (WDS) response, we aimed to functionally characterize its roles in transgenic Arabidopsis, overexpressing OsSAP10. OsSAP10 transgenics showed improved tolerance to water-deficit stress at seed germination, seedling and mature plant stages. At physiological and biochemical levels, OsSAP10 transgenics exhibited a higher survival rate, increased relative water content, high osmolyte accumulation (proline and soluble sugar), reduced water loss, low ROS production, low MDA content and protected yield loss under WDS relative to wild type (WT). Moreover, transgenics were hypersensitive to ABA treatment with enhanced ABA signaling and stress-responsive genes expression. The protein-protein interaction studies revealed that OsSAP10 interacts with proteins involved in proteasomal pathway, such as OsRAD23, polyubiquitin and with negative and positive regulators of stress signaling, i.e., OsMBP1.2, OsDRIP2, OsSCP and OsAMTR1. The A20 domain was found to be crucial for most interactions but insufficient for all interactions tested. Overall, our investigations suggest that OsSAP10 is an important candidate for improving water-deficit stress tolerance in plants, and positively regulates ABA and WDS signaling via protein-protein interactions and modulation of endogenous genes expression in ABA-dependent manner.

Do extracellular vesicles have specific target cells?; Extracellular vesicle mediated embryo maternal communication.

Extracellular vesicles (EVs) serve as messengers for intercellular communication, yet the precise mechanisms by which recipient cells interpret EV messages remain incompletely understood. In this study, we explored how the origin of EVs, their protein cargo, and the recipient cell type influence the cellular response to EVs within an embryo implantation model. We treated two types of EVs to 6 different recipient cell types and expression of zinc finger protein 81 (ZNF81) gene expression in the recipient cells were quantified using quantitative polymerase chain reaction (qPCR). The proteomic contents of the EV cargos were also analyzed. The results showed that downregulation of the ZNF81 gene was a specific cellular response of receptive endometrial epithelial cells to trophoblast derived EVs. Protein cargo analysis revealed that the proteomic profile of EVs depends on their cell of origin and therefore may affect the recipient cell response to EVs. Furthermore, trophoblastic EVs were found to be specifically enriched with transcription factors such as CTNNB1 (catenin beta-1), HDAC2 (histone deacetylase 2), and NOTCH1 (neurogenic locus notch homolog protein 1), which are known regulators of ZNF81 gene expression. The current study provided compelling evidence supporting the existence of EV specificity, where the characteristics of both the EVs and the recipient cell type collectively contribute to regulating EV target specificity. Additionally, EV protein cargo analysis suggested a potential association between transcription factors and the specific functionality of trophoblastic EVs. This in vitro embryo implantation model and ZNF81 read-out provides a unique platform to study EV specific functionality in natural cell-cell communication.

Development of Locus-Directed Editing of the Epigenome from Basic Mechanistic Engineering to First Clinical Applications.

The introduction of CRISPR/Cas systems has resulted in a strong impulse for the field of gene-targeted epigenome/epigenetic reprogramming (EpiEditing), where EpiEditors consisting of a DNA binding part for targeting and an enzymatic part for rewriting of chromatin modifications are applied in cells to alter chromatin modifications at targeted genome loci in a directed manner. Pioneering studies preceding this era indicated causal relationships of chromatin marks instructing gene expression. The accumulating evidence of chromatin reprogramming of a given genomic locus resulting in gene expression changes opened the field for mainstream applications of this technology in basic and clinical research. The growing knowledge on chromatin biology and application of EpiEditing tools, however, also revealed a lack of predictability of the efficiency of EpiEditing in some cases. In this perspective, the dependence of critical parameters such as specificity, effectivity, and sustainability of EpiEditing on experimental settings and conditions including the expression levels and expression times of the EpiEditors, their chromatin binding affinity and specificity, and the crosstalk between EpiEditors and cellular epigenome modifiers are discussed. These considerations highlight the intimate connection between the outcome of epigenome reprogramming and the details of the technical approaches toward EpiEditing, which are the main topic of this volume of Methods in Molecular Biology. Once established in a fully functional "plug-and-play" mode, EpiEditing will allow to better understand gene expression control and to translate such knowledge into therapeutic tools. These expectations are beginning to be met as shown by various in vivo EpiEditing applications published in recent years, several companies aiming to exploit the therapeutic power of EpiEditing and the first clinical trial initiated.

Participant-derived cell line transcriptomic analyses and mouse studies reveal a role for ZNF335 in plasma cholesterol statin response.

BACKGROUND: Statins lower circulating low-density lipoprotein cholesterol (LDLC) levels and reduce cardiovascular disease risk. Though highly efficacious in general, there is considerable inter-individual variation in statin efficacy that remains largely unexplained. METHODS: To identify novel genes that may modulate statin-induced LDLC lowering, we used RNA-sequencing data from 426 control- and 2 µM simvastatin-treated lymphoblastoid cell lines (LCLs) derived from European and African American ancestry participants of the Cholesterol and Pharmacogenetics (CAP) 40 mg/day 6-week simvastatin clinical trial (ClinicalTrials.gov Identifier: NCT00451828). We correlated statin-induced changes in LCL gene expression with plasma LDLC statin response in the corresponding CAP participants. For the most correlated gene identified (ZNF335), we followed up in vivo by comparing plasma cholesterol levels, lipoprotein profiles, and lipid statin response between wild-type mice and carriers of a hypomorphic (partial loss of function) missense mutation in Zfp335 (the mouse homolog of ZNF335). RESULTS: The statin-induced expression changes of 147 human LCL genes were significantly correlated to the plasma LDLC statin responses of the corresponding CAP participants in vivo (FDR = 5%). The two genes with the strongest correlations were zinc finger protein 335 (ZNF335 aka NIF-1, rho = 0.237, FDR-adj p = 0.0085) and CCR4-NOT transcription complex subunit 3 (CNOT3, rho = 0.233, FDR-adj p = 0.0085). Chow-fed mice carrying a hypomorphic missense (R1092W; aka bloto) mutation in Zfp335 had significantly lower non-HDL cholesterol levels than wild-type C57BL/6J mice in a sex combined model (p = 0.04). Furthermore, male (but not female) mice carrying the Zfp335R1092W allele had significantly lower total and HDL cholesterol levels than wild-type mice. In a separate experiment, wild-type mice fed a control diet for 4 weeks and a matched simvastatin diet for an additional 4 weeks had significant statin-induced reductions in non-HDLC (-43 ± 18% and -23 ± 19% for males and females, respectively). Wild-type male (but not female) mice experienced significant reductions in plasma LDL particle concentrations, while male mice carrying Zfp335R1092W allele(s) exhibited a significantly blunted LDL statin response. CONCLUSIONS: Our in vitro and in vivo studies identified ZNF335 as a novel modulator of plasma cholesterol levels and statin response, suggesting that variation in ZNF335 activity could contribute to inter-individual differences in statin clinical efficacy.

Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer.

Zinc finger protein 180 (ZNF180) is a multifunctional protein that interacts with nucleic acids and regulates various cellular processes; however, the function of ZNF180 in colorectal cancer (CRC) remains unclear. The present study investigated the role and function of ZNF180 in CRC, and aimed to reveal the underlying molecular mechanism. The results revealed that ZNF180 was downregulated in CRC tissues and was associated with a good prognosis in patients with CRC. Additionally, the expression of ZNF180 was downregulated by methylation in CRC. In vivo and in vitro experiments revealed that ZNF180 overexpression was functionally associated with the inhibition of cell proliferation and the induction of apoptosis. Mechanistically, chromatin immunoprecipitation­PCR and luciferase assays demonstrated that ZNF180 markedly regulated the transcriptional activity of methyltransferase 14, N6­adenosine­methyltransferase non­catalytic subunit (METTL14) by directly binding to and activating its promoter region. Simultaneous overexpression of ZNF180 and knockdown of METTL14 indicated that the reduction of METTL14 could suppress the effects of ZNF180 on the induction of apoptosis. Clinically, the present study observed a significant positive correlation between ZNF180 and METTL14 expression levels, and low expression of ZNF180 and METTL14 predicted a poor prognosis in CRC. Overall, these findings revealed a novel mechanism by which the ZNF180/METTL14 axis may modulate apoptosis and cell proliferation in CRC. This evidence suggests that this axis may serve as a prognostic biomarker and therapeutic target in patients with CRC.

Zinc finger translocation­associated protein promotes ferroptosis through the upregulation of ACSL4 expression in vascular endothelial cells.

Numerous studies have reported the potential involvement of ferroptosis in the development of atherosclerosis (AS). Acyl-CoA synthetase long chain family member 4 (ACSL4) is an essential component in the promotion of ferroptosis. The present study aimed to investigate the role of ACSL4 and zinc finger translocation-associated protein (ZFTA) in the regulation of endothelial cell ferroptosis in AS. Human umbilical vein endothelial cells (HUVECs) with ACSL4 knockout were generated using CRISPR/Cas9 technology. To assess ferroptosis, malondialdehyde concentration, iron content and reactive oxygen species levels were quantified in the present study. In addition, western blot analysis was conducted to explore the potential mechanisms underlying ACSL4 and ZFTA in the modulation of ferroptosis in HUVECs. The results of the present study demonstrated that the expression levels of ACSL4 and ZFTA were significantly increased in human atherosclerotic plaques. In addition, ACSL4 knockout led to a reduced susceptibility to ferroptosis, while ZFTA contributed to ferroptosis in HUVECs. Results of the present study also demonstrated that ZFTA overexpression upregulated ACSL4 expression in HUVECs, whereas ZFTA knockdown led to decreased ACSL4 expression. Co-transfection experiments demonstrated that the ZTFA overexpression-mediated increase in ferroptosis was reversed following ACSL4 knockdown. Collectively, results of the present study highlighted that ACSL4 mediated the effects of ZFTA on the ferroptosis of HUVECs. Thus, the present study demonstrated the potential role of ACSL4 and ZFTA in the regulation of ferroptosis, and highlighted that ferroptosis-related pathways may act as potential targets in the treatment of AS.

Transmembrane protein TMEM230, regulator of metalloproteins and motor proteins in gliomas and gliosis.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

Pan-Cancer Bioinformatics Indicates ZNF207 is a Promising Prognostic Biomarker and Immunotherapeutic Target.

In the era of personalized cancer treatment, understanding the complexities of tumor biology and immune modulation is paramount. This comprehensive analysis delves into the multifaceted role of Zinc Finger Protein 207 (ZNF207) in pan-cancer, shedding light on its involvement in tumorigenesis, immune evasion, and therapeutic implications. Through integrated genomic and clinical data analysis, we reveal consistent upregulation of ZNF207 across diverse cancer types, highlighting its potential as a prognostic marker and therapeutic target, particularly for liver cancers. Notably, ZNF207 demonstrates intricate associations with clinical-pathological features, immune subtypes, and molecular pathways, indicating its pervasive influence in cancer biology. Furthermore, our study uncovers ZNF207's involvement in immune escape mechanisms, suggesting its potential as a modulator of immune responses within the tumor microenvironment. These findings underscore the significance of ZNF207 in shaping cancer progression and immune landscape, presenting promising avenues for targeted therapy and immunomodulation. Recognizing ZNF207's multifaceted contributions to cancer progression and immune evasion suggests its central role in understanding tumor immunology, beyond mere therapeutic targeting. Nevertheless, further mechanistic studies are imperative to elucidate ZNF207's precise molecular mechanisms and therapeutic implications in cancer treatment. This study primarily utilized various bioinformatics tools such as TIMER 2.0, cProSite, UALCAN, SangerBox, GEPIA2, TISIDB and TIDE to analyze the expression of ZNF207 in multiple cancer samples from the TCGA database.

C(P)XCG Proteins of Haloferax volcanii with Predicted Zinc Finger Domains: The Majority Bind Zinc, but Several Do Not.

In recent years, interest in very small proteins (µ-proteins) has increased significantly, and they were found to fulfill important functions in all prokaryotic and eukaryotic species. The halophilic archaeon Haloferax volcanii encodes about 400 µ-proteins of less than 70 amino acids, 49 of which contain at least two C(P)XCG motifs and are, thus, predicted zinc finger proteins. The determination of the NMR solution structure of HVO_2753 revealed that only one of two predicted zinc fingers actually bound zinc, while a second one was metal-free. Therefore, the aim of the current study was the homologous production of additional C(P)XCG proteins and the quantification of their zinc content. Attempts to produce 31 proteins failed, underscoring the particular difficulties of working with µ-proteins. In total, 14 proteins could be produced and purified, and the zinc content was determined. Only nine proteins complexed zinc, while five proteins were zinc-free. Three of the latter could be analyzed using ESI-MS and were found to contain another metal, most likely cobalt or nickel. Therefore, at least in haloarchaea, the variability of predicted C(P)XCG zinc finger motifs is higher than anticipated, and they can be metal-free, bind zinc, or bind another metal. Notably, AlphaFold2 cannot correctly predict whether or not the four cysteines have the tetrahedral configuration that is a prerequisite for metal binding.

Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals.

Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities.

The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors (EEs) enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus-specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here, we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to: account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus-specificity by considering concentration, affinity, avidity, and sequestration effects.

Zinc finger 259 gene polymorphisms in Egyptian patients with metabolic syndrome and its association with dyslipidemia.

BACKGROUND: Zinc finger protein 1 (ZPR1), encoded by the ZNF259 gene, plays crucial roles in transcriptional regulation and cell cycle progression. Despite its known functions, its specific involvement in Metabolic Syndrome (MetS) remains debated. Genome-wide association studies have identified several genes, including ZNF259, implicated in lipid metabolism and associated with MetS. Single nucleotide polymorphisms (SNPs) in ZNF259 have been linked to altered lipid metabolism during the development of MetS. This study aims to investigate the association between MetS in Egyptian patients and three specific ZNF259 SNPs: rs964184, rs2075294, and rs2075290. The objective is to explore how these SNPs correlate with MetS development, other health outcomes, and their interaction with dyslipidemia biomarkers. METHODS: 200 Egyptian participants were enrolled, and divided into two groups: 100 patients diagnosed with dyslipidemia and 100 healthy controls. The study involved comprehensive assessments, including lipid profile analysis, anthropometric measurements, and genotyping of rs964184, rs2075290, and rs2075294 in the ZNF259 gene using Real-Time Polymerase Chain Reaction (PCR). RESULTS: The findings indicate that rs964184 SNP correlates significantly with elevated plasma triacylglycerol (TG) levels, while rs2075290 and rs2075294 are associated with higher total serum cholesterol (TC) and TG levels. Among these SNPs, rs2075294 showed the highest predictive value (area under the curve of 0.748), followed by rs2075290 (0.738), and rs964184 (0.583), suggesting rs2075294 as the most influential SNP in MetS prediction. CONCLUSION: This study underscores the predictive role of ZNF259 SNPs in MetS risk among Egyptians. Future research should further explore the implications of ZNF259 in MetS pathogenesis and its potential as a biomarker for personalized health interventions.

Emergence of the ZNF675 Gene During Primate Evolution-Influenced Human Neurodevelopment Through Changing HES1 Autoregulation.

In this study, we investigated recurrent copy number variations (CNVs) in the 19p12 locus, which are associated with neurodevelopmental disorders. The two genes in this locus, ZNF675 and ZNF681, arose via gene duplication in primates, and their presence in several pathological CNVs in the human population suggests that either or both of these genes are required for normal human brain development. ZNF675 and ZNF681 are members of the Krüppel-associated box zinc finger (KZNF) protein family, a class of transcriptional repressors important for epigenetic silencing of specific genomic regions. About 170 primate-specific KZNFs are present in the human genome. Although KZNFs are primarily associated with repressing retrotransposon-derived DNA, evidence is emerging that they can be co-opted for other gene regulatory processes. We show that genetic deletion of ZNF675 causes developmental defects in cortical organoids, and our data suggest that part of the observed neurodevelopmental phenotype is mediated by a gene regulatory role of ZNF675 on the promoter of the neurodevelopmental gene Hes family BHLH transcription factor 1 (HES1). We also find evidence for the recently evolved regulation of genes involved in neurological disorders, microcephalin 1 and sestrin 3. We show that ZNF675 interferes with HES1 auto-inhibition, a process essential for the maintenance of neural progenitors. As a striking example of how some KZNFs have integrated into preexisting gene expression networks, these findings suggest the emergence of ZNF675 has caused a change in the balance of HES1 autoregulation. The association of ZNF675 CNV with human developmental disorders and ZNF675-mediated regulation of neurodevelopmental genes suggests that it evolved into an important factor for human brain development.

Production of stable and pure ZC3H11A - An extensively disordered RNA binding protein.

Human ZC3H11A is an RNA-binding zinc finger protein involved in mRNA export and required for the efficient growth of human nuclear replicating viruses. Its biochemical properties are largely unknown so our goal has been to produce the protein in a pure and stable form suitable for its characterization. This has been challenging since the protein is large (810 amino acids) and with only the N-terminal zinc finger domain (amino acids 1-86) being well structured, the remainder is intrinsically disordered. Our production strategies have encompassed recombinant expression of full-length, truncated and mutated ZC3H11A variants with varying purification tags and fusion proteins in several expression systems, with or without co-expression of chaperones and putative interaction partners. A range of purification schemes have been explored. Initially, only truncated ZC3H11A encompassing the zinc finger domain could successfully be produced in a stable form. It required recombinant expression in insect cells since expression in E. coli gave a protein that aggregated. To reduce problematic nucleic acid contaminations, Cys8, located in one of the zinc fingers, was substituted by Ala and Ser. Interestingly, this did not affect nucleic acid binding, but the full-length protein was stabilised while the truncated version was insoluble. Ultimately, we discovered that when using alkaline buffers (pH 9) for purification, full-length ZC3H11A expressed in Sf9 insect cells was obtained in a stable and >90 % pure form, and as a mixture of monomers, dimers, tetramers and hexamers. Many of the challenges experienced are consistent with its predicted structure and unusual charge distribution.