DNA Binding Activity Research Articles

Non-canonical role for the ataxia-telangiectasia-Rad3 pathway in STAT3 activation in human multiple myeloma cells.

The goal of this study was to characterize the relationship between ATR and STAT3 interactions in human multiple myeloma (MM) cells. Various MM cell lines, including IL-6-dependent cells were exposed to ATR inhibitors and effects on STAT3 Tyr705 and Ser727 were monitored by WB analysis and ImageStream analysis. Parallel studies examined induction of cell death, STAT3 DNA binding activity, and expression of STAT3 downstream targets (BCL-XL, MCL-1, c-MYC). Validation was obtained in ATR shRNA knock-down cells, and in cells ectopically expressing BCL-XL, MCL-1, or c-MYC. Analogous studies were performed in primary MM cells and in a MM xenograft model. Multiple pharmacologic ATR inhibitors inhibited STAT3 Tyr705 (but not Ser727) phosphorylation at low uM concentrations and down-regulated BCL-XL, MCL-1, c-MYC in association with cell death induction. Compatible results were observed in ATR shRNA knock-down cells. Cell death induced by ATR inhibitors was significantly attenuated in cells ectopically expressing constitutively active STAT3, BCL-XL, MCL-1, or c-MYC. Concordant results were observed in primary human MM cells and in an in vivo MM xenograft model. Collectively, these findings argue for a non-canonical role for the ATR kinase in STAT3 activation in MM cells, and suggest that STAT3 inactivation contributes to the lethal actions of ATR inhibitors in MM.

Transcription factor HNF4A activity is suppressed by microbiota and inhibits IFN beta production

Intestinal epithelial cells (IECs) separate the intestinal lumen from lamina propria resident immune cells, positioning IECs as mediators of microbial signals to the immune system. Understanding these microbe-IEC-immune interactions may represent potential therapeutic targets for immunological diseases. We previously discovered that microbiota regulate the activity of the nuclear receptor transcription factor Hepatocyte nuclear factor 4 alpha (HNF4A). Microbiota suppress HNF4A-activated genes in zebrafish and lead to genome-wide reduction of HNF4A DNA occupancy in mouse IECs. HNF4A activates lipid metabolism and epithelial differentiation gene programs, while also suppressing inflammatory and immune response gene programs in the intestine. We therefore hypothesized that microbial suppression of HNF4A contributes to activation of immunological pathways. We conducted an unbiased coimmunoprecipitation-mass-spectrometry analysis comparing HNF4A interacting partners in IECs from germ-free and conventionalized (germ-free mice colonized with SPF feces for 2 weeks) mice. HNF4A interacted with significantly more proteins in germ-free IECs, including several proteins involved in promotion of transcription and chromatin regulation, consistent with HNF4A being more active in germ-free mice. Our HNF4A and H3k27ac ChIP-seq data from IECs derived from germ-free and conventionalized mice identified that HNF4A binds ubiquitously to IEC enhancers, but also revealed a small number of enhancers that were activated by microbiota with no HNF4A binding. Motif enrichment analysis showed that these enhancers were enriched for binding sites for Interferon regulatory factors (IRFs). This raises the interesting possibility that there is a selective disadvantage to colocalizing IRF and HNF4A occupancy at a given enhancer, and suggests that these transcriptional regulatory pathways may otherwise communicate. To test this, we used a luciferase reporter assay in HEK293T cells to probe the impact of HNF4A on the IFNβ promoter, which is controlled by IRF3/7. HNF4A potently suppressed IFNβ promoter activity following viral stimulation. HNF4A DNA-binding activity is necessary for this suppression; however, there is no evidence HNF4A binds the IFNβ promoter, suggesting HNF4A likely induces a repressor of the IFNβ promoter. Together these results indicate that HNF4A is both a target of microbiota suppression and a potent suppressor of IFNβ production. Though IRF bound enhancers appear to be insulated against HNF4A binding in IECs, our results show that these two transcriptional pathways communicate, potentially representing a novel mechanism through which microbiota influence innate immunity. NIH P01-DK094779 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Scaffold protein RACK1A positively regulates leaf senescence by coordinating the EIN3-miR164-ORE1 transcriptional cascade in Arabidopsis.

Plants have adopted versatile scaffold proteins to facilitate the crosstalk between multiple signaling pathways. Leaf senescence is a well-programmed developmental stage that is coordinated by various external and internal signals. However, the functions of plant scaffold proteins in response to senescence signals are not well understood. Here, we report that the scaffold protein RACK1A (RECEPTOR FOR ACTIVATED C KINASE 1 A) participates in leaf senescence mediated by ethylene signaling via the coordination of the EIN3-miR164-ORE1 transcriptional regulatory cascade. RACK1A is a novel positive regulator of ethylene-mediated leaf senescence. The rack1a mutant exhibits delayed leaf senescence, whilst transgenic lines overexpressing RACK1A display early leaf senescence. Moreover, RACK1A promotes EIN3 protein accumulation, and directly interacts with EIN3 (ETHYLENE INSENSITIVE 3) to enhance its DNA-binding activity. Together, they then associate with the miR164 promoter to inhibit its transcription, leading to the release of the inhibition on downstream ORE1 (ORESARA 1) transcription and the promotion of leaf senescence. This study reveals a mechanistic framework by which RACK1A promotes leaf senescence via the EIN3-miR164-ORE1 transcriptional cascade, and provides a paradigm for how scaffold proteins finely tune phytohormone signaling to control plant development. This article is protected by copyright. All rights reserved.

Novel rRNA transcriptional activity of NhaR revealed by its growth recovery for the bipA-deleted Escherichia coli at low temperature.

The BipA protein is a universally conserved GTPase in bacterial species and is structurally similar to translational GTPases. Despite its wide distribution, BipA is dispensable for growth under optimal growth conditions but is required under stress conditions. In particular, bipA-deleted cells (ESC19) have been shown to display a variety of phenotypic changes in ribosome assembly, capsule production, lipopolysaccharide (LPS) synthesis, biofilm formation, and motility at low temperature, suggesting its global regulatory roles in cold adaptation. Here, through genomic library screening, we found a suppressor clone containing nhaR, which encodes a Na+-responsive LysR-type transcriptional regulator and whose gene product partially restored the growth of strain ESC19 at 20°C. The suppressed cells showed slightly reduced capsule production and improved biofilm-forming ability at 20°C, whereas the defects in the LPS core and swimming motility were not restored but aggravated by overexpression of nhaR. Notably, the overexpression partially alleviated the defects in 50S ribosomal subunit assembly and rRNA processing of ESC19 cells by enhancing the overall transcription of rRNA. Electrophoretic mobility shift assay revealed the association of NhaR with the promoter of seven rrn operons, suggesting that NhaR directly regulates rRNA transcription in ESC19 at 20°C. The suppressive effects of NhaR on ribosomes, capsules, and LPS were dependent on its DNA-binding activity, implying that NhaR might be a transcriptional factor involved in regulating these genes at 20°C. Furthermore, we found that BipA may be involved in adaptation to salt stress, designating BipA as a global stress-responsive regulator, as the deletion of bipA led to growth defects at 37°C and high Na+ concentrations without ribosomal defects.

Synthesis, spectral characterization, computational studies, antifungal, DNA interaction, antioxidant and fluorescence property of novel Schiff base ligand and its metal chelates

Cobalt, Copper, Nickel and Zinc coordinated metal complexes were synthesized by novel thiazole Schiff base ligand 2-ethoxy-4-((5-methylthiazol-2-ylimino)methyl) phenol derived from 3-ethoxy-4-hydroxybenzaldehyde and 2-amino- 5-methylthiazol. The synthesized compounds were spectrochemically characterized by elemental analysis, molar conductance, FT-IR, UV–Vis, Mass spectral analysis, Powdered-XRD and cyclic voltammetry. The thermal stability of synthesized complexes were investigated by using thermogravimetric analysis (TGA). Theoretical computational study were performed for all the synthesized compounds utilizing the DFT/B3LYP method at the 6-31G basic set for Schiff base ligand whereas LANL2DZ basis set for metal complexes. Molecular Electrostatic Potential (MEP), HOMO-LUMO, Mulliken charges and global reactivity descriptors, including chemical potential (μ), global softness (S), chemical hardness (η), and electrophilicity index (ω) were measured and correlated with antimicrobial activity. The synthesized thiazole Schiff base ligand and its coordinated metal complexes shows good antifungal agreement against Fusarium Oxysporum and Aspergillus Niger species. These compounds also exhibit DNA binding, DNA cleaving and antioxidant activity. All the synthesized molecules indicate potential fluorescence property.

Novel molecular requirements for CRISPR RNA-guided transposition.

CRISPR-associated transposases(CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system(VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.

RAD51–WSS1-dependent genetic pathways are essential for DNA–protein crosslink repair and pathogenesis in Candida albicans

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and proteases-dependent repair (PDR) pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated due to the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPCs repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 are known to be involved in DPCR, however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1 dependent HR and PDR pathways are essential for DPCs removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. Additionally, we observed truncation of chromosome#6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp PCNA is not a requisite for Wss1's function. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

Ruthenium biochanin-A complex ameliorates lung carcinoma through the downregulation of the TGF-β/PPARγ/PI3K/TNF-α pathway in association with caspase-3-mediated apoptosis.

Lung cancer is the most often reported cancer with a terrible prognosis worldwide. Flavonoid metal complexes have exhibited potential chemotherapeutic effects with substantially low adverse effects. This study investigated the chemotherapeutic effect of the ruthenium biochanin-A complex on lung carcinoma in both in vitro and in vivo model systems. The synthesized organometallic complex was characterized via UV‒visible spectroscopy, FTIR, mass spectrometry, and scanning electron microscopy. Moreover, the DNA binding activity of the complex was determined. The in vitro chemotherapeutic assessment was performed on the A549 cell line through MTT assay, flow cytometry, and western blot analysis. An in vivo toxicity study was performed to determine the chemotherapeutic dose of the complex, and subsequently, chemotherapeutic activity was assessed in benzo-α-pyrene-induced lung cancer mouse model by evaluating the histopathology, immunohistochemistry, and TUNEL assays. The IC50 value of the complex in A549 cells was found to be 20 µM. The complex demonstrated significant apoptosis induction, enhanced caspase-3 expression and cell cycle arrest with downregulated PI3K, PPARγ, TGF-β, and TNF-α expression in A549 cells. The in vivo study suggested that ruthenium biochanin-A therapy restored the morphological architecture of lung tissue in a benzo-α-pyrene-induced lung cancer model and inhibited the expression of Bcl2. Additionally, increased apoptotic events were identified with upregulation of caspase-3 and p53 expression. In conclusion, the ruthenium biochanin-A complex successfully amelioratedlung cancer incidence in both in vitro and in vivo models through the alteration of the TGF-β/PPARγ/PI3K/TNF-α axis with the induction of the p53/caspase-3-mediated apoptotic pathway.

The H2TH-like motif of the Escherichia coli multifunctional protein KsgA is required for DNA binding involved in DNA repair and the suppression of mutation frequencies

BackgroundDNA oxidatively damaged by reactive oxygen species is repaired by base excision repair (BER) pathway proteins, with DNA glycosylases removing damaged or mismatched bases in the first step of BER. KsgA is a multifunctional protein that exhibits the activities of two enzymes, DNA glycosylase and rRNA dimethyltransferase. The structure-function relationship of the KsgA protein in cellular DNA repair remains unclear because the domains required for KsgA to recognize DNA have not been identified.PurposeTo clarify the mechanisms by which KsgA recognizes damaged DNA and to identify the DNA-binding site, which exists in KsgA.MethodsA structural analysis and in vitro DNA-protein binding assay were performed. The C-terminal function of the KsgA protein was investigated in vitro and in vivo.ResultsThe 3D conformations of KsgA, MutM, and Nei were compared at UCSF Chimera. The root mean square deviation of KsgA (214-273) and MutM (148-212) and that of KsgA (214-273) and Nei (145-212) were 1.067 and 1.188 Å, both less than 2 Å, suggesting that the C terminal of KsgA is spatially similar to the H2TH domains of MutM and Nei. The full-length KsgA protein and KsgA lacking 1-8 or 214-273 amino acids were purified and used in gel mobility shift assays. KsgA exhibited DNA-binding activity, which was lost in the C-terminally deleted KsgA protein. Spontaneous mutation frequency was measured using a mutM mutY ksgA-deficient strain, and the results obtained showed that the mutation frequency was not suppressed by KsgA lacking the C-terminal region, whereas it was in KsgA. To assess dimethyltransferase activity, kasugamycin sensitivity was assessed in wild-type and ksgA-deficient strains. Plasmids carrying the full-length ksgA gene and C-terminal deletion gene were introduced into ksgA-deficient strains. KsgA lacking the C terminus restored dimethyltransferase activity in the ksgA-deficient strain as well as KsgA.ConclusionThe present results confirmed that one enzyme exhibited two activities and revealed that the C-terminal (214-273) amino acids of KsgA were highly similar to the H2TH structural domain, exhibited DNA-binding activity, and inhibited spontaneous mutations. This site is not essential for dimethyltransferase activity.

Preparation and spectroscopic properties of bioactive 1, 2, 3-triazole-linked boronate esters for use in antioxidant, antimicrobial, and DNA binding studies

In this article, the preparation, spectral evaluation, and biological studies of bioactive 1, 2, 3-triazole-linked boronate esters are reported. The bioactive 1, 2, 3-triazole group linked-boronate esters have been designed and obtained by azido-functionalized boronate ester with different alkyne molecules via click chemistry approach under mild conditions. The prepared boronate esters (B1-5) were characterized using NMR (1H, 13C, and 11B) spectra, FT-IR spectra, UV-Vis spectra, LC-MS/MS spectra, elemental analysis, and melting point measurement techniques. After all structures of synthesized boronate esters were characterized in detail, the antimicrobial (antioxidant and antibacterial) and DNA binding properties of newly prepared boronate esters were investigated. The bioactive 1, 2, 3-triazole-linked boronate ester (B5) showed the highest radical scavenging (71.59 ± 1.85%) and reducing power activity (0.476 ± 0.01) at 200.0 µg/mL concentration. According to the method of reducing power activity, the compound (B5) exhibited the strongest activity with 0.476 ± 0.014. The anti-bacterial activity of all boronate esters (B1-5) was determined with four pathogenic bacteria. It was determined that only the 1, 2, 3-triazole-linked boronate ester (B5) showed low activity against two bacteria (8 mm for S. aureus and 10 mm for E. hirae), while other boronate esters were found to have no activity. According to electrophoresis results using calf thymus DNA (CT-DNA), 1, 2, 3-triazole-linked boronate esters (B3-5) were determined to have DNA binding activity.

Abstract 3725: ABI1 regulates STAT3 transcription through a DNA binding activity

Abstract Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa, as evidenced by its loss or downregulation in high-grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. Cell Commun Signal. 2019). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggests the possibility that it plays a role in EMT by directly regulating transcriptional activity by DNA binding. To examine this hypothesis, we set out to analyze ABI1-DNA binding. Structural NMR studies and in-vitro protein-DNA binding assays confirmed ABI1 binding to DNA regulated by alternative spliced ABI1 Exon 4 region located on the C-terminus of ABI1 HHR. RNA-sequencing data and PCa cell lines qPCR assays reveal the alternative ABI1 Exon 4 spliced-in is enriched in high Gleason-scored PCa samples. ABI1 and STAT3 co-regulate their nuclear vs. cytoplasm localization, and the subsequent functional studies using PCa lines expressing wild type or HHR Exon 4 deletion mutant of ABI1 demonstrated that HHR regulates the STAT3 DNA binding patterns, STAT3 mediated chromatin structure programming as well as its transcription activities. We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression. Citation Format: Xiang Li, Neeru Arya, Baylee A. Porter, Allysa P. Kemraj, Xuesen Dong, Dominique Frueh, Alaji Bah, Leszek Kotula. ABI1 regulates STAT3 transcription through a DNA binding activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3725.

Abstract 514: Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice

Abstract Unlike classical Signal transducer and activator of transcription (Stat) signaling, which is transient in non-transformed cells, the aberrant activation of the family member, Stat3, occurs in malignant transformation and is implicated in breast, ovarian and many other human cancers. Thus, Stat3 remains a validated and important target for the discovery of novel anticancer drugs. Despite this, the discovery and development of potent Stat3 small molecule inhibitors has proven to be a significant challenge, and no drug is available yet in the market. We have discovered novel Stat3 irreversible azetidine inhibitors with unprecedent submicromolar potency through systematic medicinal chemistry structure activity relationship (SAR). Mechanism of action of most potent azetidine inhibitors depend on whether they are salicylic acids or not, i.e., salicylic acids preferably bind irreversibly to Stat3 Cys426 site in the DNA-binding domain (DBD), and non-salicylic acids, e.g., current lead H182, bind to Stat3 Cys468 DBD site. As expected, the inhibition of Stat3 DNA-binding activity was time dependent, with IC50 in the range of 0.27-0.87 µM at one hour incubation with active Stat3. On the other hand, azetidine salicylic acids also bind to the SH2 domain, although reversibly and at much weaker affinity, as determined in fluorescent polarization (FP) assay, with IC50 of 10-16 µM at one hour incubation, while non-salicylic acids, e.g., lead H182, present no binding affinity up to 600 µM for the SH2 domain. Despite that H182 does not bind to SH2 domain, it still inhibits its phosphorylation. Though lead compound H182 presents significant mouse in vivo efficacy, mouse in vivo pharmacokinetics shows very low plasma AUC, which correlates with quite high mouse in vitro hepatocyte CLint of 138 µL/min/106 cells; however, human in vitro hepatocyte assay gives much better results and is in the middle stability range (CLint of 14.6 µL/min/106 cells). The in vivo PK in other species, e.g., rat, is being determined. H182 represents a plausible molecule for further development. Citation Format: Yue Chen, Ning Zhai, Yinsong Zhu, Peibin Yue, Nagendra Verma, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco J. Lopez-Tapia, James Turkson. Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 514.

Abstract 3730: A new type of transcriptional reprogramming by an IRF4 mutation in lymphoma

Abstract Disease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation1-3. Such mutations can affect TF-interactions with their cognate DNA-binding motifs4,5. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation6,7. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Furthermore, IRF4-C99R thoroughly modifies IRF4 function, by blocking IRF4-dependent plasma cell induction, and up-regulating disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single arginine mutation creates a complex switch of TF specificity and gene regulation. These data open the possibility of designing specific inhibitors to block the neomorphic, disease-causing DNA-binding activities of a mutant transcription factor. Citation Format: Pierre Cauchy. A new type of transcriptional reprogramming by an IRF4 mutation in lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3730.

MP41-12 ASSOCIATION OF KIDNEY CANCER WITH BOTH NOVEL AND REPORTED MONOGENIC GENE MUTATIONS: RESULTS OF WHOLE EXOME SEQUENCING IN THE UK BIOBANK

You have accessJournal of UrologyCME1 Apr 2023MP41-12 ASSOCIATION OF KIDNEY CANCER WITH BOTH NOVEL AND REPORTED MONOGENIC GENE MUTATIONS: RESULTS OF WHOLE EXOME SEQUENCING IN THE UK BIOBANK Jun Wei, Kristian Novakovic, Zhuqing Shi, Andrew Rifkin, S. Lilly Zheng, Brian Helfand, and Jianfeng Xu Jun WeiJun Wei More articles by this author , Kristian NovakovicKristian Novakovic More articles by this author , Zhuqing ShiZhuqing Shi More articles by this author , Andrew RifkinAndrew Rifkin More articles by this author , S. Lilly ZhengS. Lilly Zheng More articles by this author , Brian HelfandBrian Helfand More articles by this author , and Jianfeng XuJianfeng Xu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003279.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Pathogenic mutations in several cancer susceptibility genes have been reported in kidney cancer patients. However, their associations with kidney cancer have not been formally tested in well-characterized case-control studies and using the same sequencing and analytical pipeline. The objective of this study was to systematically evaluate association of kidney cancer with genes in the exome in a well-characterized prospective population-based case-control cohort. METHODS: A total of 187,966 subjects from the UK Biobank (UKB) with whole exome sequencing data were included in the analysis, including 1,217 kidney cancer patients (ICD10 C64). Pathogenic/likely pathogenic (P/LP) mutations were annotated based on criteria of the American College of Medical Genetics. The robust SKAT-O, a novel gene-based analysis that properly controls for type I error rates due to unbalanced case-control ratio, was used for association tests adjusting for age at recruitment, gender and genetic background. RESULTS: Among the 21 previously reported candidate genes, three were statistically significant at P<0.05, including VHL, SDHB, and CHEK2 (Table). In addition, associations with 9 novel genes (at P<0.0001) were found, including ZNF772, PRIMPOL, HES4, MOXD1, KRTAP10-9, ZNF827, ACAN, CDK20, and LRRFIP1. While these associations were only marginally significant after adjusting for multiple testing, they have potential roles in cancer development. For example, ZNF772 is predicted to enable DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity which are all known to be dysregulated in cancer. CONCLUSIONS: Identification of genes that influence kidney cancer may advance our understanding of disease etiology and identify potential therapeutic targets. Using the same sequencing method and mutation annotation pipeline in the largest population-based cohort to date, we confirmed several previously reported genes and identified several novel candidate genes for their association with kidney cancer. Confirmation in independent study populations is required for these novel candidate genes. Source of Funding: None. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e559 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jun Wei More articles by this author Kristian Novakovic More articles by this author Zhuqing Shi More articles by this author Andrew Rifkin More articles by this author S. Lilly Zheng More articles by this author Brian Helfand More articles by this author Jianfeng Xu More articles by this author Expand All Advertisement PDF downloadLoading ...

Photophysical study on DNA & BSA binding and cytotoxic behaviour of piperidine-Pt(II) complexes: Their kinetics & mechanism and molecular docking

For the development of Pt(II) based anticancer drug candidates, carrier ligand 2-(Piperidine-1-yl)ethylamine (PIEAM) was used as a bidentate chelator to synthesize dichloro complex [Pt(PIEAM)Cl2] C-1 and its diaqua [Pt(PIEAM)(OH2)2](X)2 (C-2). The diaqua complex C-2 was further used to synthesize thiol substituted Pt(II)-sulfur complexes with l-cysteine (LC) and N-acetyl-l-cysteine (NAC). The Pt(II) complexes were characterized by multi spectroscopic methods and thermal analysis. The role of electronic and steric factors of carrier ligand PIEAM on substitution reactions of C-2 with LC and NAC was studied in aqueous medium at pH 4.0 under pseudo first-order kinetics and rate constant, equilibrium constant and thermodynamic parameters were calculated. The DNA binding activity and binding mode were confirmed by gel electrophoresis and viscosity measurement. The binding activity of Pt(II) complexes with DNA and BSA was explored by absorption and fluorometric titration methods. In vitro MTT assay of Pt(II) complexes were tested on different cancer cell lines like; MCF 7, A549, HCT 116 and also on normal human embryonic kidney cell HEK 293. The cell cycle arrest of MCF 7 cell growth and also the oxidative stress level (ROS) after treating with Pt(II) complexes were analysed. The structural optimization for molecular docking of Pt(II) complexes at B3LYP functional level were performed to correlate the experimental findings. The antineoplastic activity and drug likeness behaviour of Pt(II) complexes were further supported by PASS prediction and in silico ADME analysis.

The DELLA-ABI4-HY5 module integrates light and gibberellin signals to regulate hypocotyl elongation

Plant growth is coordinately controlled by various environmental and hormonal signals, of which light and gibberellin (GA) signals are two critical factors with opposite effects on hypocotyl elongation. Although interactions between the light and GA signaling pathways have been studied extensively, the detailed regulatory mechanism of their direct crosstalk in hypocotyl elongation remains to be fully clarified. Previously, we reported that ABA INSENSITIVE 4 (ABI4) controls hypocotyl elongation through its regulation of cell-elongation-related genes, but whether it is also involved in GA signaling to promote hypocotyl elongation is unknown. In this study, we show that promotion of hypocotyl elongation by GA is dependent on ABI4 activation. DELLAs interact directly with ABI4 and inhibit its DNA-binding activity. In turn, ABI4 combined with ELONGATED HYPOCOTYL 5 (HY5), a key positive factor in light signaling, feedback regulates the expression of the GA2ox GA catabolism genes and thus modulates GA levels. Taken together, our results suggest that the DELLA-ABI4-HY5 module may serve as a molecular link that integrates GA and light signals to control hypocotyl elongation.

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

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

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

IKBA phosphorylation governs human sperm motility through ACC-mediated fatty acid beta-oxidation

The nuclear factor-κB (NF-κB) signaling pathway regulates specific immunological responses and controls a wide range of physiological processes. NF-κB inhibitor alpha (IKBA) is an NF-κB inhibitory mediator in the cytoplasm that modulates the nuclear translocation and DNA binding activities of NF-κB proteins. However, whether the upstream cascade of the canonical NF-κB signaling pathway has physiological roles independent of IKBA-mediated transcriptional activation remains unclear. Herein we investigated the function of IKBA in mature sperm in which transcriptional and translational events do not occur. IKBA was highly expressed in human sperm. The repression of IKBA phosphorylation by its inhibitor Bay117082 markedly enhanced sperm motility. On the contrary, lipopolysaccharide-stimulated IKBA phosphorylation significantly decreased sperm motility. Nevertheless, Bay117082 treatment did not affect the motility of IKBA-knockout sperm. Further, untargeted metabolomic analysis and pharmacological blocking assays revealed that the Bay117082-induced increase in sperm motility was attributable to fatty acid β-oxidation (FAO) enhancement. In addition, we found that IKBA phosphorylation inhibition resulted in a significant reduction of acetyl-CoA carboxylase levels in the FAO metabolic pathway. Our findings indicate that IKBA-mediated signaling orchestrates sperm motility program and improves our understanding of transcription-independent NF-κB signaling pathway in cells.

Screening of potential hub genes and key pathways associated with breast cancer by bioinformatics tools.

Breast cancer (BC) remains the leading cause of cancer-related death in women worldwide. The development of new targeted therapies that may improve patient survival remains an area of growing interest. This study aimed to identify new biomarkers involved in BC progression that could be used as potential targeted therapies. DEGs were selected from three gene expression profiles, GSE55715, GSE124646, and GSE87049, using the GEO2R tool and Venn diagram software. Gene Ontology and KEGG pathways were then performed using DAVID software. Next, the PPI network was constructed using STRING and visualized using Cytoscape software, and hub genes were extracted using the cytoHubba plug-in. Survival analysis was performed using the Kaplan-Meier Plotter, while the expression of hub genes in BC was verified using the GEPIA2 tool. Finally, transcription the factors of hub genes were determined using the NetworkAnalyst database, and the TIMER tool was employed to explore the infiltration levels of tumor immune cells with related genes. A total of 146 DEGs were identified in the three datasets, including 60 upregulated genes that were enriched in the cell cycle, and 86 downregulated genes that were mainly enriched in the TNF signaling pathway and pathways in cancer. Ten genes were identified: BUB1, CDK1, HMMR, MAD2L1, CEP55, AURKA, CCNB2, TPX2, MELK, and KIF20A. The overexpression of hub genes, except CDK1, was associated with poor survival in BC and was regulated by several transcription factors involved in DNA binding activity and transcription regulation. The infiltration levels of immune cells were positively correlated with hub genes, particularly macrophages and CD4+ T cells. This study identified new reliable molecular biomarkers that can serve as potential therapeutic targets for BC treatment.