Promoter DNA Research Articles

G9a/DNMT1 co-targeting inhibits non-small cell lung cancer growth and reprograms tumor cells to respond to cancer-drugs through SCARA5 and AOX1

The treatment of non-small cell lung cancer (NSCLC) patients has significantly improved with recent therapeutic strategies; however, many patients still do not benefit from them. As a result, new treatment approaches are urgently needed. In this study, we evaluated the antitumor efficacy of co-targeting G9a and DNMT1 enzymes and its potential as a cancer drug sensitizer. We observed co-expression and overexpression of G9a and DNMT1 in NSCLC, which were associated with poor prognosis. Co-targeting G9a/DNMT1 with the drug CM-272 reduced proliferation and induced cell death in a panel of human and murine NSCLC cell lines. Additionally, the transcriptomes of these cells were reprogrammed to become highly responsive to chemotherapy (cisplatin), targeted therapy (trametinib), and epigenetic therapy (vorinostat). In vivo, CM-272 reduced tumor volume in human and murine cell-derived cancer models, and this effect was synergistically enhanced by cisplatin. The expression of SCARA5 and AOX1 was induced by CM-272, and both proteins were found to be essential for the antiproliferative response, as gene silencing decreased cytotoxicity. Furthermore, the expression of SCARA5 and AOX1 was positively correlated with each other and inversely correlated with G9a and DNMT1 expression in NSCLC patients. SCARA5 and AOX1 DNA promoters were hypermethylated in NSCLC, and SCARA5 methylation was identified as an epigenetic biomarker in tumors and liquid biopsies from NSCLC patients. Thus, we demonstrate that co-targeting G9a/DNMT1 is a promising strategy to enhance the efficacy of cancer drugs, and SCARA5 methylation could serve as a non-invasive biomarker to monitor tumor progression.

Identification of two novel variants, c.-35A>T and c.[-35A>T, 725T>G], in the FUT1 gene in a patient exhibiting the para-Bombay phenotype.

Reduced or absent H antigens on red cells with the (para-)Bombay phenotype can arise from FUT1 gene mutations, impacting the structure and function of 1,2-L-fucosyltransferase 1 (1,2-L-FucT1). Here, we identified the novel mutations in one patient displaying the para-Bombay phenotype and examined the potential molecular mechanisms underlying this phenotype. ABH antigens and antibodies were detected in patient's blood and saliva using serological methods. The genotypes of ABO, FUT1, and FUT2 were imputed using the genetic variations discovered in the whole exome sequencing data. Three-dimensional (3D) models of FUT1 variants were built using Deepmind's AlphaFold2 and HDOCK, and the possible effects of the variants were predicted to evaluate using DynaMut2 and Polyphen-2. Serological analysis confirmed the para-Bombay B phenotype producing anti-HI and exhibiting normal genotypes ABO*B.01/O.01.02 and FUT2*01.09/01.09. Remarkably, the phenotype is caused by a compound heterozygous genotype: one allele containing the novel c.-35A>T mutation and the known c.725T>G mutation (p.Leu242Arg) of FUT1, and the other allele containing the c.-35A>T mutation. From the computerized stimulation analysis, p.Arg242 of the FUT1 variant may be detrimental, destabilizing, and probably damaging to 1,2-L-FucT1, although not altering the 3D structure of the entire enzyme. The c.-35A>T promoter DNA left at the binding interface of both ZID and c-Rel transcription factors may enable regulation of 1,2-L-FucT1 function at gene promoters. We identified the two novel variants, c.-35A>T and c.[-35A>T, 725T>G], in the FUT1 causing the para-Bombay phenotype. This finding may clarify the molecular mechanisms and enhance blood transfusion safety.

NRF3-mediated mitochondrial superoxide promotes cardiomyocyte apoptosis and impairs cardiac functions by suppressing Pitx2

Abstract Background Myocardial infarction (MI) elicits mitochondria reactive oxygen species (ROS) production and cardiomyocyte apoptosis. Nrf3 (Nuclear factor erythroid 2-related factor 3) has an established role in regulating redox signaling and tissue homeostasis. Herein, we aimed to uncover the exact role and potential mechanism of Nrf3 in injury-induced cardiac remodeling. Methods Global (Nrf3-KO) and cardiomyocyte-specific (NRF3△CM) Nrf3 knockout mice were subjected to MI or ischemia/reperfusion (I/R) injury, followed by functional and histopathological analysis. Primary neonatal mice (NMVMs) and rat (NRVMs) ventricular myocytes as well as cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) were used to detect the possible impact of Nrf3 on cardiomyocyte apoptosis and mitochondrial ROS production. Chromatin immunoprecipitation sequencing and immunoprecipitation–mass spectrometry analysis were used to uncover potential targets of Nrf3. MitoPQ administration and cardiomyocyte-specific AAV vector were used to further confirm the in vivo relevance of the identified signal pathways. Results Increased level of Nrf3 was observed in cardiomyocytes within border zone of infarcted human heart and murine cardiac tissues post-MI. Both global and cardiomyocyte-specific Nrf3 knockout significantly decreased injury-induced mitochondrial ROS production and cardiomyocyte apoptosis, consequently improving cardiac functions. Additionally, cardiac-specific Nrf3 overexpression reversed the cardiac phenotypes observed in Nrf3-KO mice. Functional studies showed that Nrf3 promoted NMVM, NRVM and hiPSC-CM apoptosis by increasing mitochondrial ROS production. Critically, restoring mitochondrial ROS using MitoParaquat blunted the beneficial effects of Nrf3 deletion on cardiac functions and remodeling. Mechanistically, the cardiac redox regulator paired-like homeodomain transcription factor 2 (Pitx2) was identified as one of the main target genes of Nrf3. Specifically, Nrf3 binds to Pitx2 gene promoter where it increases Pitx2 gene promoter DNA methylation through recruiting heterogeneous nuclear ribonucleoprotein K and DNA-methyltransferase 1 complex, thereby inhibiting Pitx2 expression. Importantly, cardiomyocyte-specific knockdown of Pitx2 blunted the beneficial effects of Nrf3 deletion on cardiac functions and remodeling. Conclusions Nrf3 promotes injury-induced cardiomyocyte apoptosis and deteriorates cardiac functions by increasing mitochondrial ROS production via suppressing Pitx2 expression. Targeting Nrf3-Pitx2-mitochondrial ROS signal axis could represent novel therapeutics for treating MI patients.Graphic Abstract

The mystique of epigenetic regulation: the remarkable case of a human noncoding RNA, nc886.

nc886 is a regulatory noncoding RNA that is transcribed by RNA polymerase III (Pol III), is variably expressed in different biological contexts, and plays roles in inflammation and cancer. Epigenetic mechanisms play an intriguing role in regulating nc886 expression. As a maternally imprinted gene and metastable epiallele, nc866 exhibits polymorphic imprinting, with a methylation status that is influenced by environmental and biological factors. Consequently, the promoter DNA methylation status and the different resulting RNA expression levels of nc886 are associated with physiological and pathological conditions. In this review, we summarize the literature and explore the significance in relation to diverse roles of nc886.

Bioactive Compounds and Their Chondroprotective Effects for Osteoarthritis Amelioration: A Focus on Nanotherapeutic Strategies, Epigenetic Modifications, and Gut Microbiota

In degenerative joint disease like osteoarthritis (OA), bioactive compounds like resveratrol, epigallocatechin gallate, curcumin, and other polyphenols often target various signalling pathways, including NFκB, TGFβ, and Wnt/β-catenin by executing epigenetic-modifying activities. Epigenetic modulation can target genes of disease pathophysiology via histone modification, promoter DNA methylation, and non-coding RNA expression, some of which are directly involved in OA but have been less explored. OA patients often seek options that can improve the quality of their life in addition to existing treatment with nonsteroidal anti-inflammatory drugs (NSAIDs). Although bioactive and natural compounds exhibit therapeutic potential against OA, several disadvantages loom, like insolubility and poor bioavailability. Nanoformulated bioactive compounds promise a better way to alleviate OA since they also control systemic events, including metabolic, immunological, and inflammatory responses, by modulating host gut microbiota that can regulate OA pathogenesis. Recent data suggest gut dysbiosis in OA. However, limited evidence is available on the role of bioactive compounds as epigenetic and gut modulators in ameliorating OA. Moreover, it is not known whether the effects of polyphenolic bioactive compounds on gut microbial response are mediated by epigenetic modulatory activities in OA. This narrative review highlights the nanotherapeutic strategies utilizing bioactive compounds, reporting their effects on chondrocyte growth, metabolism, and epigenetic modifications in osteoarthritis amelioration.

Peptidylprolyl isomerase A guides SENP5/GAU1 DNA-lncRNA triplex generation for driving tumorigenesis.

The three-stranded DNA-RNA triplex hybridization is involved in various biological processes, including gene expression regulation, DNA repair, and chromosomal stability. However, the DNA-RNA triplex mediating mechanisms underlying tumorigenesis remain to be fully elucidated. Here, we show that peptidylprolyl isomerase A (PPIA) serves as anchor to recruit GAU1 lncRNA by interacting with exon 4 of GAU1 and enhances the formation of SENP5/GAU1 DNA-lncRNA triplex. Intriguingly, TFR4 region of GAU1 exon 3 and TTS4 region of SENP5 promoter DNA constitute fragments forming the SENP5/GAU1 triplex. The SENP5/GAU1 triplex subsequently triggers the recruitment of the methyltransferase SET1A to exon 1 of GAU1, leading to the enrichment of H3K4 trimethylation and the activation of SENP5 transcription for driving the tumorigenesis of gastric cancer in vitro and in vivo. Our study reveals a mechanism of PPIA-guided SENP5/GAU1 DNA-lncRNA triplex formation in tumorigenesis and providing a concept in the dynamics of isomerase assisted DNA-RNA hybridization.

A community effort to optimize sequence-based deep learning models of gene regulation.

A systematic evaluation of how model architectures and training strategies impact genomics model performance is needed. To address this gap, we held a DREAM Challenge where competitors trained models on a dataset of millions of random promoter DNA sequences and corresponding expression levels, experimentally determined in yeast. For a robust evaluation of the models, we designed a comprehensive suite of benchmarks encompassing various sequence types. All top-performing models used neural networks but diverged in architectures and training strategies. To dissect how architectural and training choices impact performance, we developed the Prix Fixe framework to divide models into modular building blocks. We tested all possible combinations for the top three models, further improving their performance. The DREAM Challenge models not only achieved state-of-the-art results on our comprehensive yeast dataset but also consistently surpassed existing benchmarks on Drosophila and human genomic datasets, demonstrating the progress that can be driven by gold-standard genomics datasets.

Mycobacterial HelD connects RNA polymerase recycling with transcription initiation

Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

LINC01116-dependent upregulation of RNA polymerase I transcription drives oncogenic phenotypes in lung adenocarcinoma

BackgroundHyperactive RNA Polymerase I (Pol I) transcription is canonical in cancer, associated with malignant proliferation, poor prognosis, epithelial-mesenchymal transition, and chemotherapy resistance. Despite its significance, the molecular mechanisms underlying Pol I hyperactivity remain unclear. This study aims to elucidate the role of long noncoding RNAs (lncRNAs) in regulating Pol I transcription in lung adenocarcinoma (LUAD).MethodsBioinformatics analyses were applied to identify lncRNAs interacting with Pol I transcriptional machinery. Fluorescence in situ hybridization was employed to examine the nucleolar localization of candidate lncRNA in LUAD cells. RNA immunoprecipitation assay validated the interaction between candidate lncRNA and Pol I components. Chromatin isolation by RNA purification and Chromatin Immunoprecipitation (ChIP) were utilized to confirm the interactions of candidate lncRNA with Pol I transcriptional machinery and the rDNA core promoter. Functional analyses, including lncRNA knock-in and knockdown, inhibition of Pol I transcription, quantitative PCR, cell proliferation, clonogenicity, apoptosis, cell cycle, wound-healing, and invasion assays, were performed to determine the effect of candidate lncRNA on Pol I transcription and associated malignant phenotypes in LUAD cells. ChIP assays and luminometry were used to investigate the transcriptional regulation of the candidate lncRNA.ResultsWe demonstrate that oncogenic LINC01116 scaffolds essential Pol I transcription factors TAF1A and TAF1D, to the ribosomal DNA promoter, and upregulate Pol I transcription. Crucially, LINC01116-driven Pol I transcription activation is essential for its oncogenic activities. Inhibition of Pol I transcription abrogated LINC01116-induced oncogenic phenotypes, including increased proliferation, cell cycle progression, clonogenicity, reduced apoptosis, increased migration and invasion, and drug sensitivity. Conversely, LINC01116 knockdown reversed these effects. Additionally, we show that LINC01116 upregulation in LUAD is driven by the oncogene c-Myc, a known Pol I transcription activator, indicating a functional regulatory feedback loop within the c-Myc-LINC01116-Pol I transcription axis.ConclusionCollectively, our findings reveal, for the first time, that LINC01116 enhances Pol I transcription by scaffolding essential transcription factors to the ribosomal DNA promoter, thereby driving oncogenic activities in LUAD. We propose the c-Myc-LINC01116-Pol I axis as a critical oncogenic pathway and a potential therapeutic target for modulating Pol I transcription in LUAD.Graphical

Mammalian D-Cysteine controls insulin secretion in the pancreas

D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Analysis of gliomas DNA methylation: Assessment of pre-analytical variables.

Precision oncology is driven by biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is an important prognostic and treatment clinical biomarker. Time consuming pre-analytical steps such as biospecimen storage, fixation, sampling, and processing are sources of data irreproducibility, and all these pre-analytical variables are confounded by intratumor heterogeneity of MGMT promoter methylation. To assess the effect of pre-analytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multi-sonicator (PIXUL), and 5-methylcytosine (5mC) DNA immunoprecipitation (Matrix MeDIP-qPCR/seq) platforms were used. MGMT promoter methylation status assayed by MeDIP-qPCR was validated with methylation specific PCR (MS-PCR). MGMT promoter methylation levels in frozen and formalin fixed paraffin embedded (FFPE) sample pairs were not statistically different, confirming reliability of FFPEs for MGMT promoter methylation analysis. Warm ex-vivo ischemia (up to 4hrs at 37oC) and 3 cycles of repeated sample thawing and freezing did not statistically impact 5mC at MGMT promoter, exon, and enhancer regions, indicating the resistance of DNA methylation to common variations in sample processing conditions that might be encountered in research and clinical settings. 26-34% of specimens exhibited intratumor heterogeneity in the MGMT DNA promoter methylation. These data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have a statistically significant impact on MGMT promoter methylation assessment. However, intratumor methylation heterogeneity underscores the value of multiple biopsies at different GBM geographic tumor sites in the evaluation of MGMT promoter methylation status. Matrix-MeDIP-seq analysis revealed that MGMT promoter methylation status clustered with other differentially methylated genomic loci (e.g. HOXA and lncRNAs) that are resilient to variation in the above pre-analytical conditions. These observations offer new opportunities to develop more granular data-based epigenetic GBM biomarkers. In this regard, the high throughput CryoGrid-PIXUL-Matrix toolbox could be useful.

Potential of quantum machine learning for solving the real-world problem of cancer classification

Quantum machine learning (QML) algorithms have demonstrated the power of quantum computing for solving complex problems and big data in certain tasks. In this study, we explore the capabilities of QML for the classification of real-world biological large datasets including ten different cancer types based on gene expression values. By comparing the classification results obtained from the quantum algorithm with those from classical approaches, we disclose that the QML algorithm overall achieves comparable and reliable results. Moreover, we identify novel biomarkers that can contribute to the understanding of cancer biology. Some of these biomarkers are consistent with DNA promoter methylation. Our findings highlight the potential of QML in cancer classification and biomarker discovery, paving the way for future advancements in other disease research and clinical applications.

Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in Mycobacterium tuberculosis.

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.

DNA methylation-mediated FGFR1 silencing enhances NF-κB signaling: implications for asthma pathogenesis.

Fibroblast growth factor receptor 1 (FGFR1) is known to play a crucial role in the pathogenesis of asthma, although the precise mechanism remains unclear. This study aims to investigate how DNA methylation-mediated silencing of FGFR1 contributes to the enhancement of NF-κB signaling, thereby influencing the progression of asthma. RT-qPCR was utilized to assess FGFR1 mRNA levels in the serum of asthma patients and BEAS-2B, HBEpiC, and PCS-301-011 cells. CCK8 assays were conducted to evaluate the impact of FGFR1 overexpression on the proliferation of BEAS-2B, PCS-301-011, and HBEpiC cells. Dual-luciferase and DNA methylation inhibition assays were performed to elucidate the underlying mechanism of FGFR1 gene in asthma. The MassARRAY technique was employed to measure the methylation levels of the FGFR1 DNA. Elevated FGFR1 mRNA levels were observed in the serum of asthma patients compared to healthy controls. Overexpression of FGFR1 in BEAS-2B cells significantly enhanced cell proliferation and stimulated NF-ĸB transcriptional activity in HERK-293T cells. Furthermore, treatment with 5-Aza-CdR, a DNA demethylating agent, markedly increased the expression of FGFR1 mRNA in BEAS-2B, PCS-301-011, and HBEpiC cells. Luciferase activity analysis confirmed heightened NF-ĸB transcriptional activity in FGFR1-overexpressing BEAS-2B cells and BEAS-2B cells treated with 5-Aza-CdR. Additionally, a decrease in methylation levels in the FGFR1 DNA promoter was detected in the serum of asthma patients using the MassARRAY technique. Our findings reveal a potential mechanism involving FGFR1 in the progression of asthma. DNA methylation of FGFR1 inactivates the NF-ĸB signaling pathway, suggesting a promising avenue for developing effective therapeutic strategies for asthma.

Discovery of KW0113 as a First and Effective PROTAC Degrader of DNMT1 Protein

DNA methyltransferase 1 (DNMT1) is an attractive therapeutic target for acute myelocytic leukemia (AML) and other malignancies. It has been reported that the genetic depletion of DNMT1 inhibited AML cell proliferation through reversing DNA methylation abnormalities. However, no DNMT1‐targeted PROTAC degraders have been reported yet. Herein, a series of proteolysis‐targeting chimera (PROTAC) degrader of DNMT1 based on dicyanopyridine scaffold and VHL E3 ubiquitin ligase ligand was developed. Among them, KW0113 (DC50 = 643/899 nM in MV4‐11/MOLM‐13 cells) exhibited optimal DNMT1 degradation. KW0113 induced DNMT1‐selective degradation in a dose‐ and time‐dependent manner through VHL engagement. Moreover, KW0113 inhibited AML cell growth by reversing promoter DNA hypermethylation and tumor‐suppressor genes silencing. In conclusion, these findings proved the capability of PROTAC strategy for inducing DNMT1 degradation, demonstrated the therapeutic potential of DNMT1‐targeted PROTACs. This work also provided a convenient chemical knockdown tool for DNMT1‐related studies.

The first embryonic landscape of G-quadruplexes related to myogenesis

BackgroundDNA G-quadruplexes (G4s) represent a distinctive class of non-canonical DNA secondary structures. Despite their recognition as potential therapeutic targets in some cancers, the developmental role of G4 structures remains enigmatic. Mammalian embryonic myogenesis studies are hindered by limitations, prompting the use of chicken embryo-derived myoblasts as a model to explore G4 dynamics. This study aims to reveal the embryonic G4s landscape and elucidate the underlying mechanisms for candidate G4s that influence embryonic myogenesis.ResultsThis investigation unveils a significant reduction in G4s abundance during myogenesis. G4s stabilizer pyridostatin impedes embryonic myogenesis, emphasizing the regulatory role of G4s in this process. G4 Cut&Tag sequencing and RNA-seq analyses identify potential G4s and DEGs influencing embryonic myogenesis. Integration of G4 and DEG candidates identifies 32 G4s located in promoter regions capable of modulating gene transcription. WGBS elucidates DNA methylation dynamics during embryonic myogenesis. Coordinating transcriptome data with DNA G4s and DNA methylation profiles constructs a G4-DMR-DEG network, revealing nine interaction pairs. Notably, the NFATC2 promoter region sequence is confirmed to form a G4 structure, reducing promoter mCpG content and upregulating NFATC2 transcriptional activity.ConclusionsThis comprehensive study unravels the first embryonic genomic G4s landscape, highlighting the regulatory role of NFATC2 G4 in orchestrating transcriptional activity through promoter DNA methylation during myogenesis.

FAM109B plays a tumorigenic role in low-grade gliomas and is associated with tumor-associated macrophages (TAMs)

BackgroundFamily with sequence similarity 109, member B (FAM109B) is involved in endocytic transport and affects genetic variation in brain methylation. It is one of the important genes related to immune cell-associated diseases. In the tumor immune system, methylation can regulate tumor immunity and influence the maturation and functional response of immune cells. Whether FAM109B is involved in tumor progression and its correlation with the tumor immune microenvironment has not yet been disclosed.MethodsA comprehensive pan-cancer analysis of FAM109B expression, prognosis, immunity, and TMB was conducted. The expression, clinical features, and prognostic value of FAM109B in low-grade gliomas (LGG) were evaluated using TCGA, CGGA, and Gravendeel databases. The expression of FAM109B was validated by qRT-PCR, immunohistochemistry (IHC), and Western blotting (WB). The relationship between FAM109B and methylation, Copy Number Variation (CNV), prognosis, immune checkpoints (ICs), and common chemotherapy drug sensitivity in LGG was explored through Cox regression, Kaplan–Meier curves, and Spearman correlation analysis. FAM109B levels and their distribution were studied using the TIMER database and single-cell analysis. The potential role of FAM109B in gliomas was further investigated through in vitro and in vivo experiments.ResultsFAM109B was significantly elevated in various tumor types and was associated with poor prognosis. Its expression was related to aggressive progression and poor prognosis in low-grade glioma patients, serving as an independent prognostic marker for LGG. Glioma grade was negatively correlated with FAM109B DNA promoter methylation. Immune infiltration and single-cell analysis showed significant expression of FAM109B in tumor-associated macrophages (TAMs). The expression of FAM109B was closely related to gene mutations, immune checkpoints (ICs), and chemotherapy drugs in LGG. In vitro studies showed increased FAM109B expression in LGG, closely related to cell proliferation. In vivo studies showed that mice in the sh-FAM109B group had slower tumor growth, slower weight loss, and longer survival times.ConclusionsFAM109B, as a novel prognostic biomarker for low-grade gliomas, exhibits specific overexpression in TAMs and may be a potential therapeutic target for LGG patients.

Identification and characterization of a small molecule BFstatin inhibiting BrpR, the transcriptional regulator for biofilm formation of Vibrio vulnificus.

Many pathogenic bacteria form biofilms that are resistant to not only host immune defenses but also antibiotics, posing a need for the development of strategies to control biofilms. In this study, to prevent biofilm formation of the fulminating foodborne pathogen Vibrio vulnificus, chemical libraries were extensively screened to identify a small molecule inhibiting the activity of BrpR, a transcriptional regulator for biofilm genes. Accordingly, the BrpR inhibitor BFstatin [N1-(2-chloro-5-fluorophenyl)-N3-propylmalonamide], with a half-maximal effective concentration of 8.01 μM, was identified. BFstatin did not interfere with bacterial growth or exhibit cytotoxicity to the human epithelial cell line. BFstatin directly bound to BrpR and interrupted its binding to the target promoter DNAs of the downstream genes. Molecular dynamics simulation of the interaction between BFstatin and BrpR proposed that BFstatin modifies the structure of BrpR, especially the DNA-binding domain. Transcriptomic analyses revealed that BFstatin reduces the expression of the BrpR regulon including the cabABC operon and brp locus which contribute to the production of biofilm matrix of V. vulnificus. Accordingly, BFstatin diminished the biofilm levels of V. vulnificus by inhibiting the matrix development in a concentration-dependent manner. Altogether, BFstatin could be an anti-biofilm agent targeting BrpR, thereby rendering V. vulnificus more susceptible to host immune defenses and antibiotics.

Transcriptional activation domains interact with ATPase subunits of yeast chromatin remodelling complexes SWI/SNF, RSC and INO80

Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.

Association of a Promoter DNA Methyltransferase 3 Gene Variant with DNA Methylation and Anthropometrics in Children from 4 to 12 Years Old

The global prevalence of obesity among adults, adolescents, and children has increased to alarming levels, making this disease a serious public health problem. The etiology of obesity is complex and multifactorial. Currently, epigenetic alterations are being investigated to understand the mechanisms of interaction between genes and environmental and behavioral risk factors involved in the genesis of obesity. In this study, we examined the association of the DNA methyltransferase 3 (DNMT3B) gene-149 C>T variant (rs2424913) genotypes with global DNA methylation and the changes in anthropometric parameters in a cohort of 171 children followed from birth to 12 years old. Genotypes were obtained using real-time polymerase chain reaction, and global DNA methylation was measured in blood samples collected at 4 years old through enzyme-linked immunosorbent assays. Our results showed that the TT genotype is associated with an increase in global methylation levels at 4 years old and higher changes in body mass index, waist circumference, subscapular subcutaneous fat, body fat mass, body lean mass, and basal metabolic rate from 4 to 12 years. Our results suggest that this promoter DNMT3B gene variant and DNA methylation can be factors relevant to the increased risk of children developing obesity at an early age.