Search Results (335)

Progesterone receptor (PR) regulates gene expression through recruiting coregulators and general transcription factors by activation functions AF1 and AF2. AF1 localizes to the non-conserved and disordered N-terminal domain and is believed to facilitate tissue- and gene-specific activity. Our previous proteomic analysis identified three key residues (K464, K481 and R492) in AF1 that are monomethylated. Methylation mimic mutations KKR → FFF created hypoactive PR, whereas the KKR → QQQ mutation generated hyperactive PR in gene reporter assays. The current study used these mutants to determine the roles of AF1 in PR regulation of cellular activities and global gene regulation in breast cancer cells MCF-7. AF1-FFF mutation attenuated PR regulation of cell proliferation and apoptosis in response to progestin, whereas AF1-QQQ mutation enhanced these effects. AF1-FFF mutation attenuated gene regulation by progestin in ~60% of PR target genes, including genes involved in cell proliferation, hypoxia and TNFα signaling. However, the AF1-FFF mutation had little effect on ligand-independent gene regulation, suggesting distinct mechanisms of gene regulation by liganded and unliganded PR. Intriguingly, impaired activity of methylation mimic mutant PRB-FFF is associated with greater chromatin binding in ChIP-Seq analysis, corresponding to a stronger association between PRB-FFF and Steroid Receptor Coactivator-1 (SRC-1), a member of the p160 family of nuclear receptor coactivators, as was previously reported. In conclusion, PR AF1 is important for the core activities of liganded PR in regulating ~half of target genes and cell proliferation. AF1 monomethylation may modulate PR-chromatin interactions through stronger association with coregulators, thereby decelerating chromatin binding kinetics. This is supported by PRODIGY’s prediction of higher binding affinities of monomethylated AF1 and methylation mimic mutant with SRC-1. Full article

Eukaryotic DNA is wrapped around octamers of four core histones, forming nucleosomes. Histone post-translational modifications (PTMs) influence chromatin structure and the recruitment of regulatory factors, thereby affecting gene expression and DNA repair, including the response to DNA double-strand breaks (DSBs). Here, we describe a robust chromatin immunoprecipitation protocol combined with micrococcal nuclease digestion and DNA sequencing (MNase-ChIP-seq) to map histone modifications and their genome-wide distribution after the induction of a single DSB by the HO endonuclease in Saccharomyces cerevisiae. We validate the method by detecting changes in histone H3 methylation following HO transcriptional activation and DSB induction. This protocol enables reliable analysis of histone PTMs across mutant strains or stress conditions, supporting studies of chromatin dynamics in yeast. Full article

Background/Objectives: The annual ~36,000 prostate cancer (PCa) deaths represent a large clinical unmet need and a call for deeper understanding of PCa metastasis. Epithelial–mesenchymal-transition (EMT) has been used to model metastatic behaviors in numerous cancers including PCa. One hallmark of EMT is cell cycle suppression, but how EMT impacts PCa proliferation remains unclear primarily due to the lack of appropriate models. Methods: We transiently induced Snail1 (SNAI1) expression, an EMT driver expressed in PCa, at physiological levels in three PCa cells lines, C4-2B, 22Rv1, and DU145. We used RNA-seq, ChIP-Seq, bioinformatics, qRT-PCR, shRNA, and immunoblotting to identify mechanisms of Snail1-driven inhibition of proliferation. Results: Snail1 suppressed proliferation and G2/M cell cycle progression, without affecting cell death. Mechanistically, Snail1 upregulated expression of CEBPγ, ERG1, FOXO1, cyclin G1, p21, stress genes SESN3 and SOD3, apoptotic programmers Puma, Bax, and Noxa, and senescence-related laminB1, and downregulated Ki67, cyclins A2 and B2. ChIP-Seq data identified Snail1 direct binding to p21, cyclin B2 and G1, EGR1, and CEPBγ promoters. EGR1 induced FOXO1, and EGR1 was required for Snail1-induced SOD3 and Puma, and suppression of Caspase 3 to prevent apoptosis. The EGR1/FOXO1 axis induced BAX, Noxa, and SESN3. CEBPγ was required for Snail1 induction of Lamin B1 to block Snail1-induced senescence. Conclusions: We identified three new major downstream targets of Snail1 that improve our understanding of the role of EMT in limiting stress signaling, apoptosis, and senescence during cell cycle suppression to create a vulnerability for therapeutic targeting. Full article

Vitamin D deficiency is associated with the risk of atopic diseases and respiratory infections. The activated vitamin D receptor (VDR) forms a dimer with the retinoid X receptor alpha (RXRA) and binds to VDR/RXRA composite elements (CEs) in enhancers of target genes. However, VDR/RXRA CEs are identified in only 11.5% of cases in ChIP-Seq peaks. Our hypothesis was that VDR could form a VDR-Partner complex with transcription factor for which CEs have not yet been identified. We utilized Web-MCOT to search for novel VDR/Partner CEs in regulatory DNA. The potential formation of the VDR-Partner protein complex was assessed using the AlphaFold machine learning model. Through real-time RT-PCR, we measured the expression of immune system genes in a culture of U937 macrophage-like cells incubated with the active metabolite of vitamin D, calcitriol. We have predicted novel VDR/NR2C2 and VDR/PPARG CEs in the regulatory regions of immune system genes. We found potential synergism of VDR/NR2C2 and VDR/RXRA CEs in relation to the IRF5 gene, as well as potential synergism of VDR/PPARG and VDR/RXRA CEs for MAPK13. Predicting new regulatory relationships through the identification of new potential VDR/Partner CEs may provide insight into the deep mechanisms of vitamin D involvement in the pathogenesis of atopic dermatitis, bronchial asthma, allergic rhinitis, and pulmonary infections. Full article

The dynamic remodeling of the fungal cell wall depends on a balance between chitin synthesis and degradation. Chitinases are critical for nutrient acquisition, cell wall remodeling, and defense; yet, the upstream regulatory mechanisms controlling chitinase gene expression remain poorly understood. Here, Tandem Affinity Purification–Mass Spectrometry (TAP–MS) with the Penicillium oxalicum Snf1 kinase (PoSnf1) as bait identified the zinc finger transcription factor (TF) PoCon7 as a putative target of the Snf1 kinase complex. This complex comprises the catalytic α subunit Snf1, one of three alternative β subunits Gal83, and the γ subunit Snf4. Although PoCon7 does not directly bind PoSnf1 or PoSnf4, it specifically interacts with PoGal83. Phylogenetic analysis indicates that PoCon7 is a conserved, nuclear-localized C2H2-type TF in filamentous fungi. PoCon7 is likely essential for fungal viability, as only a truncated mutant (con7-B) could be generated, while full deletion was lethal. The con7-B mutant displayed delayed hyphal extension, reduced conidiation, downregulation of developmental genes, and upregulation of cell wall-degrading enzyme (CWDE) genes. DNA Affinity Purification Sequencing (DAP-seq) revealed that PoCon7 binds target gene promoters via the motif 5′-TATTWTTAT-3′. ChIP-qPCR confirmed PoCon7 enrichment at specific sites within the chitinase genes chi18A and chi18C, and the disruption of PoCon7 markedly reduced their expression. Thus, PoCon7 represents the first TF shown to directly regulate chitinase gene expression in filamentous fungi. Full article

Environmental allergens trigger epithelial reactive oxygen species (ROS) production and cellular senescence, contributing to airway inflammation. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor responsive to environmental stimuli, may modulate this process. Single-cell transcriptomics from allergen-challenged bronchoalveolar brushings of allergic asthma and non-asthmatic allergic control subjects were analyzed for ROS, senescence, and AhR activity. Club cell-specific p16 knockout (p16^ΔScgb1a1) and AhR-deficient (AhR^ΔScgb1a1) mice were used to assess epithelial senescence and AhR function. Single-cell analysis revealed epithelial senescence as a hallmark of allergen-induced asthma. p16^ΔScgb1a1 mice exhibited reduced ROS levels and airway inflammation. Single-cell analysis also demonstrated increased AhR activity and ROS generation in airway epithelial cells of allergen-treated asthmatics, and ROS correlated positively with AhR activity and senescence. It was documented that the regulation of AhR on senescence was attenuated by VAF347, whereas AhR deficiency exacerbated ROS generation and inflammation in AhR^ΔScgb1a1 mice. RNA-seq identified senescence as a key AhR-regulated pathway, implicating c-Myc, TGF-β2, and SERPINE1 as major targets. AhR binding to the c-Myc promoter was confirmed by ChIP-PCR, and pharmacologic inhibition of c-Myc with EN4 reduced allergen-induced ROS, senescence, and inflammation. These findings demonstrate that epithelial AhR suppresses allergen-induced ROS generation and cellular senescence via direct regulation of c-Myc. Full article

Stress-induced mental disorders, including depression and anxiety disorders, constitute a global issue in contemporary society due to treatment complexity and the diversity of manifestations. Understanding the molecular mechanisms of these disorders presents a significant challenge for neurobiology. We investigated the effects of social defeat stress (SDS) of varying durations (10 and 30 days) on behavioral patterns and the H3K4me3 (trimethylation at the 4th lysine residue of histone H3) landscape in the prefrontal cortex of C57BL/6 mice. Furthermore, we compared these data with previously published H3K4me3 landscape data obtained after 15 days of SDS and transcriptomic data collected after 10, 15, and 30 days. We discovered that a 30-day period of stress results in more pronounced depressive-like behavior. SDS induces slight alterations in the H3K4me3 density across numerous nucleosomal peaks. The analysis of differential enrichment peaks of H3K4me3 in promoter regions following varying durations of SDS revealed that the aggregation of multiple H3K4me3 nucleosome peaks in the promoter region functions as a QR code, likely affecting the promoter’s state regarding the accessibility of transcription factors. Furthermore, we identified a cluster of genes in the promoter regions exhibiting differential enrichment peaks of H3K4me3 following SDS of any duration. This cluster includes genes encoding transcription factors such as Mef2c and Nr4a3, as well as postsynaptic density proteins (Shank2, Shank1, and Gria2), which are associated with stress sensitivity and the onset of depression; their protein products are involved in synaptic transmission and signal transduction mechanisms. The comparison of ChIP-seq and RNA-seq data following varying durations of SDS enabled a deeper insight in to the dynamics of SDS-induced changes. Together, these findings provide a better understanding of the molecular mechanisms of SDS in the prefrontal cortex. Full article

Ligand-activated Retinoid X Receptors (RXRs) regulate gene networks essential for neural development, neuroinflammation, and metabolism. Understanding how RXR activation influences chromatin architecture and gene expression may reveal mechanisms relevant to neurodegenerative diseases. We used Bexarotene-treated APP/PS1ΔE9 mice to study RXR-mediated regulatory mechanisms by integrating single-nucleus ATAC-seq (snATAC-seq) with single-cell RNA-seq (scRNA-seq) and validating differentially accessible chromatin peaks using RXR ChIP-seq. Transcription factor (TF) footprinting analysis mapped regulatory networks activated by ligand-bound RXR. Our integrated analyses revealed a multilayered transcriptional cascade initiated by RXR signaling. We identified RXR-centered regulatory circuits involving heterodimer activation, upregulation of downstream TFs, and induction of metabolic pathways relevant to neural function. Detailed analysis of neuronal TF networks revealed that Bexarotene modulates RXR’s role through existing regulatory scaffolds rather than creating new ones. This study demonstrates that combining scRNA-seq, snATAC-seq, and ChIP-seq enables comprehensive analysis of RXR-mediated transcriptional regulation. RXR activation orchestrates cell-type-specific chromatin remodeling of gene networks controlling neuroinflammation, lipid metabolism, and synaptic signaling, providing mechanistic insights into RXR-dependent transcriptional programs in Alzheimer’s disease pathology. Full article

Asexual sporulation (conidiogenesis) in filamentous fungi is a complex developmental process that requires precise coordination with primary metabolism and environmental stress responses. In the model fungus Aspergillus nidulans, we demonstrate that the bZIP-type transcription factor AtfA plays a central role in integrating conidiogenesis with the underlying metabolic and regulatory networks. Using combined ChIP-seq and RNA-seq analyses in wild-type, ∆atfA, and atfA-complemented strains under stress-free and oxidative stress (menadione) conditions, we identify a conserved AtfA binding motif and map its functional targets genome-wide. Our data reveal that AtfA binding to its target promoters is largely stress-independent, suggesting a preemptive regulatory mechanism in conidial development. AtfA directly binds to the promoters of genes involved in the MAPK signaling cascade, light-dependent sporulation, antioxidant defense, eisosome biogenesis, and the biosynthesis of trehalose and polyols—key metabolites supporting spore maturation and dormancy. Importantly, AtfA acts predominantly as a transcriptional activator, and its regulatory scope extends beyond stress adaptation to the orchestration of metabolic processes essential for spore integrity and germination. These findings position AtfA as a master integrator that synchronizes morphological development with metabolic preparedness during asexual reproduction in A. nidulans. Full article

(1) Background: Epigenetic mechanisms play a significant role in plant architecture. Histone deacetylases, as crucial epigenetic regulators, shape plant architecture by modifying chromatin structure and regulating gene expression. (2) Methods: This study combined bioinformatic identification of BpHST1 with its functional characterization in transgenic birch overexpressing 35S::BpHST1::FLAG, including phenotypic and cytological analyses. The putative direct targets of BpHST1 were further identified by integrating RNA-seq and ChIP-seq data. (3) Results: Phylogenetic analysis revealed that the HST1 orthologs from birch and peach form a distinct clade, consistent with their high degree of protein sequence conservation. BpHST1 exhibited light-inducible and leaf-preferential expression, with transcript levels elevated under light versus dark conditions, enriched in leaves relative to roots, and promoter activity confirming this spatial patterning. Overexpression of BpHST1 significantly suppressed plant height, cell length, cell width, and photosynthetic capacity. Integrated RNA-seq and ChIP-seq analysis suggested that BpLHCA2 possible functions as a direct downstream target of BpHST1, mediating plant growth and development. (4) Conclusions: Our findings delineated the role of BpHST1 in regulating plant architecture through comprehensive expression and functional analyses, and identified a candidate target gene. This study provided a novel insight into the molecular mechanisms governing plant architecture and offers potential strategies for future epigenetic breeding. Full article

Soybean cyst nematode (SCN, Heterodera glycines) is one of the major pathogens of soybean worldwide. We utilized the CHIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) data from the transgenic GmMYB29 strain (Glycine Max roots). We then performed enrichment analysis using KEGG and GO to identify potential candidate genes within the promoter-binding region. A targeted regulatory relationship between the GmMYB29 and GmPP2C-37like genes was further identified using the dual-luciferase Assay (Luciferase, LUC) and yeast one-hybrid Assay (Y1H). Hairy roots with target gene overexpression and gene-edited hairy roots were generated, and their resistance to soybean cyst nematode (SCN) was evaluated. Meanwhile, the presence of reciprocal genes with GmPP2C-37like was determined by the yeast two-hybrid library screening method. The targeting relationship between GmMYB29 and GmPP2C-37like genes was further validated through the Y1H assay and LUC assay. Based on phenotypic assessments of SCN, transgenic soybean roots overexpressing GmPP2C-37like exhibited significantly enhanced resistance to SCN 3 compared to wild-type. Further analysis revealed that GmPP2C-37like collaborates with other regulatory factors to modulate soybean resistance against SCN. Yeast two-hybrid library (Y2H) screening identified 18 interacting proteins. These findings not only illuminate the functional role of GmPP2C-37like but also provide a foundation for dissecting its molecular network. Moreover, the results offer promising candidate genes for enhancing SCN resistance and optimizing soybean resilience through targeted genetic strategies. Full article

As reported in earlier work, when a pea apyrase, psNTP9 (PS), and a modified version of it, psNTP9-DM (DM), are expressed in Saccharomyces cerevisiae, they localize to nuclei, binding to largely non-overlapping promoter regions of chromatin. PS- and DM-expressing yeast also exhibit different expression profiles for potentially regulated target genes, consistent with observed phenotypes. In the present study, we use ChIP-seq assays to show that PS and DM also associate with largely different promoter regions of Arabidopsis genes, with similar non-overlapping expression profiles for potential target genes. Functional studies, using electrophoretic mobility shift assays (EMSA), verified PS-specific binding to yeast or plant promoter binding sites. DM binding to both heterologous dsDNA and to PS-specific binding site sequences was minimal. AlphaFold3 modeling of PS protein binding to a yeast PHM6 promoter sequence identified potential DNA-binding residues and a potential binding site motif (5′-(G/T)GG(G/T)A-3′) that is also present in two Arabidopsis promoter binding sites. These novel findings extend the previously known functions of PS and other plant apyrases in the Golgi or extracellular matrix, and support their potential function as DNA-binding proteins that can regulate gene expression in both yeast and Arabidopsis. Full article

Seed oil content and fatty acid (FA) composition collectively determine the quality and economic value of Brassica napus. Little is known about the role of R2R3-MYB transcription factors (TFs) in regulating FA biosynthesis in B. napus. Here, BnaC04.MYB89 was found to be expressed primarily in developing seeds. Overexpression of BnaC04.MYB89 consistently decreased FA levels, as evidenced by its effect in both the Arabidopsis thaliana myb89-1 mutant and B. napus seeds. RNA-seq of developing seeds at 30 DAP (days after pollination) revealed marked suppression of FA biosynthetic genes in BnaC04.MYB89-overexpressing plants compared to the K407 control. ChIP (Chromatin immunoprecipitation) analysis revealed that BnaC04.MYB89 directly inhibited the expression of BnaA03.BCCP1 and BnaC03.HD while indirectly regulating that of BnaA09.BADC1, BnaA03.BADC3, BnaA03.MOD1, and BnaA08.FAT8, thereby reducing seed FA accumulation. Collectively, these results elucidate the role for BnaC04.MYB89 and provide new insights into the transcriptional regulatory network controlling seed oil accumulation in B. napus. Full article

Extrachromosomal circular DNA (eccDNA) has been recognized as a key player in tumorigenesis and progression. However, eccDNA transcriptional regulatory mechanisms under DNA damage in cancer remain poorly characterized. Here, we used doxorubicin to induce DNA damage in the hepatocellular carcinoma cell line HepG2 and performed Circle-seq to profile eccDNAs before and after the damage. We observed a significant increase in the number, length, and chromosomal distribution density of eccDNAs following DNA damage. RNA-seq revealed that the expression of genes carried on eccDNA was positively correlated with eccDNA copy number under DNA damage. Further ATAC-seq profiling identified distinct chromatin characteristics at eccDNA breakpoint regions compared to other regions of eccDNA and linear genomic regions. Additionally, eccDNAs generated under DNA damage preferentially originated from linear genomic regions characterized by low GC content and hypomethylation. Finally, by integrating Hi-C and H3K27ac ChIP-seq, we uncovered that eccDNAs with mobile enhancer activity (ME-eccDNAs) display significantly enhanced chromatin interactions and H3K27ac enrichment after DNA damage. Overall, our findings systematically elucidate the DNA damage-driven mechanisms underlying eccDNA biogenesis, chromatin characteristics and transcriptional regulation in HCC HepG2 cells. Full article

Epigenetic modifications are emerging as key regulators of plant stress responses. However, their role in eggplant (Solanum melongena)–western flower thrips (WFTs; Frankliniella occidentalis) interactions remains elusive. WFTs cause substantial economic losses in eggplant cultivation worldwide. Understanding the molecular mechanisms underlying eggplants’ defense is critical for developing resistant varieties. We investigated the function of histone H3 lysine 27 trimethylation (H3K27me3) in modulating the early transcriptional reprogramming of eggplants during WFT infestation. We performed ChIP-seq and RNA-seq on eggplant leaves at an early stage of WFT infestation to elucidate the epigenetic landscape and associated gene expression alterations. ChIP-seq analysis showed that genome-wide enrichment of H3K27me3 was mainly at the transcription start sites, with a notable decrease in WFT-infested plants. Concurrently, RNA-seq analysis identified 2822 genes that were upregulated following WFT infestation. Many of these genes associated with abscisic acid, jasmonic acid, and salicylic acid pathways were upregulated, underscoring their central role in early plant defense. Integrated analysis revealed six genes with decreased H3K27me3 levels and concurrent upregulation, potentially involved in ABA and JA signaling. Thus, removal of the repressive H3K27me3 mark may facilitate the transcriptional activation of early defense genes in eggplants that are crucial in their response to insect herbivory. Full article