Search Results (275)

The role of G-quadruplexes (G4s) in gene regulation has been widely documented, especially in gene promoters. However, the transcriptional mechanisms involving G4s in other regulatory regions remain largely unexplored. In this study, we integrated the G4-DNA data derived from 22 breast cancer patient-derived tumor xenograft (PDTX) models and MCF7 cell line as potential breast cancer-associated G4s (BC-G4s). Genome-wide analysis showed that BC-G4s are more prevalent in gene promoters and the first introns. The genes accommodating promoter or intronic BC-G4s show significantly higher transcriptional output than their non-G4 counterparts. The biased distribution of BC-G4s in close proximity to the transcription start site (TSS) is associated with an enrichment of transcription factor (TF) interactions. A significant negative correlation was detected between the G4–TF interactions within the first introns and their cognate promoters. These different interactions are complementary rather than redundant. Furthermore, the differentially expressed genes (DEGs) harboring promoter and first intron BC-G4s are significantly enriched in the cell cycle pathway. Notably, promoter BC-G4s of DEGs could be a central hub for TF–TF co-occurrence. Our analysis also revealed that G4-related single nucleotide variants (SNVs) affect the stability of G4 structures and the transcription of disease-related genes. Collectively, our results shed light on how BC-G4s within promoters and first introns regulate gene expression and reinforce the critical role of G4s and G4-related genes in breast cancer-associated processes. Full article

Aging in females affects the ovaries before any other organ. This has a significant impact on women’s health. Aging results in the gradual depletion of ovarian follicles and a decline in oocyte quality. Studies have shown that cellular changes within ovaries manifest before the depletion of ovarian follicles. To understand the molecular mechanisms underlying these changes, we conducted a comprehensive analysis of gene expression changes in aging mouse ovaries. When RNA sequencing data from 6-month-old mice were compared to those from 12-month-old mice, we identified numerous differentially expressed genes, as well as transcript variants. Transcript variants arise from alternative transcription start sites (TSSs) and alternative pre-mRNA processing. Therefore, we further analyzed a specific set of regulators for these cellular processes. Our findings indicate that ovarian aging alters the expression of epigenetic regulators (ERs) and transcription factors (TFs) that are involved in alternative TSS usage. Ovarian aging also affects the expression of RNA-binding proteins (RBPs) and spliceosome components (SPs), which are essential for pre-mRNA processing. We noticed that variations in transcript variants were more pronounced than those found through gene expression analysis. While 8% of the known TFs and ERs were differentially expressed at the gene level, this increased to 30% at the transcript variant level. Similarly, 3% of the known RBPs but no known SPs were differentially expressed at the gene level, while this increased to 30% at the transcript variant level. These observations highlight the importance of focusing on transcript variants and their functions in aging research, as they may provide insight into the underlying biological processes involved. Full article

Cortisol, the main glucocorticoid in teleost, plays a central role in mediating the physiological response to stress by regulating metabolism, immune function, and growth. While its transcriptional effects are well known, its role in modulating chromatin accessibility in fish skeletal muscle remains poorly understood. In this study, we investigated the epigenomic and transcriptomic changes induced by cortisol in a juvenile rainbow trout’s (Oncorhynchus mykiss) skeletal muscle using ATAC-seq and RNA-seq. Fish were treated with a single intraperitoneal dose of cortisol (10 mg/kg) or vehicle, and muscle samples were collected 3 h post-treatment. ATAC-seq analysis revealed a total of 163,802 differentially accessible regions (DARs), with an important enrichment of open regions near transcription start sites and promoters. A total of 1612 and 1746 differentially accessible genes (DAGs) were identified in the cortisol and control groups, respectively. Motif enrichment analysis identified 89 transcription factors to be significantly enriched, among which key stress-responsive regulators such as Fos, AP-1, FoxO1/3, Mef2a/b/c, Klf5/10, and ATF4 were prominently represented. RNA-seq analysis identified 4050 differentially expressed genes (DEGs), with 2204 upregulated genes involved in autophagy, mitophagy, and FoxO signaling, while 1864 downregulated genes were enriched in spliceosome and chromatin remodeling pathways. Integrative analysis revealed 174 overlapping genes between ATAC-seq and RNA-seq datasets, highlighting pathways linked to autophagy and ATP-dependent chromatin remodeling. Four selected DEGs (sesn1, sesn2, cullin3, samtor) were validated by qPCR, showing high concordance with transcriptomic data. These findings provide new insights into cortisol-mediated regulation of chromatin dynamics and gene expression in teleost skeletal muscle and underscore the importance of epigenetic mechanisms in fish stress responses. Full article

Background: Glucocorticoid insensitivity is a problem for the therapy of chronic inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Both are non-communicable chronic inflammatory lung diseases with worldwide increasing incidences. Only symptoms can be controlled by inhaled or systemic glucocorticoids, often combined with β2 agonists and/or muscarinic receptor antagonists. The therapeutic effect of glucocorticoids varies between individuals, and a significant number of patients do not respond well. It is believed that only protein-free circulating unbound glucocorticoids can enter cells by diffusion and achieve their therapeutic effect by binding to the intracellular glucocorticoid receptor (GR), encoded by the NR3C1 gene, for which over 3000 single-nucleotide polymorphisms have been described. In addition, various GR protein isoforms result from 11 transcription start sites, and differential mRNA splicing leads to further GR protein variants; each can be modified post-translational and alter steroid response. To add more variety, some GR isoforms are expressed cell-type specific or in a sub-cellular location. The GR only functions when it forms a complex with other intracellular proteins that regulate ligand binding, cytosol-to-nuclear transport, and nuclear and cytosolic action. Importantly, the timing of the GR activity can be cell type, time, and condition specific. These factors are rarely considered when assessing disease-specific loss or reduced GR response. Conclusions: Future studies should analyze the timing of the availability, activity, and interaction of all components of the glucocorticoid signaling cascade(s) and compare these factors between non-diseased and diseased probands, applying the combination of all omics methods (250). Full article

Sorghum (Sorghum bicolor L.) is the fifth largest cereal crop in the world and widely used in the fields of food, feed, brewing, and fuel, while knowledge is mostly limited for sorghum grain development, including starch biosynthesis. B3 family transcription factors (TFs) play a crucial role in plant development, including grain development, dormancy, and storage of nutrients. In the present study, a comprehensive analysis of sorghum B3 genes was performed, and a total of 76 related genes were identified to be distributed on 10 chromosomes across the whole sorghum genome. According to the sequence features, the sorghum B3 family members were divided into four sub-families of ARF, RAV, LAV, and REM. Multiple elements, i.e., light-responsive elements, phytohormone-responsive elements, growth and development-related elements, and stress-responsive elements, were discovered to be located within the 2000 bp upstream of the translation start site. Results of expression analysis across multiple tissues suggested significantly different expression patterns of sorghum B3 genes. Further assays confirmed that SbLAV1, which belonged to the LAV subfamily of B3, co-expressed with 15 key starch biosynthesis-related genes (SBRGs), and the corresponding product of SbLAV1 could activate the promoter activities of multiple key SBRGs. Collectively, the integrative results of the present study indicate that B3 family members, including SbLAV1, might play critical roles in starch biosynthesis and grain development in sorghum. Full article

All beta- and gamma-herpesviruses utilize a set of six viral proteins to facilitate transcription from specific promoters that become active late in the viral life cycle. These proteins form a complex that interacts with a TA-rich sequence upstream of the late transcription start sites and recruits RNA polymerase II (Pol II). The structure of any of the late transcription factors (LTFs) alone or in complexes has not been solved by standard means yet, but a fair amount is known about how the proteins interact and where the complex is positioned over the late promoters. Here, AlphaFold3 was used to predict and analyze the LTF complex using proteins from the beta-herpesviruses HCMV, MCMV, HHV6, and HHV7, and from the gamma-herpesviruses EBV and KSHV. The predicted structures had high levels of confidence and were remarkably similar even though there is little sequence conservation in the LTFs across the viruses. The results are consistent with most of the previously determined information concerning the interaction of the factors with each other and with DNA. A conserved threonine phosphorylation in one of the subunits that is critical to the function of the LTFs is predicted to be at the junction of five subunits. AlphaFold 3 predicts seven metal ion binding sites in each of the four beta-herpesviruses and either five or six in the gamma-herpesviruses created by conserved residues in three of the subunits. The structures also provide insights into the function of the subunits and which host general transcription factors (GTFs) may or may not be utilized during initiation. Full article

Transposable elements (TEs) are major drivers of plant genome plasticity, but the immediate molecular consequences of new TE insertions remain poorly understood. In this study, we generated a wild-type Arabidopsis thaliana population with novel insertions of ONSEN retrotransposon to investigate early epigenomic and transcriptomic changes using whole-genome and cDNA nanopore sequencing. We found that novel ONSEN insertions were distributed non-randomly, with a strong preference for genic regions, particularly in chromatin enriched for H2A.Z, H3K27me3, and H3K4me2. Most full-length ONSEN insertions within genes were rapidly recognized and spliced out as new introns (intronization), thereby mitigating potential deleterious effects on transcript isoforms. In some cases, ONSEN insertions provided alternative transcription start or termination sites, generating novel transcript isoforms. Genome-wide methylation analysis revealed that new ONSEN copies were efficiently and precisely targeted by DNA methylation. Independently on the location of the original ONSEN element, the euchromatic and heterochromatic insertions display distinct methylation signatures, reflecting the action of different epigenetic pathways. In conclusion, our results demonstrate that DNA methylation and alternative splicing are effective control mechanisms safeguarding the plant genome and transcriptome integrity after retrotransposition burst. Full article

The present Perspective analyzes the remarkable evolution of the Amyloid Cascade Hypothesis 2.0 (ACH2.0) theory of Alzheimer’s disease (AD) since its inception a few years ago, as reflected in the diminishing role of amyloid-beta (Aβ) in the disease. In the initial iteration of the ACH2.0, Aβ-protein-precursor (AβPP)-derived intraneuronal Aβ (iAβ), accumulated to neuronal integrated stress response (ISR)-eliciting levels, triggers AD. The neuronal ISR, in turn, activates the AβPP-independent production of its C99 fragment that is processed into iAβ, which drives the disease. The second iteration of the ACH2.0 stemmed from the realization that AD is, in fact, a disease of the sustained neuronal ISR. It introduced two categories of AD—conventional and unconventional—differing mainly in the manner of their causation. The former is caused by the neuronal ISR triggered by AβPP-derived iAβ, whereas in the latter, the neuronal ISR is elicited by stressors distinct from AβPP-derived iAβ and arising from brain trauma, viral and bacterial infections, and various types of inflammation. Moreover, conventional AD always contains an unconventional component, and in both forms, the disease is driven by iAβ generated independently of AβPP. In its third, the current, iteration, the ACH2.0 posits that proteolytic production of Aβ is suppressed in AD-affected neurons and that the disease is driven by C99 generated independently of AβPP. Suppression of Aβ production in AD seems an oxymoron: Aβ is equated with AD, and the later is inconceivable without the former in an ingrained Amyloid Cascade Hypothesis (ACH)-based notion. But suppression of Aβ production in AD-affected neurons is where the logic leads, and to follow it we only need to overcome the inertia of the preexisting assumptions. Moreover, not only is the generation of Aβ suppressed, so is the production of all components of the AβPP proteolytic pathway. This assertion is not a quantum leap (unless overcoming the inertia counts as such): the global cellular protein synthesis is severely suppressed under the neuronal ISR conditions, and there is no reason for constituents of the AβPP proteolytic pathway to be exempted, and they, apparently, are not, as indicated by the empirical data. In contrast, tau protein translation persists in AD-affected neurons under ISR conditions because the human tau mRNA contains an internal ribosomal entry site in its 5′UTR. In current mouse models, iAβ derived from AβPP expressed exogenously from human transgenes elicits the neuronal ISR and thus suppresses its own production. Its levels cannot principally reach AD pathology-causing levels regardless of the number of transgenes or the types of FAD mutations that they (or additional transgenes) carry. Since the AβPP-independent C99 production pathway is inoperative in mice, the current transgenic models have no potential for developing the full spectrum of AD pathology. What they display are only effects of the AβPP-derived iAβ-elicited neuronal ISR. The paper describes strategies to construct adequate transgenic AD models. It also details the utilization of human neuronal cells as the only adequate model system currently available for conventional and unconventional AD. The final alteration of the ACH2.0, introduced in the present Perspective, is that AβPP, which supports neuronal functionality and viability, is, after all, potentially produced in AD-affected neurons, albeit not conventionally but in an ISR-driven and -compatible process. Thus, the present narrative begins with the “omnipotent” Aβ capable of both triggering and driving the disease and ends up with this peptide largely dislodged from its pedestal and retaining its central role in triggering the disease in only one, although prevalent (conventional), category of AD (and driving it in none). Among interesting inferences of the present Perspective is the determination that “sporadic AD” is not sporadic at all (“non-familial” would be a much better designation). The term has fatalistic connotations, implying that the disease can strike at random. This is patently not the case: The conventional disease affects a distinct subpopulation, and the basis for unconventional AD is well understood. Another conclusion is that, unless prevented, the occurrence of conventional AD is inevitable given a sufficiently long lifespan. This Perspective also defines therapeutic directions not to be taken as well as auspicious ways forward. The former category includes ACH-based drugs (those interfering with the proteolytic production of Aβ and/or depleting extracellular Aβ). They are legitimate (albeit inefficient) preventive agents for conventional AD. There is, however, a proverbial snowball’s chance in hell of them being effective in symptomatic AD, lecanemab, donanemab, and any other “…mab” or “…stat” notwithstanding. They comprise Aβ-specific antibodies, inhibitors of beta- and gamma-secretase, and modulators of the latter. In the latter category, among ways to go are the following: (1) Depletion of iAβ, which, if sufficiently “deep”, opens up a tantalizing possibility of once-in-a-lifetime preventive transient treatment for conventional AD and aging-associated cognitive decline, AACD. (2) Composite therapy comprising the degradation of C99/iAβ and concurrent inhibition of the neuronal ISR. A single transient treatment could be sufficient to arrest the progression of conventional AD and prevent its recurrence for life. Multiple recurrent treatments would achieve the same outcome in unconventional AD. Alternatively, the sustained reduction/removal of unconventional neuronal ISR-eliciting stressors through the elimination of their source would convert unconventional AD into conventional one, preventable/treatable by a single transient administration of the composite C99/iAβ depletion/ISR suppression therapy. Efficient and suitable ISR inhibitors are available, and it is explicitly clear where to look for C99/iAβ-specific targeted degradation agents—activators of BACE1 and, especially, BACE2. Directly acting C99/iAβ-specific degradation agents such as proteolysis-targeting chimeras (PROTACs) and molecular-glue degraders (MGDs) are also viable options. (3) A circumscribed shift (either upstream or downstream) of the position of transcription start site (TSS) of the human AβPP gene, or, alternatively, a gene editing-mediated excision or replacement of a small, defined segment of its portion encoding 5′-untranslated region of AβPP mRNA; targeting AβPP RNA with anti-antisense oligonucleotides is another possibility. If properly executed, these RNA-based strategies would not interfere with the protein-coding potential of AβPP mRNA, and each would be capable of both preventing and stopping the AβPP-independent generation of C99 and thus of either preventing AD or arresting the progression of the disease in its conventional and unconventional forms. The paper is interspersed with “validation” sections: every conceptually significant notion is either validated by the existing data or an experimental procedure validating it is proposed. Full article

Dehydrins are stress-inducible proteins with protective functions, characterized by high hydrophilicity, thermostability, and a low degree of secondary structure. They stabilize cellular membranes, preserve macromolecule conformation, and support enzymatic and structural protein functions. Their accumulation in plant tissues under drought is regulated by abscisic acid (ABA)-dependent and ABA-independent pathways. Ethylene plays a key role in stress adaptation, but its relationship with dehydrin accumulation remains unclear. This study investigates how ethylene influences dehydrin expression in Arabidopsis thaliana during prolonged dehydration using transcript profiling and immunodetection in wild-type (Col-0), ethylene-constitutive (ctr1-1), and ethylene-insensitive (ein3eil1) mutants. Comparative analyses showed increased survival of ctr1-1 plants under dehydration stress, likely due to reduced oxidative damage. Analysis of dehydrin-coding genes identified multiple Ethylene Response Factor (ERF) binding sites, flanking the transcription start sites, which suggests a fine-tuned ethylene-dependent regulation. The ability of ethylene signaling to either suppress or stabilize particular dehydrins was demonstrated by RT-qPCR and immunodetection experiments. Under drought stress, ethylene signaling appeared to suppress root-specific dehydrins. A Y-segment-containing protein with approximate molecular weight of 20 kDa showed decreased levels in ctr1-1 and higher accumulation in ein3eil1, indicating that ethylene signaling acts as a negative regulator. These results provide new information on the dual role of ethylene in dehydrin control, highlighting its function as a molecular switch in stress adaptive responses. Full article

Growth rate of bovine skeletal muscle has a major impact on beef yield. Cadherin-11 (CDH11) was found to be a potential candidate gene for growth and development in beef cattle. This study confirmed the high expression of CDH11 in bovine longissimus dorsi using a tissue expression analysis. To understand the transcriptional regulation mechanism of CDH11 gene, we constructed a double luciferase vector for the promoter region of CDH11 gene and determined that the core transcriptional regulatory region was located at −129/+55 bp relative to the transcription start site (TSS). In addition, we confirmed that skeletal muscle growth and development-related transcription factor-specific protein 1 (SP1) and glucocorticoid receptor (GR) bind to the CDH11 gene promoter region at −36/−27 bp and −20/−11 bp, respectively, to regulate CDH11 expression. These interactions provide valuable information for understanding the mechanism of transcriptional regulation of the bovine CDH11 gene during muscle growth and development. Full article

Understanding the genetic regulation of gene expression and splicing in muscle tissues is critical for elucidating the molecular mechanisms of meat quality traits. In this study, we integrated large-scale whole-genome sequencing and strand-specific RNA-seq data from 582 F2 hybrid pigs (White Duroc × Erhualian) to characterize the expression and splicing quantitative trait loci (eQTLs/sQTL) in longissimus dorsi muscle. We identified 11,058 cis-eQTL-associated genes (eGenes) and 5139 cis-sQTL-associated genes (sGenes), of which 29% of eGenes and 80% of sGenes were previously unreported in the PigGTEx database. Functional analyses revealed distinct genomic features: eQTLs were enriched near transcription start sites (TSSs) and associated with active TSS-proximal transcribed regions and enhancers, whereas sQTLs clustered at splice junctions, underscoring their distinct roles in gene expression and splicing regulation. Colocalization analysis of e/sQTLs with GWAS signals prioritized PHKG1 as a key candidate gene (PPH4 > 0.9) for glycogen metabolism. Notably, we confirmed that an sQTL-driven alternative splicing event in exon 10 of PHKG1 was significantly correlated with phenotypic variation (R = −0.39, p = 9.5 × 10⁻²¹). Collectively, this study provides novel insights into the genetic regulation of gene expression and alternative splicing in porcine muscle tissue, advancing our understanding of the molecular mechanisms underlying economically important meat quality traits. Full article

Retrozymes are a class of non-autonomous plant retrotransposons that have long terminal repeats (LTRs) containing hammerhead ribozymes (HHRs) that facilitate the circularization of the retrozyme RNA. The LTR of Nicotiana benthamiana retrozyme 1 (NbRZ1) has been shown to contain a promoter that directs transcription of this retroelement. In this study, we identified the transcription start site of the promoter contained in the LTR of NbRZ1 and mapped the promoter region essential for its transcriptional activity. Using transgenic Arabidopsis thaliana plants carrying the GUS gene under the control of the NbRZ1 LTR, the NbRZ1 transcript was demonstrated to potentially encode a protein targeted for proteasomal degradation in the plant cell. Overexpression of this protein in plants using a viral expression vector was found to cause severe necrosis. The data presented suggest a tight regulation of the expression of the NbRZ1-encoded polypeptide in plants and its potential functional importance, although further research is needed to determine whether circular and/or linear retrozyme RNA forms can be translated in plants. Full article

G-quadruplexes play a crucial role in transcription, translation, and DNA replication in plant genomes. Here, we comprehensively examined the prevalence and functions of G-quadruplexes in Vitis vinifera. A total of 467,813 G-quadruplexes were identified in grapevine genome, with enrichment in the promoter (0.54/kbp) and near transcription start sites (TSSs, 1.00/kbp), and showed conservative strand preference. The G-quadruplex density in centromeres exhibited heterogeneity. The differentially expressed genes (DEGs) under two-day drought stress manifested high G-quadruplex density in the promoter and TSS regions. The upregulated DEGs showed template strand-biased G-quadruplex enrichment, while downregulated DEGs displayed coding strand dominance linked to metal ion homeostasis and sugar–acid metabolism pathways, respectively. G-quadruplexes were enriched in key sugar–acid metabolism genes, including pyruvate kinase and sucrose synthase. The number of G-quadruplexes in sucrose transferase VINV genes was higher than that in the CWINV and NINV genes. This study revealed G-quadruplexes as regulatory elements of stress response and berry development, providing abundant genetic targets for precision breeding and the quality improvement of grapevines. Full article

Background: In humans, vitamin D₃ synthesis follows a day–night rhythm due to its UV-B-dependent production. Results: As part of the VitDHiD intervention study, we identified 87 in vivo vitamin D target genes with circadian expression patterns in immune cells, forming a regulatory network centered on transcription factors and membrane receptors. These genes exhibit a narrow basal expression range, with 80% downregulated upon vitamin D₃ supplementation. Clustering analysis revealed six distinct gene groups, with the two most prominent clusters driven by the transcription factor CSRNP1 (cysteine- and serine-rich nuclear protein 1) and GAS7 (growth arrest-specific 7), a known differentiation inducer. Among the 25 VitDHiD study participants, we identified two subgroups distinguished by significant differences in the responsiveness of 14 in vivo vitamin D target genes. These genes encode transcription factors like CSRNP1, as well as metabolic enzymes and transporters, including NAMPT (nicotinamide phosphoribosyltransferase), PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), and SLC2A3 (solute carrier family 2 member 3). Notably, all 14 genes possess a vitamin D receptor-binding enhancer within a reasonable distance of their transcription start site. Conclusions: These findings highlight a novel link between vitamin D signaling and circadian gene regulation, with potential implications for personalized supplementation strategies. Full article

Most eukaryotic genes express more than one mature mRNA, defined as transcript variants. This complex phenomenon arises from various mechanisms, such as using alternative transcription start sites and alternative post-transcriptional processing events. The resulting transcript variants can lead to synthesizing proteins that possess distinct functional domains or may even generate noncoding RNAs, each with unique roles in cellular processes. The generation of these transcript variants is not merely a random occurrence; it is cell-type specific and varies with developmental stages, aging processes, or pathogenesis of diseases. This highlights the biological significance of transcript variants in regulating gene expression and their potential impact on cellular functionality. Despite the biological importance, investigating transcript variants has been hampered by challenges associated with detecting their expression. This review article addresses the advancements in molecular techniques in detecting transcript variants. Traditional methods such as RT-PCR and RT-qPCR can easily detect known transcript variants using primers that target unique exons associated with the variants. Other techniques like RACE-PCR and hybridization-based methods, including Northern blotting, RNase protection assays, and microarrays, have also been utilized to detect transcript variants. Nevertheless, RNA sequencing (RNA-Seq) has emerged as a powerful technique for identifying transcript variants, especially those with previously unknown sequences. The effectiveness of RNA sequencing in transcript variant detection depends on the specific sequencing approach and the precision of data analysis. By understanding the strengths and weaknesses of each laboratory technique, researchers can develop more effective strategies for detecting mRNA transcript variants. This ability will be crucial for our comprehensive understanding of gene regulation and the implications of transcript diversity in various biological contexts. Full article