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Cell-specific DNA methylation in human alpha and beta cells regulates gene expression in type 2 diabetes.

Epigenome-wide studies of pancreatic islets provide valuable insights into type 2 diabetes (T2D) but lack methylomes from individual cell types. Here we show changes to alpha and beta cell-specific methylomes and transcriptomes from people with or without T2D, using whole-genome bisulfite sequencing and RNA sequencing. We discover 22,544 differentially methylated regions annotated to 7,975 genes in alpha versus beta cells, such as INS, GCG, PDX1 and PCSK1, with ~50% showing differential expression. CRISPR-dCas9-DNMT3A-based epigenetic editing increases INS and TH DNA methylation, while CRISPR-dCas9-TET1-based editing decreases GCG methylation, each altering INS, TH or GCG expression and content in beta cells. Pre-T2D/T2D-associated differentially methylated regions in alpha and beta cells overlap 12-18% of T2D-associated genome-wide association study candidates. Additionally, ONECUT2 is epigenetically upregulated in beta cells from people with pre-T2D/T2D and elevated in male Goto-Kakizaki rat islets. ONECUT2 overexpression in beta cells/islets downregulates gene sets impacting insulin secretion and glucose homeostasis, and reduces mitochondrial activity, ATP/ADP ratio and insulin secretion. We also provide 'alpha-beta-methylome' ( https://alpha-beta-methylome.serve.scilifelab.se/app/alpha-beta-methylome/ ), a resource exploring T2D, age and sex associations on methylation, highlighting cell-specific epigenetic regulation and dysfunctions contributing to T2D.

Humans

Genomic context-dependent roles of 5-hydroxymethylcytosine in regulating gene expression during rice drought response.

DNA methylation (5-methylcytosine, 5mC) is a key epigenetic regulator of genome stability and stress adaptation in plants. However, the functional role of its oxidative derivative, 5-hydroxymethylcytosine (5hmC), remains poorly understood in plant systems, largely due to its low abundance and unresolved enzymatic origins. Here, we integrated ACE-seq (APOBEC-coupled epigenetic sequencing) with an optimized Tn5mC-seq (transposase-based library preparation in the context of whole-genome bisulfite sequencing, WGBS) approach to generate the first single-base resolution map of 5hmC in rice (Oryza sativa), unveiling its stress-responsive dynamics and regulatory interplay with 5mC during drought adaptation. Genome-wide profiling revealed a basal 5hmC level of ~0.03 (defined as the ratio of C/(C + T) at each site), with drought triggering a pronounced reduction in 5hmC abundance and locus number, followed by incomplete recovery post-rehydration. Unlike 5mC, which accumulates in heterochromatin, 5hmC preferentially localized to euchromatic regions, including promoters, exons, and intergenic elements, and exhibited enrichment at ABA-responsive transcription factors (e.g., OsATAF1, bZIP50). Strikingly, drought induced an antagonistic relationship between 5hmC and 5mC, with the latter increasing globally to reinforce transposon silencing. Multi-omics analyses demonstrated that 5hmC depletion in promoters correlated with transcriptional downregulation, while its accumulation in gene bodies (notably 5'-UTRs) suppressed stress-responsive genes. These findings highlight 5hmC's bifunctional regulatory capacity, contingent on genomic context, and its role in balancing transcriptional plasticity with genome stability during stress. Our work establishes 5hmC as a dynamic epigenetic mark in plant environmental adaptation and provides a foundation for leveraging DNA hydroxymethylation in crop resilience engineering.

Oryza

Interplay between the role of DNA methylation in regulating gene expression and TE-silencing in a reptilian methylome.

DNA methylation is a major component of eukaryotic genomes with an important role in the defence against transposable elements, to transcriptionally silence their activity and prevent transposition. DNA methylation also plays a major role in the regulation of gene expression. This dual role can come into conflict, where DNA methylation in gene regulatory regions becomes perturbed due to transposable element transposition, leading to disruption of gene expression. Here, we describe how this conflict is reflected in DNA methylation patterns in the sand lizard genome where there is recent transposable element activity. Using long-read sequencing technology we show that CpG islands in gene transcriptional start sites are typically hypomethylated and associated with higher gene expression. Outside transcriptional start sites, a majority of CpG islands overlapped transposable elements and were associated with hypermethylation, consistent with a host-defence role in suppressing transposition activity. We identify 605 instances where transcriptional start sites were associated with transposable elements (4.3% of all genes). These instances were far rarer in conjunction with a CpG island, when methylation signatures would be in conflict. Transposable elements were found to be closer to and at higher density the more hypermethylated a transcriptional start site was, suggesting strong selection against selfish genetic elements transposing into hypomethylated transcriptional start sites.

CpG islands

CsMYB219 and CsMYB196 influence epigallocatechin gallate biosynthesis in tea plant (Camellia sinensis) by regulating CsSCPL1A gene expression.

Epigallocatechin gallate (EGCG) is the most abundant and biologically active catechin in tea leaves and has been widely utilized in the development of functional foods. EGCG is catalyzed by serine carboxypeptidase-like 1A (CsSCPL1A) acyltransferases in tea plants. Although CsSCPL family genes are regulated by several transcription factors (TFs), systematic studies on their regulation by MYB TFs are lacking. This study integrates targeted metabolomics, transcriptomics, DNA-protein, and protein-protein interaction analyses to elucidate the transcriptional regulation of EGCG biosynthesis-related genes CsSCPL4 and CsSCPL5-1 by R2R3-MYB TFs. CsMYB219 and CsMYB196 can specifically bind to CsSCPL4 and CsSCPL5-1 promoters and activate their expression. CsMYB196 also interacted with CsTT8a and CsTTG1 to activate the transcription activity of CsSCPL4 and CsSCPL5-1 promoters by forming a MYB/bHLH/WD40 (MBW) complex. Promoter truncation assays delineated MYB-responsive cis-elements in CsSCPL4 (-613 to -1 bp with enhancers at -1967 to -1622) and CsSCPL5-1 (-503 to -296 bp). Silencing of CsMYB219 and CsMYB196 by virus-induced gene silencing (VIGS) assay significantly reduced the expression levels of CsSCPL4 and CsSCPL5-1 and EGCG content in tea leaves. Transient overexpression of CsMYB219 and CsMYB196 in tea leaves upregulated CsSCPL4 and CsSCPL5-1 expression and elevated EGCG content. These findings enhance our understanding of the regulatory network underlying EGCG biosynthesis in tea plants and provide a solid foundation for future genetic improvement of tea plant cultivars.

Catechin

eVOC: a controlled vocabulary for unifying gene expression data.

Expression data contribute significantly to the biological value of the sequenced human genome, providing extensive information about gene structure and the pattern of gene expression. ESTs, together with SAGE libraries and microarray experiment information, provide a broad and rich view of the transcriptome. However, it is difficult to perform large-scale expression mining of the data generated by these diverse experimental approaches. Not only is the data stored in disparate locations, but there is frequent ambiguity in the meaning of terms used to describe the source of the material used in the experiment. Untangling semantic differences between the data provided by different resources is therefore largely reliant on the domain knowledge of a human expert. We present here eVOC, a system which associates labelled target cDNAs for microarray experiments, or cDNA libraries and their associated transcripts with controlled terms in a set of hierarchical vocabularies. eVOC consists of four orthogonal controlled vocabularies suitable for describing the domains of human gene expression data including Anatomical System, Cell Type, Pathology and Developmental Stage. We have curated and annotated 7016 cDNA libraries represented in dbEST, as well as 104 SAGE libraries,with expression information,and provide this as an integrated, public resource that allows the linking of transcripts and libraries with expression terms. Both the vocabularies and the vocabulary-annotated libraries can be retrieved from http://www.sanbi.ac.za/evoc/. Several groups are involved in developing this resource with the aim of unifying transcript expression information.

Animals

MucR protein: Three decades of studies have led to the identification of a new H-NS-like protein.

MucR belongs to a large protein family whose members regulate the expression of virulence and symbiosis genes in α-proteobacteria species. This protein and its homologs were initially studied as classical transcriptional regulators mostly involved in repression of target genes by binding their promoters. Very recent studies have led to the classification of MucR as a new type of Histone-like Nucleoid Structuring (H-NS) protein. Thus this review is an effort to put together a complete and unifying story demonstrating how genetic and biochemical findings on MucR suggested that this protein is not a classical transcriptional regulator, but functions as a novel type of H-NS-like protein, which binds AT-rich regions of genomic DNA and regulates gene expression.

Bacterial Proteins

Dynamic allelic expression in mouse mammary glands across the adult developmental cycle.

The mammary gland, which primarily develops postnatally, undergoes significant changes during pregnancy and lactation to facilitate milk production. Through the generation and analysis of 480 transcriptomes, we provide the most detailed allelic expression map of the mammary gland, cataloguing cell-type-specific expression from ex-vivo purified cell populations over 10 developmental stages, enabling comparative analysis. The work identifies genes involved in the mammary gland cycle, parental-origin-specific and genetic background-specific expression at cellular and temporal resolution, genes associated with human lactation disorders and breast cancer. Genomic imprinting, a mechanism regulating gene expression based on parental origin, is crucial for controlling gene dosage and stem cell potential throughout development. The analysis identified 25 imprinted genes monoallelically expressed in the mammary gland, with several showing allele-specific expression in distinct cell types. No novel imprinted genes were identified and the absence of biallelically expressed imprinted genes suggests that, unlike in brain, selective absence of imprinting does not regulate gene dosage in the mammary gland. This research highlights transcriptional dynamics within mammary gland cells and identifies novel candidate genes potentially significant in the tissue during pregnancy and lactation. Overall, this comprehensive atlas represents a valuable resource for future studies on expression and transcriptional dynamics in mammary cells.

Animals

Genome imbalance modulates the expression of long non-coding RNAs in maize.

Genome imbalance, resulting from varying the dosage of individual chromosomes (aneuploidy), has a more detrimental effect than changes in complete sets of chromosomes (haploidy/polyploidy). This imbalance is likely due to disruptions in stoichiometry and interactions among macromolecular assemblies. Previous research has shown that aneuploidy causes global modulation of protein-coding genes (PCGs), microRNAs, and transposable elements (TEs), affecting both the varied chromosome (cis-located) and unvaried genome regions (trans-located) across various taxa. While long non-coding RNAs (lncRNAs) are important gene expression regulators, their roles in the context of genomic imbalance remain largely unexplored. In this study, we analyzed and compared the impact of aneuploidy and haploidy/polyploidy on lncRNA expression using RNA-seq data from maize mature leaf tissue. Our results indicate that cis-located lncRNAs are modulated from dosage compensation to a gene dosage effect, while trans-located lncRNAs exhibit trends ranging from an inverse effect to a positive correlation with chromosomal dosage. Remarkably, the ploidy series showed a lesser degree of lncRNA modulation. LncRNAs and TEs display a similar trend of inverse modulation but exhibit greater sensitivity to dosage changes compared to PCGs. The construction of cis-acting and trans-acting lncRNA co-expression networks indicates that lncRNAs likely function as dosage-sensitive regulators of gene expression under conditions of genomic imbalance. Overall, this study not only elucidates the dosage effect of plant lncRNAs but also serves as a valuable resource for exploring potential regulators of PCGs that play significant biological functions.

Zea mays

DNA methylation controls the expression of tanshinone synthesis genes and the tanshinone accumulation in Salvia miltiorrhiza and Salvia bowleyana.

DNA methylation plays pivotal roles in regulating gene expression and the secondary metabolism in plants. Salvia miltiorrhiza and Salvia bowleyana are traditional Chinese medicinal plants with roots enriched with tanshinone components. However, the regulatory mechanism of DNA methylation on tanshinone production remains elusive. Here, we analyzed 30-day-old hairy roots of S. miltiorrhiza and S. bowleyana using targeted high-performance liquid chromatography analysis and found significantly higher tanshinone content in S. miltiorrhiza. Whole-genome bisulfite sequencing revealed elevated DNA methylation levels in S. miltiorrhiza, potentially due to the upregulation of methylation-related genes, including DOMAINS REARRANGED METHYLTRANSFERASE 1 (DRM1), DECREASE IN DNA METHYLATION 1 (DDM1), CHROMOMETHYLASE 2 (CMT1), and CHROMOMETHYLASE 3 (CMT3), alongside the low expression of the demethylase gene REPRESSOR OF SILENCING 1 (ROS1) in S. miltiorrhiza. Additionally, four genes that are involved in tanshinone biosynthesis, including 1-DEOXY-D-XYLULOSE-5-PHOSPHATE REDUCTASE (DXS1), GERANYLGERANYL DIPHOSPHATE SYNTHASE (GGPPS2), 4-HYDROXY-3-METHYLBUT-2-ENYL PYROPHOSPHATE REDUCTASE (HDR2), and COPALYL PYROPHOSPHATE SYNTHASE (CPS3), showed lower methylation levels in the promoters of DXS1, GGPPS2, and CPS3 and a higher DNA methylation level in the gene body of HDR2 in S. miltiorrhiza, which may lead to their high expression and the accumulation of tanshinones. Consistently, overexpression of the SmCMT3 in S. miltiorrhiza significantly reduced the contents of cryptotanshinone, tanshinone I, and tanshinone IIA. Transcriptomic and methylome analyses confirmed that the expression levels of the tanshinone biosynthesis-related genes, including SmMK, SmCPS1, SmDXS2, and SmAACT1, were correlated with their promoter or gene body DNA methylation levels. Our findings reveal that DNA methylation critically regulates tanshinone biosynthesis in S. miltiorrhiza and S. bowleyana, offering valuable insights for breeding.

Abietanes

Nuclear speckles: a fundamental layer of gene regulation.

Within the cell nucleus, non-DNA structures called nuclear bodies interact with chromatin to regulate gene expression and organize our genetic material. Among nuclear bodies, nuclear speckles are prominent. They interact with broad genomic regions, serve as major gene-activating structures, and are implicated in viral infection, cancer, neurodegeneration, stress response, and development. Advances that integrate genomics with imaging are leading to a deeper understanding of how nuclear speckles fit into our current knowledge of chromatin biology, genome organization, and the central dogma of biology. Collectively, these recent studies emphasize nuclear speckles as key gene regulatory structures within the cell nucleus, offering new perspectives on how gene expression dysregulation is linked to disease.

Humans

Conserved miRNA regulators of PD-1/PD-L1 in glioblastoma and colorectal cancer.

Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have transformed cancer therapy, but their efficacy remains limited in glioblastoma (GBM) and heterogeneous in colorectal cancer (CRC). MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level, including immune checkpoint molecules, yet conserved regulatory miRNA networks across distinct cancers remain poorly defined. Five conserved miRNAs (miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-20b-5p, miR-138-5p) fulfilled the selection criteria and were consistently dysregulated in GBM and CRC. MiR-106a-5p and miR-106b-5p were upregulated in both cancers and showed favourable prognostic associations, with higher expression correlating with improved survival. miR-20a-5p and miR-20b-5p were preferentially expressed in microsatellite-stable (MSS) CRC and correlated with favourable outcomes in both cancers, whereas miR-138-5p was downregulated in both tumours compared to normal tissue, but showed opposite survival associations, with higher levels linked to worse prognosis. Correlation analysis revealed significant inverse associations between several miRNAs and checkpoint gene expression, including moderate inverse correlations for CD274-miR-106a-5p in GBM, CD274-miR-20a-5p in CRC and PDCD1LG2-miR-20a-5p in both cancers. Pan-cancer profiling demonstrated broad and heterogeneous dysregulation, with expression absent in ovarian cancer for four of the five miRNAs. Pathway enrichment implicated the TGF-β, Hippo, FoxO, and cell cycle pathways, consistent with their known roles in tumour immune evasion. We identified a conserved set of miRNAs that are dysregulated in both GBM and CRC, correlate with survival, and display inverse relationships with PD-1/PD-L1/PD-L2 expression. These miRNAs represent candidate regulators of the PD-1/PD-L1/PD-L2 axis and potential biomarkers of tumour biology that may influence immune checkpoint signalling.

Humans

How advances in chromosome conformation capture (3C) methods are reshaping our understanding of gene regulation in hematopoiesis.

The three-dimensional organization of the DNA within the nucleus plays a key role in regulating gene expression. Over the past two decades, advances in chromosome conformation capture (3C) technologies, in tandem with other methods, have shown that the genome forms a complex structure at multiple scales. Early studies identified large-scale structures such as chromosome territories, compartments and topologically associating domains (TADs). As the resolution of 3C techniques has improved, it has become possible to identify contacts between regulatory elements in detail and more recently, it has become possible to define intricate structures within cis-regulatory elements. In this chapter, we review the development of 3C-based methodologies and discuss the strengths and limitations of the different approaches. We examine how these technologies have refined our understanding of genome organization and gene regulation. Recent high-resolution studies reveal that chromatin architecture extends beyond classical domain structures to include nanoscale organization. Integration of 3C data with super-resolution imaging and molecular dynamics simulations supports a model in which genome folding is governed by the biophysical properties of chromatin.

Animals

Functional genomic analysis of non-canonical DNA regulatory elements of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor activated by environmental toxicants like halogenated and polycyclic aromatic hydrocarbons, which then binds to DNA and regulates gene expression. AHR is implicated in numerous physiological processes, including liver and immune function, cell cycle control, oncogenesis, and metabolism. Traditionally, AHR binds a consensus DNA sequence (GCGTG), the xenobiotic response element (XRE), recruits coregulators, and modulates gene expression. Yet, recent evidence suggests AHR can also regulate gene expression via a non-consensus sequence (GGGA), termed the non-consensus XRE (NC-XRE). The prevalence and functional significance of NC-XRE motifs in the genome have remained unclear. While ChIP and reporter studies hinted at AHR-NC-XRE interactions, direct evidence for transcriptional regulation in a native context was lacking. In this study, we analyzed AHR binding to NC-XRE sequences genome-wide in mouse liver, integrating ChIP-seq and RNA-seq data to identify candidate AHR target genes containing NC-XRE motifs in their regulatory regions. We found NC-XRE motifs in 82% of AHR-bound DNA, significantly enriched compared to random regions, and present in promoters and enhancers of AHR targets. Functional genomics on the Serpine1 gene revealed that deleting NC-XRE motifs reduced TCDD-induced Serpine1 upregulation, demonstrating direct regulation. These findings provide the first direct evidence for AHR-mediated regulation via NC-XRE in a natural genomic context, advancing our understanding of AHR-bound DNA and its impact on gene expression and physiological relevance.

Journal Article

Measuring FOXO Activity by Using qPCR-Based Expression Analysis of FOXO Target Genes.

FOXO transcription factors belong to the forkhead protein family and are distinguished by their unique forkhead (FKH) DNA-binding domain. In the realm of mammals, four FOXO paralogs are recognized: FOXO1, FOXO3, FOXO4, and FOXO6. These paralogs are evolutionary counterparts of the daf-16 gene discovered in the nematode C. elegans. A key feature shared by these paralogs is a consensus binding site known as the DAF-16 family protein-binding site (DBE: 5'-TTGTTTAC-3'). The functional outcome of FOXO transcription factors primarily hinges on their affinity for these specific binding sites within the promoters of their target genes. Nevertheless, it is worth noting that many of these target genes exhibit tissue-specific expression patterns. Consequently, there is not a single FOXO target gene whose expression can reliably serve as a universal indicator of FOXO activity across all cell types and tissues or in response to all stimuli. In light of these considerations, we present a collection of target genes that, when collectively assessed, can accurately gauge FOXO activation. In this chapter, we outline a specific protocol for utilizing quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure the expression levels of these genes.

Forkhead Transcription Factors

Molecular and transcriptional regulation of plant defense responses to aphid infestation.

Aphids are one of the important agricultural pests causing substantial yield losses in crops grown across the globe. Aphids are known to cause direct feeding damages and indirect losses due to sooty mold development and plant virus transmission. Plants respond to these attacks by mounting a complex defense response at the infested sites and systemic levels. This multilayered defense response involves a highly coordinated network of phytohormones and other signalling components like Ca2+, mitogen activated protein kinases and reactive oxygen species. Key to these complex responses is a well-regulated gene expression involving several transcription factors. A wide range of transcription factors are structurally and functionally characterized across some model plants and in a few agronomically important crops. These transcription factors play diverse roles such as defense gene expression modulation, regulation of hormone signaling, secondary metabolism, oxidative stress response, cell wall modifications, and phloem-based defense. Understanding the integration of signaling pathways, hormone crosstalk, and transcription factor mediated regulation provides a framework for practical applications, including breeding, genome editing, and elicitor-based strategies. This review highlights how plant defense signaling and transcriptional regulation against aphids can be harnessed to develop sustainable and novel pest management solutions.

Aphid

Differential expression of neuronal function genes follows a tissue-specific temporal dynamic during Deformed Wing Virus infection in honey bees.

Deformed Wing Virus type A (DWV-A) is one of the primary threats to honeybees (Apis mellifera), significantly impacting their nervous system physiology and behavior. While its neurotropic nature is well-recognized, the temporal dynamics of the neuronal transcriptomic response following oral infection, the natural transmission route, remains poorly understood. In this study, we analyzed gene expression in the heads of worker bees orally inoculated with DWV-A over a 16-day time course (1, 4, 7, 10, 13, and 16 days post-inoculation). RNA-seq analysis at day 10 identified 147 differentially expressed genes associated with different biological processes that are critical to the organism, including cellular metabolism and neuronal activity. RT-qPCR validation revealed a persistent downregulation of key genes related to glutamatergic system (eaat-2, neto, and kainate) and sensory perception-related genes in the antennae. Notably, the simultaneous co-expression of nurse-associated and forager-associated marker genes suggests that DWV-A infection induces an asynchrony in behavioral maturation. Our findings demonstrate that DWV-A disrupts neuronal homeostasis and peripheral sensory perception in a tissue-specific and time-dependent manner, providing a molecular framework to understand the behavioral impairment and the loss of coordination at the colony level.

Animals

Tailored UPRE2 variants for dynamic gene regulation in yeast.

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Saccharomyces cerevisiae

Chromatin and gene regulation in archaea.

The chromatinisation of DNA by nucleoid-associated proteins (NAPs) in archaea 'formats' the genome structure in profound ways, revealing both striking differences and analogies to eukaryotic chromatin. However, the extent to which archaeal NAPs actively regulate gene expression remains poorly understood. The dawn of quantitative chromatin mapping techniques and first NAP-specific occupancy profiles in different archaea promise a more accurate view. A picture emerges where in diverse archaea with very different NAP repertoires chromatin maintains access to regulatory motifs including the gene promoter independently of transcription activity. Our re-analysis of genome-wide occupancy data of the crenarchaeal NAP Cren7 shows that these chromatin-free regions are flanked by increased Cren7 binding across the transcription start site. While bacterial NAPs often form heterochromatin-like regions across islands with xenogeneic genes that are transcriptionally silenced, there is little evidence for similar structures in archaea and data from Haloferax show that the promoters of xenogeneic genes remain accessible. Local changes in chromatinisation causing wide-ranging effects on transcription restricted to one chromosomal interaction domain (CID) in Saccharolobus islandicus hint at a higher-order level of organisation between chromatin and transcription. The emerging challenge is to integrate results obtained at microscale and macroscale, reconciling molecular structure and function with dynamic genome-wide chromatin landscapes.

Chromatin