The coordinated activities of histone modifications and chromatin-associated proteins establish chromatin states that regulate genome function and cellular identity. However, the organizational principles that distinguish chromatin states across cell types remain incompletely understood. Here, we integrated genome-wide profiles of CTCF, H3K27ac, H3K9ac, H3K27me3, and H3K9me3 at 1-kb resolution to generate a unified representation of chromatin organization in human cells. Unsupervised embedding resolved five principal chromatin states corresponding to constitutive heterochromatin, CTCF-associated architectural chromatin, transcriptionally active chromatin, mixed repressive chromatin, and Polycomb-associated chromatin. Comparative analyses of HCT116 and K562 cells revealed that cell-type-specific epigenomic differences arise predominantly through remodeling of Polycomb-associated chromatin, whereas the remaining chromatin states exhibit broadly similar epigenomic signatures and comparatively limited remodeling. Consistent with this observation, principal component analysis identified H3K27me3 as the primary contributor to genome-wide epigenomic divergence, whereas CTCF represented a secondary contributor. Integration with Hi-C data further demonstrated that CTCF-associated chromatin is strongly enriched at chromatin loop anchors and other local architectural features, linking chromatin-state organization to three-dimensional genome architecture. Together, our findings identify distinct chromatin-state classes that organize the human epigenome and reveal Polycomb-associated chromatin as a key determinant of cell-type-specific epigenomic differences.
How genes are desilenced without erasure of repressive chromatin is a poorly understood phenomenon. A dominant mode of repression occurs through methylation of lysine 9 of histone H3 (H3K9me3), a mark that engages heterochromatin protein 1 (HP1) to drive chromatin compaction and transcriptional silencing. The erasure and replacement of this repressive mark with acetyl/acyl groups recruits positive factors such as BRD4/BET to elicit gene transcription. Here we report that, in Friedreich's ataxia, a synthetic gene regulator (SynGR1/SynTEF1) licenses transcription across repressive chromatin without removal or replacement of H3K9me3 or HP1. By recruiting BRD4/BET into repressive GAA repeats in frataxin (FXN), SynGR1 creates a paradoxical state wherein gene transcription and repressive chromatin coexist. Contrary to convention, we find that BRD4 partitions into phase separated HP1 condensates in vitro and into HP1 puncta in patient-derived cells, thus offering a mechanistic explanation for desilencing transcription without the dispersal of mesoscale repressive chromatin. More broadly, our study highlights the dynamic nature of repressive chromatin and the context dependence of epigenetic marks in regulating gene expression.
Liver fibrosis is a key pathological process in the progression of chronic liver disease to cirrhosis and hepatocellular carcinoma, with core features including hepatic stellate cells (HSCs) activation and extracellular matrix (ECM) deposition. Nonetheless, the precise spatiotemporal regulatory mechanisms of its gene expression have not been fully clarified. Interestingly, chromatin accessibility, as a core level of epigenetic regulation, directly determines the expression "switch" of fibrosis-related genes by dynamically altering the chromatin open state, which is closely related to key pathological processes such as HSCs activation, hepatocyte injury, and immune cell infiltration. Chromatin accessibility is coordinately modulated by histone modifications, ATP-dependent chromatin remodelers, CpG methylation, as well as enhancers, super-enhancers, and transcription factors, which together constitute a multilayered epigenetic network. Key regulatory mediators, including histone deacetylases (HDACs), histone acetyltransferase p300 (p300), bromodomain-containing protein 4 (BRD4), DNA methyltransferases (DNMTs), and methyl-CpG-binding protein 2 (MeCP2), have emerged as promising therapeutic candidates. Relevant inhibitors or interventions can inhibit HSCs activation and ECM deposition by reversing abnormal chromatin accessibility. An in-depth study of the regulatory network of chromatin accessibility may provide new perspectives on the pathogenesis of liver fibrosis and lay a theoretical foundation for the development of novel precision-targeted drugs.
Histone post-translational modifications (hPTMs) are key regulators of chromatin states, influencing gene expression, epigenetic memory and transposable element repression across eukaryotic genomes. While many hPTMs are evolutionarily conserved, the extent to which the chromatin states they define are similarly preserved remains unclear. Here we developed a combinatorial indexing chromatin immunoprecipitation followed by sequencing method to simultaneously profile specific hPTMs across diverse eukaryotic lineages, including amoebozoans, rhizarians, discobans and cryptomonads. Our analyses revealed highly conserved euchromatin states at active gene promoters and gene bodies. In contrast, we observed diverse configurations of repressive heterochromatin states associated with silenced genes and transposable elements, characterized by various combinations of hPTMs such as H3K9me3, H3K27me3 and/or different H3K79 methylations. These findings suggest that, while core hPTMs are ancient and broadly conserved, their functional readout has diversified throughout eukaryotic evolution, shaping lineage-specific chromatin landscapes.
UHRF1 is a chromatin-binding protein essential for maintaining DNA methylation and histone modification states, yet its integrated role in vivo remains incompletely understood. To define its function, we generated conditional Uhrf1 knockout embryonic stem cells (ESCs) and embryos. Uhrf1 ⁻ / ⁻ ESCs exhibited near-complete loss of 5mC and 5hmC but maintained pluripotency, whereas Uhrf1-null embryos developed normally until E8.5 and then failed to develop further by E9.5, phenocopying Dnmt1 loss. Single-cell multi-omic (ME-seq) profiling of E8.5 embryos revealed impaired lineage stabilization, widespread hypomethylation, and disrupted chromatin architecture. Uhrf1 loss was associated with altered CTCF-associated chromatin signal, broad remodeling of chromatin contacts, altered cis -regulatory relationships, and reduced predicted BMP-related ligand-receptor communication, particularly within neural crest populations. These findings identify Uhrf1 as a central regulator that tightly couples DNA methylation maintenance to 3D genome organization during gastrulation, thereby, directing early lineage specification and positioning Uhrf1 as a pivotal mediator of epigenetic information transfer during early embryogenesis. Uhrf1 knockout ESCs show global loss of 5mC/5hmC but maintain pluripotency. Uhrf1 -null embryos develop normally until E8.5 but die by E9.5 with severe defects. Single-cell multi-omics revealed disrupted chromatin, transcription, and lineage stability upon knockout. Loss of Uhrf1 alters CTCF-associated chromatin signal, predicted BMP-related communication, and cis -regulatory relationships.
Skeletal muscle undergoes a progressive decline in mass and function with aging, a condition that in its extreme form is known as sarcopenia. This is driven by complex cellular and molecular alterations, such as shifts in myonucleus composition, increased fibrosis, and fat or immune cell infiltration. Despite extensive research, effective therapeutic interventions for sarcopenia remain limited. Recent advances in single-cell omics technologies have begun to unravel the cellular and molecular heterogeneity of mouse and human skeletal muscle across the lifespan, identifying age-enriched cell states and dynamic transcriptional changes. However, epigenetic regulation during skeletal muscle aging is less well characterized. To help address this gap, we performed single-nucleus Assay for Transposase-Accessible Chromatin using sequencing (snATAC-seq) on skeletal muscle from young adult and aged male mice, generating chromatin accessibility profiles from over 43,000 nuclei. Among other findings, our analyses reveal an age-enriched pro-atrophy subpopulation of type IIb myonuclei marked by increased chromatin accessibility at the Ampd3 locus. Furthermore, we delineate the epigenetic mechanisms underlying the transition of healthy type IIb myonuclei into Ampd3+ myonuclei, revealing key chromatin remodeling events that drive this phenotypic shift. Moreover, by integrating with an existing single-nucleus RNA sequencing dataset of the same anatomical origin, we identified thousands of cell-type-specific cis-regulatory elements related to aging programs. Within these elements, we observed a broad depletion of binding motifs for transcription factors with roles in cellular identity and muscle regeneration, concomitant with the gain of stress-responsive transcription factors. Our work helps understand the epigenetic events underlying mammalian skeletal muscle aging.
The Origin Recognition Complex (ORC) is known for initiating DNA replication in eukaryotic cells, but increasing evidence suggests that ORC has multiple functions. Here we show that the organization and function of two major nuclear components, heterochromatin and the nucleolus, depend on multivalent interactions between Orc1 and Heterochromatin Protein 1a (HP1a) in Drosophila melanogaster . Specifically, binding requires two short motifs (R1 and R2) in the intrinsically disordered region (IDR) of Orc1, and two motifs (HGM and CTE) located in the N- and C-terminal regions of HP1a. Pairing of these four motifs promotes ORC-HP1 interactions, which also requires HP1 dimerization. Disrupting ORC-HP1a interactions by mutating the R1/R2 motifs causes defects in heterochromatin functions, specifically suppression of Position-Effect Variegation (PEV), ribosomal DNA (rDNA) decondensation, increased rRNA transcription, and nucleolar expansion, without global loss of H3K9me2 epigenetic modifications. Our findings indicate that ORC acts as a structural regulator that organizes heterochromatic regions and promotes proper genome regulation through multivalent interactions with HP1 and chromatin.
Dentinogenesis, a process essential for tooth function, relies on the precise differentiation of dental papilla cells into odontoblasts. This lineage commitment is governed by complex transcription factor networks. RUNX2 and KLF4, known key TFs co-expressed in this process, were proven to operate synergistically, but the specific cofactors coordinating their activity at the chromatin level remain unknown. Here, we identify the chromatin remodeller BRD9 as a novel interactor of both RUNX2 and KLF4 during odontoblastic differentiation. To probe their shared function, we generated neural crest-specific conditional knockout mice. Wnt1-Cre; Brd9fl/fl mice exhibited a disordered odontoblast layer with reduced secretion of extracellular matrix proteins (DMP1 and DSPP), strikingly phenocopying the compound Wnt1-Cre; Runx2fl/wt; Klf4fl/fl mutants. In vitro, chemical BRD9 degradation suppressed odontoblastic differentiation and mineral deposition. Integrative analyses of RNA-seq and ATAC-seq revealed that BRD9 maintained chromatin accessibility at odontogenesis-associated regions enriched with RUNX2 and KLF4 motifs, specifically at the Fam20c enhancer, thereby facilitating RUNX2 and KLF4 binding for transcriptional activation. Crucially, exogenous supplementation of FAM20C protein partially rescued the odontoblastic differentiation defects upon BRD9 loss. These findings establish BRD9 as a critical epigenetic orchestrator that coordinates RUNX2-KLF4 synergy to activate key odontogenic genes like Fam20c, thus affecting odontoblastic differentiation and dentinogenesis.
Limited oxygen supply or hypoxia can impair fetal development and lead to developmental disorders, but the molecular mechanism underlying this phenomenon remains poorly understood. It is also well known that hypoxia results in transcriptomic alterations and epigenetic reprogramming. Drosophila melanogaster (fruit fly) has been used for decades as a powerful model to dissect the molecular mechanisms regulating development. To better understand the role of early hypoxic stress on development, we performed single-cell joint analysis of chromatin accessibility and transcriptome to characterize the influence of hypoxia on Drosophila embryonic development. We identified hypoxia-induced alterations in both gene expression and chromatin accessibility across 22 cell groups, especially in the genes regulating organogenesis and development of neuronal, tracheal, and muscular systems, including a reduction of germ cells, suggesting a long-lasting influence of hypoxic stress at an early embryonic stage on development and reproduction. In summary, this study demonstrates that early embryonic hypoxia induces cell type- and dose-dependent changes in chromatin accessibility and gene expression, leading to distinct developmental phenotypic responses, such as reduced number of germ cells under both 3% and 5% O₂. We further conclude that the tramtrack ( ttk ) gene is critical in germ cell development and reproduction in Drosophila melanogaster .
Hepatitis B virus (HBV) e antigen (HBeAg) plays a critical role in inducing macrophage activation and subsequent immune tolerance, which facilitates viral immune escape. However, the underlying spatial 3D genomic and epigenetic mechanisms driving this macrophage dysfunction remain largely unknown. To map the topological and transcriptional regulatory landscape, we integrated RNA-sequencing (RNA-seq), high-throughput chromosome conformation capture (Hi-C), and chromatin immunoprecipitation-sequencing (ChIP-seq) to comprehensively analyze control and HBeAg-stimulated macrophages. Key findings were cross-validated using human HBV-infected datasets. HBeAg robustly activated pro-inflammatory transcriptional programs, prominently featuring the TNF signaling pathway as a central node. Hi-C analysis revealed profound global 3D chromatin reorganization accompanying this phenotypic shift. Specifically, inactive-to-active (B-to-A) compartment switching, coupled with de novo H3K27ac enhancer accumulation, directly drove the upregulation of functional genes such as MET and FHOD3. Furthermore, topologically associating domain (TAD) restructuring, particularly TAD merging, exposed new regulatory elements to upregulate FLNB and SESN2. Conversely, the disruption of specific intrachromosomal loops resulted in the targeted downregulation of genes like KBTBD11 and BLVRB. Mechanistically, targeted epigenetic reprogramming drove this 3D structural rewiring: H3K27ac enhancer deposition was enriched for AP-1/STAT motifs, CTCF coordinated with FOX-family factors to alter structural boundaries, and HLTF mediated targeted H3K27me3-associated gene silencing. HBeAg orchestrates a highly coordinated hierarchical restructuring of the 3D genome and targeted epigenetic reprogramming to induce macrophage dysfunction. The identified structural variants and core spatial-target genes (such as MET, FLNB, and BLVRB) provide novel mechanistic insights and represent potential therapeutic targets for HBV-related liver diseases.
Chromatin regulators (CRs), as key components of the epigenetic machinery, participate in tumor progression and therapeutic resistance by modulating chromatin structure and gene expression. Nonetheless, the precise functions of CRs in cancer immunotherapy and the tumor microenvironment (TME) remain largely unexplored. Here, we integrated multi-omics data to characterize genomic alterations, transcriptomic profiles, and copy number variations (CNVs) of 215 CRs and identified survival-associated CRs using Cox regression analysis. Subsequently, based on CR expression patterns, esophageal squamous cell carcinoma (ESCC) patients were classified into CR-active and CR-silent subgroups, which exhibited distinct survival outcomes and TME characteristics. Importantly, the CR-Score developed here was associated with treatment response-related endpoints and post-treatment disease-free survival in patients receiving neoadjuvant PD-1 blockade therapy. In summary, we systematically characterized the molecular and immune features of CR clusters in ESCC and demonstrated that the CR-Score is a novel biomarker for prognostic prediction, with potential clinical utility and theoretical relevance for neoadjuvant immunotherapy.
Atrial fibrillation (AF) represents the most common sustained cardiac arrhythmia in clinical practice and imposes a growing global disease burden. However, current therapeutic strategies remain considerably limited in efficacy, durability, and safety. Recently, the role of epigenetic mechanisms in the pathogenesis of AF, as key regulators of atrial structural and electrical remodeling, has garnered considerable attention. This review systematically elucidates the central role of multiple epigenetic regulatory mechanisms, including deoxyribonucleic acid (DNA) methylation, histone modifications, non-coding ribonucleic acids (RNAs) (microRNAs, long non-coding RNAs, circular RNAs, and transfer RNA-derived small RNAs), chromatin remodeling, and RNA methylation, in the atrial structural and electrical remodeling underlying AF. These reversible molecular modifications are widely implicated in the pathophysiological processes of AF through the regulation of key gene expression. In addition, circulating epigenetic markers (e.g., methylated DNA fragments and specific miRNAs) show substantial potential as biomarkers for AF diagnosis, risk stratification, and relapse prediction. Furthermore, targeted epigenetic intervention strategies (e.g., histone deacetylase (HDAC) inhibitors, demethylating agents) offer novel avenues for the precision therapy of AF. Thus, this review seeks to comprehensively summarize recent advances in the epigenetic regulation of AF pathogenesis, provide a theoretical basis for an in-depth understanding of the underlying mechanisms, and inform the development of innovative preventive and therapeutic strategies.
Aberrant epigenetic reprogramming together with dysregulated mTOR signaling are hallmarks of cancer, where altered chromatin methylation and nutrient-sensing pathways cooperate to drive tumor progression. S-adenosylmethionine (SAM), the universal methyl donor, is essential for these processes, yet how tumors sustain elevated SAM availability to support oncogenic transmethylation reactions remains poorly defined. Here, using prostate cancer (PCa) as a model system, we identify nicotinamide N-methyltransferase (NNMT) as a critical metabolic-epigenetic regulator and tumor suppressor. Using a prostate-specific Nnmt knockout mouse model, we demonstrate that NNMT loss accelerates PCa progression, particularly in the context of Pten deletion, resulting in infiltrating carcinoma and reduced survival. Mechanistically, NNMT functions as a "SAM-sink," and its loss increases intracellular SAM abundance, thereby activating mTORC1 signaling through SAMTOR-dependent sensing and broadly enhancing chromatin methylation. In human PCa, recurrent genomic deletions of NNMT occur in up to 7% of cases, and NNMT protein expression is largely absent in primary tumors and metastases. NNMT-deficient PCa cells exhibit elevated SAM:SAH ratios, increased histone methylation, and heightened mTORC1 activity, enabling sustained tumor growth even under dietary methionine-restriction (MR). Notably, combined MR and pharmacologic mTORC1 inhibition synergistically suppresses the growth of NNMT-deficient tumors, revealing a previously unrecognized therapeutic vulnerability. Collectively, these findings establish NNMT as a key tumor suppressor that constrains SAM-driven epigenetic and signaling programs in PCa and suggest a rational, diet-based therapeutic strategy for advanced cancers with NNMT loss.
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Centromeres ensure chromosome segregation, but their chromatin organization within repetitive alpha-satellite DNA has been difficult to resolve. To address this, we generated haplotype-resolved satellite DNA annotations for the complete diploid T2T-HG002 human genome assembly, then we mapped centromere protein A (CENP-A), H3K9me3, and CpG methylation on ultra-long, adaptively sampled nanopore reads using directed methylation with long-read sequencing (DiMeLo-seq). We find that CENP-A occupies multiple discrete subdomains within hypomethylated centromere dip regions (CDRs), with constrained aggregate size and balanced CENP-A dosage between homologous chromosomes despite extensive satellite array variation. We also show that extended lymphoblastoid cell culture and induced pluripotent stem cell (iPSC) reprogramming remodel DNA methylation and alter CENP-A abundance and CDR subdomain organization. These results define a single-molecule, haplotype-resolved framework for studying human centromere plasticity, epigenetic inheritance, and chromosomal instability in development and disease.
The tumor microenvironment (TME) critically regulates cancer progression by providing biochemical and biophysical cues that shape cellular behavior. However, how defined physical microenvironments govern cancer stemness and chemoresistance through mechanotransduction remains poorly understood. Here, we systematically engineered eight tumor-mimetic microenvironments by integrating serum, oxygen, and 3D compacted culture to investigate their effects on A549 non-small cell lung cancer cells. Among all conditions, cells cultured under 3D culture (PM4C) exhibited reduced cellular stiffness, enhanced expression of cancer stemness markers (EpCAM and CD44), and significantly increased resistance to cisplatin in both in vitro and nude mouse xenograft models. Transcriptomic analysis revealed that differentially expressed genes in the PM4C group were predominantly enriched in cell adhesion, mechanotransduction, stemness, and cisplatin resistance pathways. Metabolomic profiling further revealed a substantial accumulation of anaerobic metabolites associated with the maintenance of stemness. Mechanistically, the PM4C microenvironment remodeled matrix production, cell-ECM interactions, and cytoskeletal organization while inducing epigenetic reprogramming (reduced H3K9 acetylation), collectively promoting a stem-like and chemoresistant phenotype. These findings establish a direct mechanistic link between TME and cancer cell stemness, demonstrating that TME can reprogram stemness and drug responsiveness through mechano-epigenetic regulation. This work provides a mechanobiological framework for engineering physiologically relevant tumor organoids and offers new strategies for developing TME-targeted drugs and therapies.
Transcriptional activation of cell-type-specific genes is achieved by multiple mechanisms that guarantee chromatin accessibility at cis-regulatory elements and the long-range interactions between them. Generally, DNA methylation counteracts these processes and promotes gene silencing in vertebrates. CTCF is a conserved and essential multifunctional transcription factor relevant for establishing and maintaining chromatin architecture and accessibility, while its precise role in regulating DNA methylation remains to be determined. We focused on the highly abundant and previously studied erythroid-specific αD gene (HBAD) to systematically investigate the role of CTCF binding on DNA methylation, chromatin accessibility, and gene expression using a chicken erythroid cell differentiation system. The perturbation of CTCF binding at the intergenic region between the embryonic π and the adult αD gene resulted in DNA methylation propagation towards the αD gene, which was accompanied by decreased chromatin accessibility, GATA-1 binding, and gene expression. Notably, chromatin conformation analyses revealed that CTCF binding enables αD transcriptional activation independently of its architectural function. We observed a similar role of CTCF on DNA methylation and gene expression in the human orthologous gene HBA2. Our findings support a conserved role of CTCF in preventing DNA methylation spreading and gene silencing of cell-type-specific genes along differentiation.
The retinal pigment epithelium (RPE) is crucial for visual function, and its dysfunction contributes to retinal diseases such as age-related macular degeneration. Despite the translational potential of iPSC-derived RPE (iPSC-RPE) in cell replacement therapy, the functional visual gains achieved to date are modest. A key challenge is that the molecular and epigenomic signatures underlying functional RPE are yet to be fully elucidated. By integrating multiomics data, we systematically benchmarked the 3D epigenomic landscapes of primary human RPE (hRPE), iPSC-RPE, and the immortalized ARPE-19 cell line. Our analysis reveals that iPSC-RPE exhibits a mixed molecular state. iPSC-RPE recapitulates hRPE-like transcription and chromatin looping, but its histone modification states remain incompletely matured, and its chromatin accessibility and higher-order chromatin organization do not fully converge to hRPE. Furthermore, we found that hRPE exhibits strong extracellular matrix (ECM) organization driven by enhancer-mediated long-range chromatin interactions and enriched RUNX1 motifs, while iPSC-RPE retains key developmental-related transcriptional signatures, marked by factors such as HAND1, OTX2, and PAX6. These findings establish a multiomics benchmark for RPE maturity, pinpoint key regulatory nodes like ECM organization and RUNX1 for therapeutic targeting, and provide a roadmap for optimizing differentiation protocols and scaffold design in retinal regenerative medicine.
Recent experimental evidence suggests that aging may arise from the progressive deterioration of the epigenetic landscape, while reversing the trend can result in cell and tissue rejuvenation. A mechanistic understanding of how restoration of a key component of this landscape - the 3D structure of the genome - can be accomplished is lacking. Here we investigate lamina-dependent disruption and recovery of the 3D architecture of the Drosophila melanogaster genome at TAD resolution (~ 100 kb), using a model of the entire nucleus; weakening of chromatin-lamina interactions mimics an aging-associated loss of chromatin organization. We characterize this loss using the Shannon entropy of appropriately normalized Hi-C contact matrices. Our main finding is that lamina-depletion-induced increases in Hi-C map disorder, deterioration of chromosome territories, and cell-to-cell conformational heterogeneity are largely reversible when WT-like LAD-nuclear-envelope interactions are restored. The original and recovered conformational states of chromatin are nearly indistinguishable by bulk Hi-C contact matrix; the corresponding Pearson correlation coefficient is 0.999902. The direct experimentally testable prediction is that restoration of functional LAD-lamina interactions will promote recovery of young/WT-like 3D chromatin architecture after lamina-dependent architectural disruption.
Chromatin remodeling plays a central role in regulating plant development and physiology by shaping the gene expression patterns that drive biological processes. Among epigenetic modifications, histone acetylation is particularly relevant as it alters chromatin structure and influences transcriptional activity. MYST-type histone acetyltransferases (HAT) are evolutionarily conserved components of the Nucleosome Acetyltransferase of histone H4 (NuA4) complex, a key regulator that acetylates histones H4, H2A, and the histone variant H2A.Z. Growing evidence supports the presence of a canonical NuA4-C in plants, similar to that described in yeast. In this review, we summarize recent studies that have begun to uncover its broad role in plant biology, highlighting its involvement in diverse processes such as the skoto- to photomorphogenesis switch, chloroplast development, shade avoidance responses, thermomorphogenesis, the vegetative-to-reproductive transition, plant growth, reproduction and hormonal signalling. In addition, we discuss recent advances in understanding the crosstalk of NuA4-C-mediated H4ac and H2A.Z deposition with other chromatin remodeling complexes in plants. Although significant progress has been made, a full understanding of the complex functions remains unavailable. Current evidence indicates that NuA4-C in yeast and TIP60 in humans are central regulators of transcription, acting not only through histone acetylation but also by influencing transcription elongation and RNA splicing, although direct evidence for similar functions in plant NuA4-C still remains limited. This regulatory role might be critical for integrating developmental programs with environmental signalling pathways. While initial insights into the recruitment of NuA4-C to target genes have emerged, further research is needed to clarify how its activity is controlled and modulated in different biological contexts.