bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2025–03–30
38 papers selected by
Connor Rogerson, University of Cambridge



  1. Nat Commun. 2025 Mar 27. 16(1): 3007
      The establishment of germ layers during early development is crucial for body formation. The Drosophila zygote serves as a model for investigating these transitions in relation to the chromatin landscape. However, the cellular heterogeneity of the blastoderm embryo poses a challenge for gaining mechanistic insights. Using 10× Multiome, we simultaneously analyzed the in vivo epigenomic and transcriptomic states of wild-type, E(z)-, and CBP-depleted embryos during zygotic genome activation at single-cell resolution. We found that pre-zygotic H3K27me3 safeguards tissue-specific gene expression by modulating cis-regulatory elements. Furthermore, we demonstrate that CBP is essential for cell fate specification functioning as a transcriptional activator by stabilizing transcriptional factors binding at key developmental genes. Surprisingly, while CBP depletion leads to transcriptional arrest, chromatin accessibility continues to progress independently through the retention of stalled RNA Polymerase II. Our study reveals fundamental principles of chromatin-mediated gene regulation essential for establishing and maintaining cellular identities during early embryogenesis.
    DOI:  https://doi.org/10.1038/s41467-025-57719-4
  2. Nat Commun. 2025 Mar 26. 16(1): 2941
      Chromatin topology can impact gene regulation, but how evolutionary divergence in chromatin topology has shaped gene regulatory landscapes for distinctive human traits remains poorly understood. CTCF sites determine chromatin topology by forming domains and loops. Here, we show evolutionary divergence in CTCF-mediated chromatin topology at the domain and loop scales during primate evolution, elucidating distinct mechanisms for shaping regulatory landscapes. Human-specific divergent domains lead to a broad rewiring of transcriptional landscapes. Divergent CTCF loops concord with species-specific enhancer activity, influencing enhancer connectivity to target genes in a concordant yet constrained manner. Under this concordant mechanism, we establish the role of human-specific CTCF loops in shaping transcriptional isoform diversity, with functional implications for disease susceptibility. Furthermore, we validate the function of these human-specific CTCF loops using human forebrain organoids. This study advances our understanding of genetic evolution from the perspective of genome architecture.
    DOI:  https://doi.org/10.1038/s41467-025-58275-7
  3. Cell Rep. 2025 Mar 27. pii: S2211-1247(25)00257-8. [Epub ahead of print]44(4): 115486
      Cell identity maintenance faces many challenges during mitosis, as most DNA-binding proteins are evicted from DNA and transcription is virtually abolished. How cells maintain their identity through division and faithfully re-initiate gene expression during mitotic exit is unclear. Here, we develop a novel reporter system enabling cell cycle synchronization-free separation of pluripotent stem cells in temporal bins of <30 min during mitotic exit. This allows us to quantify genome-wide reactivation of transcription, sequential changes in chromatin accessibility and transcription factor footprints, and re-binding of the pluripotency transcription factors OCT4, SOX2, and NANOG (OSN). We find that transcriptional activity progressively ramps up after mitosis and that OSN rapidly reoccupy the genome during the anaphase-telophase transition. We also demonstrate transcription factor-specific, dynamic relocation patterns and a hierarchical reorganization of the OSN binding landscape governed by OCT4 and SOX2. Our study sheds light on the dynamic orchestration of transcriptional reactivation after mitosis.
    Keywords:  ATAC-seq; CP: Cell biology; CP: Stem cell research; ChIP-seq; NANOG; OCT4; RNA-seq; SOX2; chromatin; mitosis; pluripotent stem cells; transcription factors
    DOI:  https://doi.org/10.1016/j.celrep.2025.115486
  4. Mol Cell. 2025 Mar 21. pii: S1097-2765(25)00194-7. [Epub ahead of print]
      Amplification-mediated oncogene overexpression is a critical and widespread driver event in cancer, yet our understanding of how amplification and dosage mediate oncogene regulation is limited. Here, we find that the most significant focal amplification event in lung adenocarcinoma (LUAD) targets a lineage "super-enhancer" near the NKX2-1 lineage transcription factor. The NKX2-1 super-enhancer is targeted by focal and co-amplification with NKX2-1 and controls NKX2-1 expression and regulation. We find that NKX2-1 directly controls enhancer accessibility to drive a lineage-addicted state in LUAD. We precisely map the effects of NKX2-1 dosage modulation upon both overexpression and knockdown and identify both linear and non-linear regulation by NKX2-1 dosage. We find that NKX2-1 is a widespread dependency in LUAD cell lines and that NKX2-1 confers persistence to EGFR inhibitors. Our data suggest a defining role for dosage in the oncogenic regulation of amplified NKX2-1 and that amplified NKX2-1 lineage addiction defines LUAD tumors.
    Keywords:  EGFR; NKX2-1; amplification; copy number; dosage; enhancer; gene regulation; lung adenocarcinoma; oncogene; transcription factor
    DOI:  https://doi.org/10.1016/j.molcel.2025.03.001
  5. J Biol Chem. 2025 Mar 22. pii: S0021-9258(25)00285-6. [Epub ahead of print] 108436
      Spt6-Spn1 is an essential histone chaperone complex that associates with RNA Polymerase II (RNAPII) and reassembles nucleosomes during gene transcription. While the interaction between Spt6 and Spn1 is important for its histone deposition and transcription functions, a precise mechanistic understanding is still limited. Here, using temperature sensitive alleles of spt6 and spn1 that disrupt their interaction in yeast, we show that Spt6-Spn1 association is important for its stable interaction with the elongating RNAPII complex and nucleosomes. Using micrococcal nuclease (MNase)-based chromatin occupancy profiling, we further find that Spt6-Spn1 interaction is required to maintain a preferred nucleosome positioning at actively transcribed genes; in the absence of Spt6-Spn1 interaction, we observe a return to replication-dependent phasing. In addition to positioning defects, Spt6-Spn1 disrupting mutants also resulted in an overall shift of nucleosomes towards the 5' end of genes that was correlated with decreased RNAPII levels. As loss of Spt6-Spn1 association results in cryptic transcription at a subset of genes, we examined these genes for their nucleosome profiles. These findings revealed that the chromatin organization at these loci is similar to other active genes, thus underscoring the critical role of DNA sequence in mediating cryptic transcription when nucleosome positioning is altered. Taken together, these findings reveal Spt6-Spn1 interaction is key to its association with elongating RNAPII and for its ability to precisely organize nucleosomes across transcription units.
    Keywords:  Spn1; Spt6; chromatin; histone chaperone; histones; nucleosomes; transcription; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2025.108436
  6. Sci Adv. 2025 Mar 28. 11(13): eadt4770
      Pioneer transcription factors (TFs) initiate chromatin remodeling, which is crucial for gene regulation and cell differentiation. In this study, we investigated how the sequential expression of neurogenin 3 (NGN3) and NEUROD1 affects their pioneering functions during pancreatic endocrine differentiation. Using a genetically engineered mouse model, we mapped NGN3-binding sites, confirming the pivotal role of this molecule in regulating chromatin accessibility. The pioneering function of NGN3 involves dose tolerance, and low doses are sufficient. Although NEUROD1 generally acts as a conventional TF, it can assume a pioneering role in the absence of NGN3. The sequential expression of NeuroD1 and Ngn3 predominantly drives α cell generation, which may explain the inefficient β cell induction observed in vitro. Our findings demonstrate that pioneer activity is dynamically shaped by temporal TF expression and inter-TF interactions, providing insights into transcriptional regulation and its implications for disease mechanisms and therapeutic targeting and enhancing in vitro differentiation strategies.
    DOI:  https://doi.org/10.1126/sciadv.adt4770
  7. Neuro Oncol. 2025 Mar 23. pii: noaf081. [Epub ahead of print]
       BACKGROUND: Atypical teratoid rhabdoid tumor (ATRT) is a deadly central nervous system embryonal tumor caused by loss of SMARCB1, a core subunit of SWI/SNF chromatin remodeling complexes. SMARCB1-deficient cancers are defined by loss of cell differentiation-associated enhancers, but how SWI/SNF interacts with other arbiters of cell differentiation (specifically lineage-specific transcription factors (TFs)) remains poorly understood.
    METHODS: We leveraged a multi-omics approach, patient-derived ATRT cells and patient-derived orthotopic xenografts to investigate the interplay of SWI/SNF with lineage-specific TFs in a clinically relevant setting.
    RESULTS: We observe that an activating protein 1 (AP-1)-dependent transcriptional regulatory network is lost in ATRT, and AP-1 and lineage-specific TFs TEAD1 and ZIC2 require SMARCB1 for enhancer binding. SMARCB1-dependent SWI/SNF integrates transcriptional functions of lineage-specific TFs into a core regulatory circuit that depends on the AP-1 subunit cJUN, whose expression is determined by a SMARCB1-dependent super enhancer that is lost in ATRT-MYC. In the absence of SMARCB1, lineage-specific TFs are sequestered to promoters, where they maintain core transcriptional programs necessary for cell survival. Targeting residual, promoter-proximal TF activity by a protein degrader of the SWI/SNF ATPase SMARCA4 or small molecule inhibitors that indirectly inhibit AP-1 and TEAD activity abrogates expression of these networks, reducing cell viability in vitro and prolonging survival in an orthotopic patient-derived xenograft model.
    CONCLUSIONS: These results demonstrate SWI/SNF complexes are critical for lineage-specific TF binding and activity at both promoters and enhancers. In the context of ATRT, these findings reveal a previously underappreciated therapeutic vulnerability in targeting residual promoter-proximal TF function in ATRT.
    Keywords:  ATRT; chromatin; epigenetics; rhabdoid tumor; transcription factors
    DOI:  https://doi.org/10.1093/neuonc/noaf081
  8. Science. 2025 Mar 28. 387(6741): eadp4319
      In mammals, fertilized eggs undergo genome-wide epigenetic reprogramming to generate the organism. However, our understanding of epigenetic dynamics during preimplantation development at single-cell resolution remains incomplete. Here, we developed scNanoATAC-seq2, a single-cell assay for transposase-accessible chromatin using long-read sequencing for scarce samples. We present a detailed chromatin accessibility landscape of mouse preimplantation development, revealing distinct chromatin signatures in the epiblast, primitive endoderm, and trophectoderm during lineage segregation. Differences between zygotes and two-cell embryos highlight reprogramming in chromatin accessibility during the maternal-to-zygotic transition. Single-cell long-read sequencing enables in-depth analysis of chromatin accessibility in noncanonical imprinting, imprinted X chromosome inactivation, and low-mappability genomic regions, such as repetitive elements and paralogs. Our data provide insights into chromatin dynamics during mammalian preimplantation development and lineage differentiation.
    DOI:  https://doi.org/10.1126/science.adp4319
  9. Mol Cell. 2025 Mar 20. pii: S1097-2765(25)00197-2. [Epub ahead of print]
      Most human transcription factor (TF) genes encode multiple protein isoforms differing in DNA-binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators," both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.
    Keywords:  alternative splicing; gene regulation; isoforms; transcription factors; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molcel.2025.03.004
  10. Front Cell Dev Biol. 2025 ;13 1561540
      Transcription factors (TFs) play a crucial role in the regulation of gene expression and the structural organization of chromatin. They interact with proteins, RNA, and chromatin DNA to exert their functions. Therefore, an efficient and straightforward experimental approach that simultaneously captures the interactions of transcription factors with DNA, RNA, and proteins is essential for studying these regulatory proteins. In this study, we developed a novel method, TF-chRDP (Transcription Factor binding Chromatin-associated RNA, DNA, and Protein), which allows for the concurrent capture of these biomolecules in a single experiment. We enriched chromatin complexes using specific antibodies and divided the chromatin into three fractions: one for DNA library preparation to analyze the genomic binding sites of transcription factors, another for RNA library preparation to investigate the RNA associated with transcription factor binding, and the third for proteomic analysis to identify protein cofactors interacting with transcription factors. We applied this method to study the transcription factor p53 and its associated chromatin complexes. The results demonstrated high specificity in the enrichment of DNA, RNA and proteins. This method provides an efficient tool for simultaneously capturing chromatin-associated RNA, DNA and protein bound to specific TF, making it particularly useful for analyzing the role of protein-DNA-RNA complexes in transcriptional regulation.
    Keywords:  DNA; TF-chRDP; chromatin-associated RNA; protein; transcription factor
    DOI:  https://doi.org/10.3389/fcell.2025.1561540
  11. Nat Commun. 2025 Mar 25. 16(1): 2931
      Aging is characterized by changes in gene expression, some of which can drive deleterious cellular phenotypes and senescence. The transcriptional activation of senescence genes has been mainly attributed to epigenetic shifts, but the changes in chromatin accessibility and its underlying mechanisms remain largely elusive in natural aging. Here, we profiled chromatin accessibility in human dermal fibroblasts (HDFs) from individuals with ages ranging from neonatal to octogenarian. We found that AP-1 binding motifs are prevalent in elderly-specific accessible chromatin regions while neonatal-specific regions are highly enriched for TEAD binding motifs. We further show that TEAD4 and FOXM1 share a conserved transcriptional regulatory landscape controlled by a not previously described and age-dependent enhancer that loses accessibility with aging and whose deletion drives senescence. Finally, we demonstrate that FOXM1 ectopic expression in elderly cells partially resets chromatin accessibility to a youthful state due to FOXM1's repressive function on several members of the AP-1 complex, which is known to trigger the senescence transcriptional program. These results place FOXM1 at a top hierarchical level in chromatin remodeling required to prevent senescence.
    DOI:  https://doi.org/10.1038/s41467-025-57503-4
  12. Sci Adv. 2025 Mar 28. 11(13): eadu8400
      A string of nucleosomes, where genomic DNA is wrapped around histones, is organized in the cell as chromatin, ranging from euchromatin to heterochromatin, with distinct genome functions. Understanding physical differences between euchromatin and heterochromatin is crucial, yet specific labeling methods in living cells remain limited. Here, we have developed replication-dependent histone (Repli-Histo) labeling to mark nucleosomes in euchromatin and heterochromatin based on DNA replication timing. Using this approach, we investigated local nucleosome motion in the four known chromatin classes, from euchromatin to heterochromatin, of living human and mouse cells. The more euchromatic (earlier-replicated) and more heterochromatic (later-replicated) regions exhibit greater and lesser nucleosome motions, respectively. Notably, the motion profile in each chromatin class persists throughout interphase. Genome chromatin is essentially replicated from regions with greater nucleosome motions, although the replication timing is perturbed. Our findings, combined with computational modeling, suggest that earlier-replicated regions have more accessibility, and local chromatin motion can be a major determinant of genome-wide replication timing.
    DOI:  https://doi.org/10.1126/sciadv.adu8400
  13. J Virol. 2025 Mar 25. e0208624
      Herpes simplex virus 1 (HSV-1) infection induces a loss of host transcriptional activity and widespread disruption of host transcription termination, which leads to an induction of open chromatin downstream of genes. In this study, we show that lytic HSV-1 infection also leads to an extension of chromatin accessibility at promoters into downstream regions. This is most prominent for highly expressed genes and independent of the HSV-1 proteins ICP0, ICP22, ICP27, and vhs. ChIPmentation of the noncanonical histone variant H2A.Z, which is strongly enriched at +1 and -1 nucleosomes, indicated that these chromatin accessibility changes are linked to a downstream shift of +1 nucleosomes. In yeast, downstream shifts of +1 nucleosomes are induced by RNA polymerase II (Pol II) degradation. Accordingly, irreversible depletion of Pol II from genes in human cells using α-amanitin altered +1 nucleosome positioning similar to lytic HSV-1 infection. Consequently, treatment with phosphonoacetic acid and knockout of ICP4, which both prevent viral DNA replication and alleviate the loss of Pol II from host genes, largely abolished the downstream extension of accessible chromatin in HSV-1 infection. In the absence of viral genomes, doxycycline-induced expression of ICP27, which redirects Pol II from gene bodies into intergenic regions by disrupting transcription termination, induced an attenuated effect that was further enhanced by co-expression of ICP22. In summary, our study provides strong evidence that HSV-1-induced depletion of Pol II from the host genome leads to a downstream shift of +1 nucleosomes at host promoters.IMPORTANCELytic herpes simplex virus 1 (HSV-1) infection leads to a profound host transcription shutoff. Loss of RNA polymerase II (Pol II) in yeast has previously been shown to relax +1 nucleosome positioning to more thermodynamically favorable sites downstream of transcription start sites. Here, we show that a similar phenomenon is likely at play in lytic HSV-1 infection. Sequencing of accessible chromatin revealed a widening of nucleosome-free regions at host promoters into downstream regions. By mapping genome-wide positions of the noncanonical histone variant H2A.Z enriched at +1 and -1 nucleosomes, we demonstrate a downstream shift of +1 nucleosomes for most cellular genes in lytic HSV-1 infection. As chemical depletion of Pol II from genes also leads to a downstream shift of +1 nucleosomes in human cells, changes in chromatin architecture at promoters in HSV-1 infection are likely a consequence of HSV-1-induced loss of Pol II activity from the host genome.
    Keywords:  +1 nucleosome; ATAC-seq; H2A.Z; HSV-1; RNA polymerases; chromatin accessibility; chromatin remodeling; herpes simplex virus
    DOI:  https://doi.org/10.1128/jvi.02086-24
  14. Cell. 2025 Mar 19. pii: S0092-8674(25)00255-7. [Epub ahead of print]
      How genomic DNA is folded during cell division to form the characteristic rod-shaped mitotic chromosomes essential for faithful genome inheritance is a long-standing open question in biology. Here, we use nanoscale DNA tracing in single dividing cells to directly visualize how the 3D fold of genomic DNA changes during mitosis at scales from single loops to entire chromosomes. Our structural analysis reveals a characteristic genome scaling minimum of 6-8 megabases in mitosis. Combined with data-driven modeling and molecular perturbations, we can show that very large and strongly overlapping loops formed by condensins are the fundamental structuring principle of mitotic chromosomes. These loops compact chromosomes locally and globally to the limit set by chromatin self-repulsion. The characteristic length, density, and increasingly overlapping structure of mitotic loops we observe in 3D fully explain how the rod-shaped mitotic chromosome structure emerges by self-organization during cell division.
    Keywords:  cell division; chromatin tracing; chromosome compaction; condensins; genome organization; loop extrusion; mitosis
    DOI:  https://doi.org/10.1016/j.cell.2025.02.028
  15. Mol Cell. 2025 Mar 21. pii: S1097-2765(25)00200-X. [Epub ahead of print]
      Dysregulation of enhancer-promoter communication in the three-dimensional (3D) nucleus is increasingly recognized as a potential driver of oncogenic programs. Here, we profiled the 3D enhancer-promoter networks of patient-derived glioblastoma stem cells to identify central regulatory nodes. We focused on hyperconnected 3D hubs and demonstrated that hub-interacting genes exhibit high and coordinated expression at the single-cell level and are associated with oncogenic programs that distinguish glioblastoma from low-grade glioma. Epigenetic silencing of a recurrent hub-with an uncharacterized role in glioblastoma-was sufficient to cause downregulation of hub-connected genes, shifts in transcriptional states, and reduced clonogenicity. Integration of datasets across 16 cancers identified "universal" and cancer-type-specific 3D hubs that enrich for oncogenic programs and factors associated with worse prognosis. Genetic alterations could explain only a small fraction of hub hyperconnectivity and increased activity. Overall, our study provides strong support for the potential central role of 3D regulatory hubs in controlling oncogenic programs and properties.
    Keywords:  3D chromatin organization; CRISPRi; HiChIP; clonogenicity; enhancer hubs; enhancer-promoter interactions; glioblastoma; oncogenic program; regulatory hubs; single-cell RNA-seq; structural variants
    DOI:  https://doi.org/10.1016/j.molcel.2025.03.007
  16. Plant J. 2025 Mar;121(6): e70073
      Cis-regulatory elements (CREs) are enriched in accessible chromatin regions (ACRs) of eukaryotes. Despite extensive research on genome-wide ACRs in various plant tissues, the global impact of these changes on developmental processes in maize seeds remains poorly understood. In this study, we employed the assay for transposase-accessible chromatin sequencing (ATAC-seq) to reveal the chromatin accessibility profile throughout the genome during the early stages of maize seed development. We identified a total of 37 952 to 59 887 high-quality ACRs in maize seeds at 0 to 8 days after pollination (DAP). Furthermore, we examined the correlation between the identified ACRs and gene expression. We observed a positive correlation between the open degree of promoter-ACRs and the expression of most genes. Moreover, we identified binding footprints of numerous transcription factors (TFs) within chromatin accessibility regions and revealed key TF families involved in different stages. Through the footprints of accessible chromatin regions, we predicted transcription factor regulatory networks during early maize embryo development. Additionally, we discovered that DNA sequence diversity was notably reduced at ACRs, yet trait-associated SNPs were more likely to be located within ACRs. We edited the ACR containing the trait-associated SNP of NKD1. Both NKD1pro-1 and NKD1pro-2 showed phenotypes corresponding to the trait-associated SNP. Our results suggest that alterations in chromatin accessibility play a crucial role in maize seed development and highlight the potential contribution of open chromatin regions to advancements in maize breeding.
    Keywords:  Zea mays; accessible chromatin region (ACR); maize seed development; stage‐specific ACRs; transcription factor footprints; transcription regulatory network
    DOI:  https://doi.org/10.1111/tpj.70073
  17. Cell Genom. 2025 Mar 21. pii: S2666-979X(25)00075-8. [Epub ahead of print] 100819
      The attachment of the kinetochore to the centromere is essential for genome maintenance, yet the highly repetitive nature of satellite regional centromeres limits our understanding of their chromatin organization. We demonstrate that single-molecule chromatin fiber sequencing (Fiber-seq) can uniquely co-resolve kinetochore and surrounding chromatin architectures along point centromeres, revealing largely homogeneous single-molecule kinetochore occupancy. In contrast, the application of Fiber-seq to regional centromeres exposed marked per-molecule heterogeneity in their chromatin organization. Regional centromere cores uniquely contain a dichotomous chromatin organization (dichromatin) composed of compacted nucleosome arrays punctuated with highly accessible chromatin patches. CENP-B occupancy phases dichromatin to the underlying alpha-satellite repeat within centromere cores but is not necessary for dichromatin formation. Centromere core dichromatin is conserved between humans and primates, including along regional centromeres lacking satellite repeats. Overall, the chromatin organization of regional centromeres is defined by marked per-molecule heterogeneity, buffering kinetochore attachment against sequence and structural variability within regional centromeres.
    Keywords:  Alpha-satellite; CENP-B; Fiber-seq; centromere; chromatin; kinetochore; single molecule
    DOI:  https://doi.org/10.1016/j.xgen.2025.100819
  18. Nat Commun. 2025 Mar 26. 16(1): 2964
      Extrachromosomal circular DNA (ecDNA) has been found in most types of human cancers, and ecDNA incorporating viral genomes has recently been described, specifically in human papillomavirus (HPV)-mediated oropharyngeal cancer (OPC). However, the molecular mechanisms of human-viral hybrid ecDNA (hybrid ecDNA) for carcinogenesis remains elusive. We characterize the epigenetic status of hybrid ecDNA using HPVOPC cell lines and patient-derived tumor xenografts, identifying HPV oncogenes E6/E7 in hybrid ecDNA are flanked by previously unrecognized somatic DNA enhancers and HPV L1 enhancers, with strong cis-interactions. Targeting of these enhancers by clustered regularly interspaced short palindromic repeats interference or hybrid ecDNA by bromodomain and extra-terminal inhibitor reduces E6/E7 expression, and significantly inhibites in vitro and/or in vivo growth only in ecDNA(+) models. HPV DNA in hybrid ecDNA structures are associated with previously unrecognized somatic and HPV enhancers in hybrid ecDNA that drive HPV ongogene expression and carcinogenesis, and can be targeted with ecDNA disrupting therapeutics.
    DOI:  https://doi.org/10.1038/s41467-025-57447-9
  19. Nat Commun. 2025 Mar 22. 16(1): 2833
      What features of transcription can be learnt by fitting mathematical models of gene expression to mRNA count data? Given a suite of models, fitting to data selects an optimal one, thus identifying a probable transcriptional mechanism. Whilst attractive, the utility of this methodology remains unclear. Here, we sample steady-state, single-cell mRNA count distributions from parameters in the physiological range, and show they cannot be used to confidently estimate the number of inactive gene states, i.e. the number of rate-limiting steps in transcriptional initiation. Distributions from over 99% of the parameter space generated using models with 2, 3, or 4 inactive states can be well fit by one with a single inactive state. However, we show that for many minutes following induction, eukaryotic cells show an increase in the mean mRNA count that obeys a power law whose exponent equals the sum of the number of states visited from the initial inactive to the active state and the number of rate-limiting post-transcriptional processing steps. Our study shows that estimation of the exponent from eukaryotic data can be sufficient to determine a lower bound on the total number of regulatory steps in transcription initiation, splicing, and nuclear export.
    DOI:  https://doi.org/10.1038/s41467-025-58127-4
  20. Sci Rep. 2025 Mar 22. 15(1): 9918
      Cohesin and condensin, two related protein complexes, play essential roles in ensuring the accurate segregation of the genome into daughter cells during cell division. However, the interaction between cohesin and condensin in embryonic stem cells remains unclear, as does the specific function of the meiosis-specific cohesin complex. Cohesin maintains the cohesion of replicated sister chromatids until their separation at anaphase, whereas condensin facilitates the reorganization of chromosomes into a highly compact structure characteristic of mitosis. First, we found via ChIP-seq analysis that cohesins (SMC3, RAD21, and REC8) and condensin (SMC4) share DNA binding sites in close proximity and directly interact with the insulator protein CTCF. Second, siRNA-regulated SMC3 depletion led to nuclear accumulation of SMC4. Third, embryonic stem (ES) cells uniquely harbor cohesin complexes containing the meiotic kleisin subunit REC8. RAD21 knockdown increased the proportion of SMC3-REC8 complexes. Our findings indicate that cohesin and condensin make important contributions to the functions of the chromosomal organization, and that meiotic cohesin may be specifically required for the mitotic program in ES cells.
    Keywords:  Cohesin; Condensin; Embryonic stem cell; Meiosis; Mitosis
    DOI:  https://doi.org/10.1038/s41598-025-94533-w
  21. Nat Commun. 2025 Mar 25. 16(1): 2890
      Enhancer RNAs (eRNAs) are a pivotal class of enhancer-derived non-coding RNAs that drive gene expression. Here we identify the SNAI1 enhancer RNA (SNAI1e; SCREEM2) as a key activator of SNAI1 expression and a potent enforcer of transforming growth factor-β (TGF-β)/SMAD signaling in cancer cells. SNAI1e depletion impairs TGF-β-induced epithelial-mesenchymal transition (EMT), migration, in vivo extravasation, stemness, and chemotherapy resistance in breast cancer cells. SNAI1e functions as an eRNA to cis-regulate SNAI1 enhancer activity by binding to and strengthening the enrichment of the transcriptional co-activator bromodomain containing protein 4 (BRD4) at the local enhancer. SNAI1e selectively promotes the expression of SNAI1, which encodes the EMT transcription factor SNAI1. Furthermore, we reveal that SNAI1 interacts with and anchors the inhibitory SMAD7 in the nucleus, and thereby prevents TGF-β type I receptor (TβRI) polyubiquitination and proteasomal degradation. Our findings establish SNAI1e as a critical driver of SNAI1 expression and TGF-β-induced cell plasticity.
    DOI:  https://doi.org/10.1038/s41467-025-58032-w
  22. Cell Stem Cell. 2025 Mar 20. pii: S1934-5909(25)00088-8. [Epub ahead of print]
      Non-energetic roles for glucose are largely unclear, as is the interplay between transcription factors (TFs) and ubiquitous biomolecules. Metabolomic analyses uncovered elevation of intracellular glucose during differentiation of diverse cell types. Human and mouse tissue engineered with glucose sensors detected a glucose gradient that peaked in the outermost differentiated layers of the epidermis. Free glucose accumulation was essential for epidermal differentiation and required the SGLT1 glucose transporter. Glucose affinity chromatography uncovered glucose binding to diverse regulatory proteins, including the IRF6 TF. Direct glucose binding enabled IRF6 dimerization, DNA binding, genomic localization, and induction of IRF6 target genes, including essential pro-differentiation TFs GRHL1, GRHL3, HOPX, and PRDM1. These data identify a role for glucose as a gradient morphogen that modulates protein multimerization in cellular differentiation.
    Keywords:  IRF6; differentiation; glucose
    DOI:  https://doi.org/10.1016/j.stem.2025.02.017
  23. Nat Commun. 2025 Mar 24. 16(1): 2749
      Red blood cell development from erythroid progenitors requires profound reshaping of metabolism and gene expression. How these transcriptional and metabolic alterations are coupled is unclear. Nprl3 (an inhibitor of mTORC1) has remained in synteny with the α-globin genes for >500 million years, and harbours most of the a-globin enhancers. However, whether Nprl3 serves an erythroid role is unknown. We found that while haematopoietic progenitors require basal Nprl3 expression, erythroid Nprl3 expression is further boosted by the α-globin enhancers. This lineage-specific upregulation is required for sufficient erythropoiesis. Loss of Nprl3 affects erythroblast metabolism via elevating mTORC1 signalling, suppressing autophagy and disrupting glycolysis. Broadly consistent with these murine findings, human NPRL3-knockout erythroid progenitors produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Therefore, we propose that the anciently conserved linkage of NprI3, α-globin and their associated enhancers has coupled metabolic and developmental control of erythropoiesis.
    DOI:  https://doi.org/10.1038/s41467-025-57683-z
  24. J Immunol. 2025 Mar 27. pii: vkaf026. [Epub ahead of print]
      Increased myeloid lineage production, termed myeloid skewing, leading to decreased tumor immunity, is a hallmark of aberrant hematopoiesis associated with cancer. It is believed that myeloid skewing may occur at the hematopoietic stem and progenitor cells (HSPCs) level to elicit hematopoietic changes. However, our understanding of the underlying molecular mechanisms remains incomplete. Here, we characterize the transcriptional and chromatin accessibility landscapes of bone marrow and splenic hematopoietic progenitors in the MMTV-PyMT mouse model of breast cancer using single-cell ATAC + RNA sequencing. We show that HSPCs in the bone marrow (BM) of the tumor-bearing mice show a modest upregulation of the myeloid-bias transcriptional signature without significant chromatin accessibility changes. By contrast, dendritic cell (DC) progenitors exhibit the most prominent transcriptional and chromatin changes, showing a signature of STAT3, CEBP, and non-DC myeloid gene activation. Compared to BM, splenic HSPCs exhibit a Notch signaling signature associated with erythroid commitment rather than further upregulation of the myeloid-bias signature. In addition, we also identify a cluster of splenic HSPCs in tumor-bearing animals with a transcriptional signature of mobilization. Our paired chromatin data suggest that AP-1 factors play a crucial role in driving this HSPC mobilization signature. Overall, we provide a comprehensive dataset for understanding the hematopoietic consequences of cancer.
    Keywords:  cancer; chromatin; hematopoiesis; single-cell multiome; transcription
    DOI:  https://doi.org/10.1093/jimmun/vkaf026
  25. Nat Commun. 2025 Mar 25. 16(1): 2883
      The Mediator complex facilitates interactions between transcription factors and RNA polymerase II, a process that is required for host gene transcription, including in response to viral infections. Among the many subunits in the Mediator complex, the MED25 subunit has been shown to be a target for viral activators during infection. Here we provide the molecular basis for the interaction between human respiratory syncytial virus (hRSV) nonstructural 1 protein (NS1) and the activator interaction domain (ACID) of MED25. The X-ray crystal structure of the complex revealed that NS1 straddles and binds two faces of MED25 ACID. This interaction is distinct from previously known viral activators. Importantly, our data support the conformational flexibility of viral transcriptional regulators. Furthermore, ChIP-seq and RNA-seq analysis identified the ATF3 transcription factor and a role for NS1/Mediator/ATF3 interaction in host gene regulation in hRSV infections. Our findings provide a molecular basis for hRSV NS1-based regulation of host gene transcription and reveal how viruses exploit the conformational heterogeneity at fuzzy transcription activator interfaces.
    DOI:  https://doi.org/10.1038/s41467-025-58216-4
  26. Nucleic Acids Res. 2025 Mar 20. pii: gkaf188. [Epub ahead of print]53(6):
      Effective transcriptional activation relies on the spatial interaction between specific DNA elements. DNA interactions have also been observed between DNA viruses and their hosts, with limited understanding of the involved details. Baculovirus is a representative species of DNA virus and has been reported to interact with the host genome in our previous study. However, the biological significance of the baculovirus-host trans-species DNA interaction and its underlying mechanisms remain elusive. Here, using Bombyx mori nucleopolyhedrovirus (BmNPV) as the model virus, we combine epigenome, transcriptome, and biochemical assays to investigate the baculovirus-host DNA interaction. Our data show that BmNPV hijacks the transcriptional regulatory capacity of host super-enhancers (SEs) by physically interacting with these regions on the host genome. This results in the usurpation of the activating capacity of an SE-binding transcription factor GATA by the virus, thereby impairing the SE-induced specific transcriptional activation of the target antiviral genes. Moreover, the hijacked regulatory capacity is spread on BmNPV genome through cis-interaction of viral DNA, leading to enhanced viral gene expression. Overall, our results provide novel insights into the intricate interplay of viruses with host gene expression regulatory networks and broaden the vision in the mechanisms of viral exploitation on cellular machinery.
    DOI:  https://doi.org/10.1093/nar/gkaf188
  27. Nucleic Acids Res. 2025 Mar 20. pii: gkaf173. [Epub ahead of print]53(6):
      Single-cell multi-omics methods enable the study of cell state diversity, which is largely determined by the interplay of the genome, epigenome, and transcriptome. Here, we describe Gtag&T-seq, a genome-and-transcriptome sequencing (G&T-seq) protocol of the same single cells that omits whole-genome amplification (WGA) by using direct genomic tagmentation (Gtag). Gtag drastically decreases the cost and improves coverage uniformity at single-cell and pseudo-bulk levels compared to WGA-based G&T-seq. We also show that transcriptome-based DNA copy number inference has limited resolution and accuracy, underlining the importance of affordable multi-omic approaches. Applying Gtag&T-seq to a melanoma xenograft model before treatment and at minimal residual disease revealed differential cell state plasticity and treatment response between cancer subclones. In summary, Gtag&T-seq is a low-cost and accurate single-cell multi-omics method that explores genetic alterations and their functional consequences in single cells at scale.
    DOI:  https://doi.org/10.1093/nar/gkaf173
  28. Genes Cells. 2025 Mar;30(2): e70016
      In eukaryotes, genomic DNA is stored in the nucleus as nucleosomes, in which a DNA segment is wrapped around a protein octamer consisting of two each of the four histones, H2A, H2B, H3, and H4. The core histones can be replaced by histone variants or altered with covalent modifications, contributing to the regulation of chromosome structure and nuclear activities. The formation of an octameric histone core in nucleosomes is widely accepted. Recently, the H3-H4 octasome, a novel nucleosome-like structure with a histone octamer consisting solely of H3 and H4, has been reported. CENP-A is the centromere-specific histone H3 variant and determines the position of kinetochore assembly during mitosis. CENP-A is a distant H3 variant sharing approximately 50% amino acid sequence with H3. In this study, we found that CENP-A and H4 also formed an octamer without H2A and H2B in vitro. We determined the structure of the CENP-A-H4 octasome at 3.66 Å resolution. In the CENP-A-H4 octasome, an approximately 120-base pair DNA segment was wrapped around the CENP-A-H4 octameric core and displayed the four CENP-A RG-loops, which are the direct binding sites for another centromeric protein, CENP-N.
    Keywords:  CENP‐A; centromere; cryo‐EM structure; histone; nucleosome
    DOI:  https://doi.org/10.1111/gtc.70016
  29. Sci Adv. 2025 Mar 28. 11(13): eadu8116
      The DNA methyltransferase 3B (DNMT3B) plays a vital role in shaping DNA methylation patterns during mammalian development. DNMT3B is intricately regulated by histone H3 modifications, yet the dynamic interplay between DNMT3B and histone modifications remains enigmatic. Here, we demonstrate that the PWWP (proline-tryptophan-tryptophan-proline) domain within DNMT3B exhibits remarkable dynamics that enhances the enzyme's methyltransferase activity upon interactions with a modified histone H3 peptide (H3K4me0K36me3). In the presence of H3K4me0K36me3, both the PWWP and ADD (ATRX-DNMT3-DNMT3L) domains transition from autoinhibitory to active conformations. In this active state, the PWWP domain most often aligns closely with the catalytic domain, allowing for simultaneous interactions with H3 and DNA to stimulate DNA methylation. The prostate cancer-associated DNMT3B R545C mutant is even more dynamic and susceptible to adopting the active conformation, resulting in aberrant DNA hypermethylation. Our study suggests the mechanism by which conformational rearrangements in DNMT3B are triggered by histone modifications, ultimately unleashing its activity in DNA methylation.
    DOI:  https://doi.org/10.1126/sciadv.adu8116
  30. Plant J. 2025 Mar;121(6): e70116
      Whole-genome duplication is an evolutionary force that drives speciation in all living kingdoms and is notably prevalent in plants. The evolutionary history of plants involved at least two genomic duplications that significantly expanded the plant morphology and physiology spectrum. Many important crops are polyploids, showing valuable features relative to morphological and stress response traits. After genome duplication, diploidization processes facilitate genomic adjustments to restore disomic inheritance. However, little is known about the chromatin changes triggered by nuclear DNA content alterations. Here, we report that synthetically induced genome duplication leads to chromatinization and significant changes in gene expression, resulting in a transcriptional landscape resembling a natural tetraploid. Interestingly, synthetic diploidization elicits only minor alterations in transcriptional activity and chromatin accessibility compared to the more pronounced effects of tetraploidization. We identified epigenetic factors, including specific histone variants, that showed increased expression following genome duplication and decreased expression after genome reduction. These changes may play a key role in the epigenetic mechanisms underlying the phenotypic complexity after tetraploidization in plants. Our findings shed light on the mechanisms that modulate chromatin accessibility remodeling and gene transcription regulation underlying plant genome adaptation in response to changes in genome size.
    Keywords:  ATAC‐seq; Arabidopsis thaliana; RNA‐seq; chromatin accessibility; gene transcription regulation; whole genome duplication; whole genome reduction
    DOI:  https://doi.org/10.1111/tpj.70116
  31. iScience. 2025 Mar 21. 28(3): 112057
      Cancer tissues are stiffer than normal tissues. Carcinogenesis stiffens the extracellular matrix (ECM) of cancerous tissues, to which cancer cells respond by activating transcription factors, such as YAP/TAZ, Twist1, and β-catenin, which further elevate their malignancy. However, these transcription factors are also expressed in normal tissues. Therefore, inhibiting these factors in order to treat cancer may lead to severe side effects. Here, we show that activating transcription factor 5 (ATF5), highly expressed in tumors, is activated by ECM stiffness and promotes the proliferation of cancer cells, including that of pancreatic cancer cells and lung cancer cells. In addition, ATF5 suppressed the expression of early growth response 1 (EGR1), thereby accelerating cancer cell proliferation. Stiff ECMs trigger the JAK-MYC pathway which activates ATF5. JAK activation was actomyosin independent whereas MYC induction was actomyosin dependent. These results demonstrate the critical role played by ATF5 in the mechanotransduction process seen in cancers.
    Keywords:  Biophysics; Cancer; Microenvironment
    DOI:  https://doi.org/10.1016/j.isci.2025.112057
  32. Nat Commun. 2025 Mar 21. 16(1): 2789
      Heat stress inhibits photosynthesis efficiency, thereby suppressing plant growth and crop yield. However, the mechanism underlying this inhibition is not fully understood. Here, we report that the multiple organellar RNA-editing factor 8 (MORF8) forms condensates with solid-like properties in chloroplasts upon heat stress. In vitro data show that the MORF8 condensation is intrinsically heat-dependent and primarily determined by its IDR (intrinsically disordered region). Purification and characterization of MORF8 condensates show that numerous editing factors including PPR proteins and MORFs are partitioned. We provide both genetic and biochemical evidence that MORF8 condensation inhibits chloroplast RNA editing. In agreement, we find that both heat stress and MORF8 condensation lead to reduced editing of RNAs encoding NADH dehydrogenase-like (NDH) complex and impaired NDH activity and photosynthesis efficiency. These findings uncover MORF8 as a putative chloroplastic thermosensor that mediates photosynthesis inhibition by heat and highlight the functional significance of solid material properties of biomolecular condensates.
    DOI:  https://doi.org/10.1038/s41467-025-58146-1
  33. Cell Rep. 2025 Mar 25. pii: S2211-1247(25)00245-1. [Epub ahead of print]44(4): 115474
      Melanoma cells can switch from a melanocytic and proliferative state to a mesenchymal and invasive state and back again. This plasticity drives intratumoral heterogeneity, progression, and therapeutic resistance. Microphthalmia-associated transcription factor (MITF) promotes the melanocytic/proliferative phenotype, but factors that drive the mesenchymal/invasive phenotype and the mechanisms that effect the switch between cell states are unclear. Here, we identify the MITF paralog, TFE3, and the non-canonical mTORC1 pathway as regulators of the mesenchymal state. We show that TFE3 expression drives the metastatic phenotype in melanoma cell lines and tumors. Deletion of TFE3 in MITF-low melanoma cell lines suppresses their ability to migrate and metastasize. Further, MITF suppresses the mesenchymal phenotype by directly or indirectly activating expression of FNIP1, FNIP2, and FLCN, which encode components of the non-canonical mTORC1 pathway, thereby promoting cytoplasmic retention and lysosome-mediated degradation of TFE3. These findings highlight a molecular pathway controlling melanoma plasticity and invasiveness.
    Keywords:  CP: Cancer; CP: Genomics; MITF; TFE3; cell plasticity; mTORC1; melanoma; metastasis; phenotype switching; protein stability
    DOI:  https://doi.org/10.1016/j.celrep.2025.115474
  34. Nature. 2025 Mar 26.
      Regulatory T (Treg) cells, which specifically express the master transcription factor FOXP3, have a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases1-3. Although stimulation of naive CD4+ T cells in the presence of TGFβ and IL-2 can induce FOXP3+ Treg cells in vitro (iTreg cells), the resulting cells are often unstable and have thus far hampered translational efforts4-6. A systematic approach towards understanding the regulatory networks that dictate Treg differentiation could lead to more effective iTreg cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalogue gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. We identify the RBPJ-NCOR repressor complex as a novel, context-specific negative regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iTreg differentiation and function, independent of canonical Notch signalling. Repeated cytokine and T cell receptor signalling stimulation in vitro revealed that RBPJ-deficient iTreg cells exhibit increased phenotypic stability compared with control cells through DNA demethylation of the FOXP3 enhancer CNS2, reinforcing FOXP3 expression. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of FOXP3 histone acetylation by HDAC3. Finally, RBPJ-ablated human iTreg cells more effectively suppressed xenogeneic graft-versus-host disease than control iTreg cells in a humanized mouse model. Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.
    DOI:  https://doi.org/10.1038/s41586-025-08795-5
  35. Genome Biol. 2025 Mar 23. 26(1): 68
       BACKGROUND: Fork-head box protein M1 (FOXM1) plays critical roles in development and progression of multiple cancers, including hepatocellular carcinoma (HCC). However, the exact regulatory hierarchy of FOXM1 remains unclear. Here, a genome-wide screen is performed to identify intranuclear proteins that promote FOXM1 transcription activity via liquid-liquid phase separation (LLPS).
    RESULTS: Abnormal spindle-like microcephaly associated (ASPM) is identified to interact with FOXM1 protein via LLPS and enhance its stability by preventing proteasome-mediated degradation. ChIP-sequencing data show ASPM and FOXM1 co-occupy the promoters of multiple genes to promote their transcription, enhancing FOXM1-driven oncogenic progression. In functional experiments, inhibition of ASPM suppresses tumor growth of HCC cells in vivo and in vitro, while overexpression of ASPM has opposite effects. Importantly, reconstitution of FOXM1 partially compensates for the weakened proliferative capacity of HCC cells caused by ASPM silencing. Intriguingly, FOXM1 binds to the promoter region of ASPM and transcriptionally activates ASPM expression in HCC cells. Furthermore, we find that a higher co-expression of ASPM and FOXM1 significantly correlates with poor prognosis in HCC patients. It indicates a double positive feedback loop between ASPM and FOXM1 which coordinately promotes the aggressive progression of HCC.
    CONCLUSIONS: Collectively, we demonstrate that LLPS and transcriptional regulation form an oncogenic double positive feedback loop between ASPM and FOXM1. This provides a rationale strategy to treat HCC by targeting this mechanism.
    Keywords:  Double positive feedback loop; FOXM1; Human hepatocellular carcinoma; Liquid–liquid phase separation; Protein degradation
    DOI:  https://doi.org/10.1186/s13059-025-03526-5
  36. Nat Genet. 2025 Mar 24.
      N6-methyladenosine (m6A), the most abundant internal RNA modification in humans, regulates most aspects of RNA processing. Prostate cancer is characterized by widespread transcriptomic dysregulation; therefore, we characterized the m6A landscape of 162 localized prostate tumors with matched DNA, RNA and protein profiling. m6A abundance varied dramatically across tumors, with global patterns emerging via complex germline-somatic cooperative regulation. Individual germline polymorphisms regulated m6A abundance, cooperating with somatic mutation of cancer driver genes and m6A regulators. The resulting complex patterns were associated with prognostic clinical features and established the biomarker potential of global and locus-specific m6A patterns. Tumor hypoxia dysregulates m6A profiles, bridging prior genomic and proteomic observations. Specific m6A sites, such as those in VCAN, drive disease aggression, associating with poor outcomes, tumor growth and metastasis. m6A dysregulation is thus associated with key events in the natural history of prostate cancer: germline risk, microenvironmental dysregulation, somatic mutation and metastasis.
    DOI:  https://doi.org/10.1038/s41588-025-02128-y
  37. PLoS Genet. 2025 Mar 28. 21(3): e1011635
      Dynamic gene expression is crucial for mammalian organ development, influencing organ-specific functions and responses. A significant number of mammalian protein-coding genes are regulated by multiple distinct promoters, suggesting that the choice of promoter is as critical as its transcriptional output. However, the role of alternative promoters in organ development remains largely unexplored. In this study, we utilized RNA-seq data from 313 mouse samples across various developmental stages in seven major organs to identify active promoters. Our analyses revealed between 967 and 3,237 developmentally dynamic promoters (DDPs) in each organ. These DDPs encompass not only major promoters with the highest activity within a gene but also alternative promoters with lower activity, which are often overlooked in traditional gene-level analyses. Notably, we found that alternative DDPs can be independently regulated compared to their major counterparts, suggesting the involvement of unique transcriptional regulatory mechanisms. Furthermore, we observed that increased alternative promoter usage plays a pivotal role in driving organ-specific functions and gene expression alterations. Our findings underscore the importance of alternative promoter usage in shaping organ identity and function, providing new insights into the regulatory complexity of organogenesis.
    DOI:  https://doi.org/10.1371/journal.pgen.1011635
  38. Genome Biol. 2025 Mar 26. 26(1): 70
       BACKGROUND: Mitochondrial DNA (mtDNA) variants hold promise as endogenous barcodes for tracking human cell lineages, but their efficacy as reliable lineage markers are hindered by the complex dynamics of mtDNA in somatic tissues.
    RESULTS: Here, we use computational modeling and single-cell genomics to thoroughly interrogate the origin and clonal dynamics of mtDNA variants across various biological settings. Our findings reveal that the majority of mtDNA variants which are specifically present in a cell subpopulation, termed subpopulation-specific variants, are pre-existing heteroplasmies in the first cell instead of de novo somatic mutations during divisions. Moreover, subpopulation-specific variants demonstrate limited discriminatory power among different genuine lineages under weak clonal expansion; however, certain subpopulation-specific variants with consistently high frequencies among a subpopulation are capable of faithfully labeling cell lineages in scenarios of stringent clonal expansion, such as strongly expanded T cell populations in diseased conditions and clonal hematopoiesis in aged individuals. Inspired by our simulations, we introduce a lineage informative score, facilitating the identification of reliable mitochondrial lineage tracing markers across different modalities of single-cell genomic data.
    CONCLUSIONS: Combining computational modeling and single-cell sequencing, our study reveals that the performance of mitochondrial lineage tracing is highly dependent on the extent of clonal expansion, which thus should be considered when applying mitochondrial lineage tracing.
    Keywords:  Clonal dynamics; Lineage tracing; Single-cell genomics; mtDNA variants
    DOI:  https://doi.org/10.1186/s13059-025-03540-7