eLife: latest articles by subject

Linking germline telomere removal to global programmed DNA elimination in Tetrahymena genome differentiation

kazufumi.mochizuki@igh.cnrs.fr (Alix Lemoine) — Tue, 14 Jul 2026 00:00:00 +0000

In the ciliate Tetrahymena, telomeres of the germline micronucleus (MIC) are removed and replaced by de novo telomere addition during somatic macronuclear (MAC) development. In this study, we investigated the kinetics and mechanism of the MIC telomere elimination. Comparison of the MIC and MAC genome sequences indicated that the MIC telomeres are excised from chromosomes as part of larger MIC-limited sequences (MLSs) through chromosomal breakage. We confirmed this using an optimized oligo-FISH protocol and found that their elimination occurs in parallel with other programmed DNA elimination processes. CRISPR-Cas9 disruption of a MLS-associated Chromosome Breakage Sequence (CBS) showed that elimination of the MLS was not blocked but instead led to loss of its adjacent MAC-destined sequence (MDS), suggesting abnormal co-elimination. In biparental crosses of the CBS mutant, however, both MLS and MDS were retained, DNA elimination was broadly disrupted, and no viable progeny were produced. These findings indicate that chromosome breakage at MLS-associated CBSs is essential for the proper separation of MLSs and MDSs, ensuring correct DNA elimination and successful sexual progeny development. We propose that the MIC telomere elimination is subsumed within the broader process of programmed DNA elimination.

Ligand-dependent enhancer activation indirectly modulates non-target promoters in a chromatin domain

aprotim@tifrh.res.in (Aprotim Mazumder) — Tue, 07 Jul 2026 00:00:00 +0000

Transcription activation of genes by estrogen is driven by enhancers, which are often located within the same topologically associating domain (TAD) as non-targeted promoters. We investigated how acute enhancer-driven activation affects neighbouring non-target genes within the same TAD. Using single-molecule RNA FISH (smFISH), we tracked the transcription of TFF1 (enhancer-target gene) and TFF3 (non-target gene) during estrogen stimulation. We observed mutually exclusive expression patterns: TFF1 expression peaked at 1 hr, while TFF3 reached its peak at 3 hr after TFF1 activation had diminished. Chromatin looping data indicated that the enhancer loops with the TFF1 gene but not TFF3, suggesting that TFF3 upregulation is not due to direct enhancer-promoter interactions. CRISPR deletion of the enhancer affected TFF1 transcription more acutely than TFF3. 1,6-hexanediol (HD) exposure suggested that the TFF1 enhancer:promoter undergoes a potential ERα-mediated condensate formation, which sequesters the transcriptional machinery and inhibits TFF3 expression. As estrogen signaling fades at 3 hr, TFF1 expression declines while TFF3 expression increases. Our findings reveal that enhancer-driven activation can indirectly repress neighboring genes within the same TAD, highlighting a dynamic shift in gene expression as signaling progresses.

Impacts of DNA methylation on H2A.Z deposition and nucleosome stability

funabih@rockefeller.edu (Hide A Konishi) — Tue, 07 Jul 2026 00:00:00 +0000

The histone variant H2A.Z and DNA methylation are enriched at mutually exclusive genomic segments, though its mechanistic bases remain unclear. Here, we examine DNA methylation’s influence on the intrinsic stability of the H2A.Z nucleosome and chaperone-mediated H2A.Z deposition. Cryo-EM and endonuclease analyses suggest that DNA methylation subtly increases the openness and accessibility of the H2A.Z nucleosome on satellite II-derived DNA sequences. In transcriptionally silent Xenopus egg extracts, H2A.Z preferentially associates with unmethylated DNA though a substantial proportion of H2A.Z is recruited to methylated DNA. Preferential H2A.Z deposition to unmethylated DNA depends on the SRCAP complex, whose DNA binding is suppressed by methylation, while an SRCAP-independent and DNA methylation-insensitive mechanism for H2A.Z deposition also exists. Altogether, we propose that SRCAP drives the biased association of H2A.Z to unmethylated DNA, while additional mechanisms, potentially taking advantage of the subtle DNA methylation-induced physical effects, further assist the exclusion of H2A.Z from methylated DNA.

Genome reorganization and its functional impact during breast cancer progression

Gary.Stein@med.uvm.edu (Andrew Fritz) — Tue, 16 Jun 2026 00:00:00 +0000

Cancer progression involves extensive alterations in epigenetic and gene expression programs, but the accompanying changes in higher-order genome organization remain less well understood. Using high-resolution Micro-C mapping in the MCF10 cell model of breast cancer, we profiled chromatin compartments, topologically associated domains, and chromatin loops. We find large-scale compartmental shifts occur predominantly in early stages of cancer development, with more fine-scale structural changes in topologically associating domains and loops accumulating during the later transition to metastasis. Relating these chromatin features to gene expression and enhancer-associated histone marks revealed that many differentially expressed genes are physically connected to distal regulatory elements. While enhancer–promoter contact frequency and distal enhancer activity correlated with gene expression, strong changes in chromatin looping were relatively infrequent during progression, suggesting that alterations in chromatin contacts are not globally necessary, but may facilitate gene regulation at a subset of genes. These results elucidate the connection between gene regulation and genome remodeling in a cell-based cancer progression model.

The DBD-α4 helix of EWSR1::FLI1 is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma

emily.theisen@nationwidechildrens.org (Ariunaa Bayanjargal) — Mon, 15 Jun 2026 00:00:00 +0000

Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI1, DBD-α4 helix, is critical for the transcription function of EWSR1::FLI1. In this study, we aimed to understand the mechanism by which the DBD-α4 helix promotes transcription and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatin marks, genome binding, and gene expression in cells expressing EWSR1::FLI1 constructs with and without the DBD-α4 helix. Our studies revealed DBD-α4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1, such as formation of topologically associated domains (TADs), chromatin loops, enhancers, and productive transcription hubs.

Contractile perinuclear actomyosin network promotes peripheral and polar chromosome interaction with the mitotic spindle

j.k.eykelenboom@dundee.ac.uk (Alexander JR Booth) — Thu, 11 Jun 2026 00:00:00 +0000

Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation in anaphase. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction immediately after nuclear envelope breakdown (NEBD) facilitates chromosome interaction with the mitotic spindle, the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery or behind spindle poles toward the center of cells. Detailed tracking of kinetochore movements following light-induced activation of a myosin II inhibitor reveals that this inward movement of chromosomes facilitates kinetochores’ initial interaction with spindle microtubules. It also promotes the onset of kinetochores’ congression toward the spindle mid-plane, but not congression itself once it starts. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Since some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, the absence of PANEM may contribute to numerical chromosomal instability in these cells.

Uncoupling the TFIIH Core and Kinase Modules leads to misregulated RNA polymerase II CTD Serine 5 phosphorylation

steveb@hms.harvard.edu (Célia Jeronimo) — Mon, 08 Jun 2026 00:00:00 +0000

TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked in Saccharomyces cerevisiae by the subunit Tfb3 (MAT1 in metazoans). The Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The Kinase Module phosphorylates the C-terminal domain (CTD) of RNApII subunit Rpb1, initiating a cycle of CTD modifications that coordinate the exchange of initiation and elongation factors. Why these two disparate activities are bundled into one factor is not obvious, but the connection may provide temporal coordination during early initiation. When Tfb3 is split into two parts to uncouple the TFIIH modules, the resulting cells are viable but grow very slowly. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that later became connected by Tfb3 during early eukaryotic evolution.

Interplay between cohesin and TORC1 links chromosome segregation and gene expression to environmental changes

jpaul.javerzat@ibgc.cnrs.fr (Adèle L Marston) — Mon, 01 Jun 2026 00:00:00 +0000

Cohesin is a DNA tethering complex essential for chromosome structure and function. In fission yeast, defects in the cohesin loader Mis4 result in chromosome segregation defects and dysregulated expression of genes near chromosome ends. A genetic screen for suppressors of the thermosensitive growth defect of mis4-G1487D identified several hypomorphic mutants of the Target of Rapamycin Complex 1 (TORC1), a conserved kinase that integrates cellular signals to regulate growth and metabolism through substrate-specific phosphorylation. Here, we demonstrate that the TORC1 pathway modulates cohesin functions in chromosome segregation and gene expression. In the context of compromised cohesin loading, the incidence of chromosome segregation defects was modulated by the growth medium in a TORC1-dependent manner. Pharmacological or genetic downregulation of TORC1 activity restored cohesin binding to its chromosomal sites and improved mitotic chromosome segregation. Notably, reduced TORC1 activity also increased cohesin binding and chromosome transmission fidelity in wild-type cells. These results suggest that environmental cues influence chromosome stability via TORC1. Biochemically, TORC1 co-purified with cohesin and reduced TORC1 activity correlated with decreased phosphorylation of specific residues on Mis4 and cohesin. Mutations in cohesin that mimic the non-phosphorylated state mirrored the effects of TORC1 downregulation, showing that TORC1 is part of the network that controls cohesin phosphorylation to modulate its functions. Finally, we show that the functional interaction between TORC1 and Mis4 extends to the regulation of stress-responsive genes. Our findings reveal a TORC1–cohesin link that may facilitate cellular adaptation to environmental changes. Given that TORC1 inhibitors and calorie restriction extend lifespan in diverse species, this connection raises the intriguing possibility that cohesin-mediated changes in chromosome structure contribute to these effects.

MORC2 mediates transcriptional regulation through liquid-liquid phase separation

huangcd@ustc.edu.cn (Chao Wang) — Wed, 20 May 2026 00:00:00 +0000

MORC2 is a chromatin-associated ATPase essential for transcriptional silencing and genome stability, yet the biophysical principles governing its regulatory activity remain elusive. Here, we demonstrate that full-length MORC2 undergoes biomolecular condensation to form dynamic nuclear assemblies, a process fundamentally required for its repressor function. Endogenous MORC2 forms discrete, dynamic condensates in neurons from Morc2a^EGFP chimeric mice, supporting the physiological relevance of these assemblies in vivo. Mechanistically, a 3.1 Å crystal structure of coiled-coil 3 (CC3) identifies a dimeric scaffold that serves as a structural hub, while multivalent ‘sticker’ interactions between an intrinsically disordered region (IDR) and a newly defined IDR-binding domain (IBD) drive condensation. We show that DNA acts as a molecular scaffold that triggers MORC2 condensation, which in turn allosterically stimulates its ATPase activity. Critically, by employing a ‘killswitch’ strategy to decouple assembly from internal fluidity, we reveal that only dynamic MORC2 condensates, not static aggregates or condensation-deficient mutants, can restore transcriptional regulation in MORC2-knockout cells. Furthermore, pathogenic variants linked to CMT2Z and SMA differentially perturb these material properties and enzymatic turnover, providing a mechanistic link between condensate dysregulation and human neuropathies. Together, our findings establish a DNA-templated condensation mechanism for MORC2 and provide a molecular framework for understanding how the material state of chromatin-associated machinery dictates gene regulation and disease pathogenesis.

Lineage priming and cell type proportioning depends on the interplay between stochastic and deterministic factors

christopher.thompson@ucl.ac.uk (Catherine Pears) — Tue, 19 May 2026 00:00:00 +0000

Isogenic cells can break symmetry and adopt different fates, even when exposed to a seemingly identical environment. This deeply conserved phenomenon allows unicellular organisms to pre-empt dynamically changing environments and is central to the evolution of multicellularity. It is thought that cells are primed towards different lineages by cell-cell variation, although the underlying mechanisms are poorly understood. To address this, we exploit the tractability of the social amoeba Dictyostelium discoideum, where cell fate choice also does not depend on spatial cues. We develop and test a model to explain quantitative experimental single-cell observations of probabilistic differentiation. The model suggests that cell cycle position affects lineage choice, as previously shown but that stochastic cell-cell variation also plays a key role. Single cell sequencing reveals genes that exhibit cell type-specific expression or genes that affect fate choice exhibit extensive stochastic cell-cell expression variation. Like lineage priming genes in ESCs, they are associated with H3K4 methylation, which when perturbed affects their expression and disrupt fate choice. We suggest the integration of stochastic and deterministic inputs represents an adaptive mechanism to increase developmental robustness against perturbations that affect deterministic signals.

Single-domain antibody inhibitors target the coiled coil arms of the Bacillus subtilis SMC complex

stephan.gruber@unil.ch (Lea M Huber-Hürlimann) — Tue, 19 May 2026 00:00:00 +0000

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane proteins. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the 14 disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the SMC-DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells.

Identification of the regulatory elements and protein substrates of lysine acetoacetylation

yzheng@uga.edu (Bhoj Kumar) — Thu, 14 May 2026 00:00:00 +0000

Short-chain fatty acylations establish connections between cell metabolism and regulatory pathways. Lysine acetoacetylation (Kacac) was recently identified as a new histone mark. However, regulatory elements, substrate proteins, and epigenetic functions of Kacac are not yet fully understood, hindering further in-depth understanding of acetoacetate-modulated (patho)physiological processes. Here, we created a chemo-immunological approach for reliable detection of Kacac, and demonstrated that acetoacetate serves as the primary precursor for histone Kacac. We report the enzymatic addition of the Kacac mark by the acyltransferases GCN5, p300, and PCAF, and its removal by the deacetylase HDAC3. Furthermore, we establish acetoacetyl-CoA synthetase as a key regulator of cellular Kacac levels. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA sequencing data reveal the broad impacts of Kacac on multifaceted cellular processes. These findings unveil pivotal regulatory mechanisms for the acetoacetate-mediated Kacac pathway, opening a new avenue for further investigation into ketone body functions in various pathophysiological states.

The long non-coding RNA Dreg1 is required for optimal ILC2 development

ajith.vasanthakumar@petermac.org (Adelynn Tang) — Wed, 13 May 2026 00:00:00 +0000

Gata3 is an essential transcription factor for the development of several distinct immune cell lineages such as T cells, natural killer (NK) cells, and innate lymphoid cells (ILCs). As such, the levels and timing of Gata3 expression are critical for directing lineage fate decisions. The Gata3 locus has a complex and dynamic distal regulatory enhancer landscape. Recently, we identified a non-coding RNA, Dreg1, located immediately upstream of the classic +280 kb T/NK cell enhancer (Tce1). To test its function, we excised the Dreg1 locus in mice and observed a selective reduction of group 2 ILCs (ILC2) across multiple tissues, but mature T, NK, and other ILC lineages remained unchanged. In bone marrow, common innate lymphoid cell progenitors (ILCPs) increased while ILC2 progenitors (ILC2P) decreased, with a modest reduction of Gata3 in upstream progenitors consistent with an early developmental bottleneck. Chromatin profiling showed the Dreg1 locus is accessible in early lymphoid progenitors and became decorated with H3K27ac in ILCP in a Tcf1-dependent manner. Furthermore, Tcf1-deficient cells did not express Dreg1 and showed alterations in the epigenetic landscape of the Dreg1 locus. Finally, we discovered that potential homologues of Dreg1 harboured in a syntenic enhancer of GATA3 are also highly expressed in human ILC2. Taken together, we conclude that Dreg1 is a Tcf1-dependent non-coding RNA critical for fine tuning the high level of Gata3 required for the optimal development of the ILC2 lineage.

Real-time transcriptomic profiling in distinct experimental conditions

buttamer@uni-mainz.de (Anna Wierczeiko) — Tue, 05 May 2026 00:00:00 +0000

Nanopore technology offers real-time sequencing opportunities, providing rapid access to sequenced data and allowing researchers to manage the sequencing process efficiently, resulting in cost-effective strategies. Here, we present focused case studies demonstrating the versatility of real-time transcriptomics analysis in rapid quality control for long-read RNA-seq. We illustrate its utility through four experimental setups: (1) transcriptome profiling of distinct human cellular populations, (2) identification of experimentally enriched transcripts, (3) transcriptional analysis of cells under heat shock conditions, and (4) identification of experimentally manipulated genes (knockout and overexpression) in several yeast strains. We show how to perform multiple layers of quality control as soon as sequencing has started, addressing both the quality of the experimental and sequencing traits. Real-time quality control measures assess sample/condition variability and determine the number of identified genes per sample/condition. Furthermore, real-time differential gene/transcript expression analysis can be conducted at various time points post-sequencing initiation (PSI), revealing dynamic changes in gene/transcript expression between two conditions. Using real-time analysis, which occurs in parallel to the sequencing run, we identified differentially expressed genes/transcripts as early as 1 hr PSI. These changes were consistently observed throughout the entire sequencing process. We discuss the new possibilities offered by real-time data analysis, which have the potential to serve as a valuable tool for rapid and cost-effective quality checks in specific experimental settings and can be potentially integrated into clinical applications in the future.

Evidence of off-target probe binding affecting 10x Genomics Xenium gene panels compromise accuracy of spatial transcriptomic profiling

jeanfan@jhu.edu (Caleb Hallinan) — Fri, 01 May 2026 00:00:00 +0000

The accuracy of spatial gene expression profiles generated by probe-based in situ spatially resolved transcriptomic technologies depends on the specificity with which probes bind to their intended target gene. Off-target binding, defined as a probe binding to something other than the target gene, can distort a gene’s true expression profile, making probe specificity essential for reliable transcriptomics. Here, we investigated off-target binding affecting the 10x Genomics Xenium technology. We developed a software tool, Off-target Probe Tracker (OPT), to identify putative off-target binding via alignment of probe target sequences and assessing whether mapped loci corresponded to the intended target gene across multiple reference annotations. Applying OPT to a Xenium human breast gene panel, we identified at least 14 out of the 313 genes in the panel potentially impacted by off-target binding to protein-coding genes. To substantiate our predictions, we leveraged a Xenium breast cancer dataset generated using this gene panel and compared results to orthogonal spatial and single-cell transcriptomic profiles from Visium CytAssist and 3′ single-cell RNA-seq derived from the same tumor block. Our findings indicate that for some genes, the expression patterns detected by Xenium demonstrably reflect the aggregate expression of the target and predicted off-target genes based on Visium and single-cell RNA-seq, rather than the target gene alone. We further applied OPT to identify potential off-target binding in custom gene panels and integrate tissue-specific RNA-seq data to assess effects. Overall, this work enhances the biological interpretability of spatial transcriptomics data and improves reproducibility in spatial transcriptomics research.

TAD boundaries and gene activity are uncoupled

mistelit@mail.nih.gov (Adib Keikhosravi) — Wed, 15 Apr 2026 00:00:00 +0000

Topologically associating domains (TADs) are prominent features of genome organization. A proposed function of TADs is to contribute to gene regulation by promoting chromatin interactions within a TAD and by suppressing interactions between TADs. Here, we directly probe the structure-function relationship of TADs by simultaneously assessing the behavior of TAD boundaries and gene activity at the single-cell and -allele level using high-throughput imaging. We find that while TAD boundaries pair more frequently than non-boundary regions, these interactions are infrequent and are uncorrelated with transcriptional activity of genes within the TAD. Similarly, acute global transcriptional inhibition or gene-specific activation does not alter TAD boundary proximity. Furthermore, while loss of the cohesin component RAD21 alters gene activity, disruption of TAD boundaries by depletion of the architectural chromatin protein CTCF is insufficient to alter expression of genes within the TAD. These results suggest that TAD boundary architecture and gene activity are largely uncoupled.

Epigenetics and chromatin structure regulate var2csa expression and the placental-binding phenotype in Plasmodium falciparum

karine.leroch@ucr.edu (Hannes Hoppe) — Wed, 15 Apr 2026 00:00:00 +0000

Plasmodium falciparum is responsible for what appears to be a never-ending public health issue in the developing world. With repeated infections, a gradual semi-immunity to severe malaria can be acquired, but this is disrupted when women become pregnant as the parasite cytoadheres in the placenta to prevent splenic clearance. This change in tissue tropism is due to specific transcription of the antigenically variable adhesin VAR2CSA. To better understand the molecular mechanisms activating var2csa and antigenic variation overall, we used a combination of phenotypic and systems biology assays. We first established phenotypically homogenous populations of VAR2CSA-expressing and placenta-binding parasites that were shown to exclusively transcribe var2csa while all other var genes remained silenced. We also confirmed that the transcriptional activation was strongly associated with distinct depletion of repressive H3K9me3 marks. Further, we used chromatin conformation capture as a high-resolution approach to determine interchromosomal interactions and established that transcriptional activation is linked to a small yet significant repositioning of var2csa relative to heterochromatic telomeric clusters. Lastly, we demonstrated that occupancy of 5-methylcytosine was present in all var genes but independent of transcriptional repression and switching. All together, these findings provide insights at high resolution into the potential role of 5-methylcytosine in P. falciparum and increase our understanding of the mechanisms regulating antigenic variation at the epigenetics and chromatin structure level.

Single-cell RNA-seq reveals trans-sialidase-like superfamily gene expression heterogeneity in Trypanosoma cruzi populations

psmircich@fcien.edu.uy (Gabriel Rinaldi) — Tue, 07 Apr 2026 00:00:00 +0000

Trypanosoma cruzi, the causative agent of Chagas disease, presents a major public health challenge in Central and South America, affecting approximately 8 million people and placing millions more at risk. The T. cruzi life cycle includes transitions between epimastigote, metacyclic trypomastigote, amastigote, and blood trypomastigote stages, each marked by distinct morphological and molecular adaptations to different hosts and environments. Unlike other trypanosomatids such as Trypanosoma brucei, T. cruzi does not employ a monoallelic model of antigenic variation; instead, it relies on a diverse repertoire of cell-surface associated proteins encoded by large multigene families, which are essential for infectivity and immune evasion. This study analyzes cell-specific transcriptomes using single-cell RNA sequencing of amastigote and trypomastigote cells to characterize stage-specific surface protein expression during mammalian infection. Through clustering and identification of cell-specific markers, we assigned cells to distinct parasite developmental forms. Analysis of individual cells revealed that surface protein-coding genes, especially members of the trans-sialidase-like superfamily (TcS), are expressed with greater heterogeneity than single-copy genes. Moreover, no recurrent combinations of TcS genes were observed between individual cells in the population. Remarkably, a small subset of TcS mRNAs, encoded by genes preferentially located in the core genomic compartment, are frequently detected across the cell population, whereas the vast majority of TcS mRNAs show low detection frequencies and are mainly encoded in the disruptive compartment. Our findings thus reveal transcriptomic heterogeneity within trypomastigote populations where each cell displays unique TcS expression profiles. Focusing on the diversity of surface protein expression, this research aims to deepen our understanding of T. cruzi cellular biology and infection strategies.

DNA O-MAP uncovers the molecular neighborhoods associated with specific genomic loci

beliveau@uw.edu (Ashley F Tsue) — Tue, 07 Apr 2026 00:00:00 +0000

The accuracy of crucial nuclear processes such as transcription, replication, and repair depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA–protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed ‘DNA O-MAP’, which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with label-free or sample multiplexed quantitative proteomics, targeted chemical perturbations, and next-generation sequencing to quantify DNA-proximal proteins and DNA–DNA interactions at specific genomic loci in human and murine cells. Furthermore, we establish that DNA O-MAP is applicable to both repetitive and unique genomic loci of varying sizes, from kilobase HOX gene clusters to megabase alpha-satellite repeats, and that DNA O-MAP can measure proximal molecular effectors in a homolog-specific manner.

UV irradiation alters TFAM binding specificity and compaction of DNA

derie@unc.edu (Alex George) — Wed, 25 Mar 2026 00:00:00 +0000

Mitochondria lack nucleotide excision repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids and binds differentially to certain forms of DNA damage. As such, TFAM has emerged as a candidate for protecting mtDNA or sensing damage. To examine the possibilities that TFAM might protect DNA from damage or act as a damage sensing protein for irreparable forms of mtDNA damage, we used live-cell imaging and HeLa cell-based assays, atomic force microscopy (AFM), and high-throughput protein-DNA binding assays to characterize the binding properties of human TFAM to ultraviolet-C (UVC) irradiated DNA and the cellular consequences of UVC irradiation. Our cell data show increased TFAM mRNA after exposure and suggest an increase in mtDNA degradation without a loss in mitochondrial membrane potential that might trigger mitophagy. Our protein-DNA binding assays indicate a reduction in sequence specificity of TFAM following UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA in vitro, we do not observe any protective effect of increased TFAM protein on DNA damage formation in cells or in vitro. Increased TFAM protein did not alter levels of mtDNA damage over time after UVC exposure in vivo, but knockdown of TFAM did alter mtDNA damage levels in HeLa cells both at baseline and after UVC exposure. Taken together, these studies indicate that UVC-induced DNA damage alters TFAM binding and promotes compaction by TFAM in vitro. We hypothesize that TFAM may act as a damage sensing protein in vivo, sequestering damaged genomes to prevent mutagenesis by facilitating removal or suppression of replication.

Dietary sulfur amino acid restriction elicits a cold-like transcriptional response in inguinal but not epididymal white adipose tissue of male mice

pmr96@cornell.edu (Aylin S Gueller) — Tue, 17 Mar 2026 00:00:00 +0000

About 1 billion people are living with obesity worldwide. GLP-1-based drugs have massively transformed care, but long-term consequences are unclear in part due to reductions in energy expenditure with ongoing use. Diet-induced thermogenesis (DIT) and cold exposure (CE) raise EE via brown adipose tissue (BAT) activation and beiging of white adipose tissue (WAT). Methionine restriction (MetR) is a candidate DIT stimulus, but its EE effect has not been benchmarked against CE, nor have their tissue-level interactions been defined. In a 2×2 design (Control vs. MetR; room temperature, RT: 22°C vs. CE: 4°C for 24 hr), we used male C57BL/6 N mice to benchmark MetR-induced thermogenesis against CE and mapped how diet and temperature interact across tissues. Bulk RNA-seq profiled liver, iBAT, iWAT, and eWAT. Differential expression was modeled with main effects and a diet × temperature interaction. KEGG GSEA was used to assess pathway-level enrichment. MetR increased EE at RT and shifted fuel use towards lipid oxidation, supporting MetR as a bona fide DIT stimulus. CE elevated EE across diets and blunted diet differences. Transcriptomic responses were tissue-specific: in liver, CE dominated gene induction while MetR and CE cooperatively repressed genes. The combination enriched glucagon/AMPK-linked and core metabolic pathways. In iBAT, CE dominated thermogenic and lipid-oxidation programs with minimal MetR contribution. In iWAT, MetR and CE acted largely additively with high concordance, enhancing fatty-acid degradation, PPAR signaling, thermogenesis, and TCA cycle pathways. In eWAT, robust co-dependent and synergistic differential expression emerged only with MetR+CE. MetR is a genuine DIT stimulus that remodels metabolism in a tissue-specific manner. Our study provides a tissue-resolved transcriptomic resource that benchmarks diet-induced (MetR) against cold-induced thermogenesis and maps their interactions across liver, iBAT, iWAT, and eWAT.

p53-induced RNA-binding protein ZMAT3 inhibits transcription of a hexokinase to suppress mitochondrial respiration in human cancer cells

ashish.lal@nih.gov (Ashish Lal) — Tue, 17 Mar 2026 00:00:00 +0000

The tumor suppressor p53 is a transcription factor that controls the expression of hundreds of genes. Emerging evidence indicates that the p53-induced RNA-binding protein ZMAT3 acts as a key splicing regulator that contributes to p53-dependent tumor suppression in vitro and in vivo. However, the mechanism by which ZMAT3 functions within the p53 pathway remains largely unclear. Here, we discovered a function of ZMAT3 in inhibiting transcription of HKDC1, a hexokinase that regulates glucose metabolism and mitochondrial respiration in human cancer cells. Quantitative proteomics revealed HKDC1 as the most significantly upregulated protein in ZMAT3-depleted colorectal cancer cells. ZMAT3 depletion resulted in increased mitochondrial respiration, which was rescued by simultaneous depletion of HKDC1, suggesting that HKDC1 is a critical downstream effector of ZMAT3. Unexpectedly, ZMAT3 did not bind to HKDC1 RNA or DNA; however, proteomic analysis of the ZMAT3 interactome identified its interaction with the oncogenic transcription factor JUN. ZMAT3 depletion enhanced JUN binding to the HKDC1 locus, leading to increased HKDC1 transcription that was rescued upon JUN depletion, suggesting that JUN activates HKDC1 transcription in ZMAT3-depleted cells. Collectively, these findings uncover a mechanism by which ZMAT3 regulates transcription through JUN and demonstrate that HKDC1 is a key component of the ZMAT3-regulated transcriptome in the context of mitochondrial respiration regulation.

EPB41L4A-AS1 long noncoding RNA acts in both cis- and trans-acting transcriptional regulation and controls nucleolar biology

igor.ulitsky@weizmann.ac.il (Alan Monziani) — Tue, 10 Mar 2026 00:00:00 +0000

Mammalian genomes are pervasively transcribed into long noncoding RNAs (lncRNAs), whose functions and modes of action remain poorly understood. EPB41L4A-AS1 is an evolutionarily conserved, broadly and highly expressed lncRNA that produces the H/ACA snoRNA SNORA13 from one of its introns. We studied the consequences of EPB41L4A-AS1 perturbation in breast cancer cells and found that it acts both in cis, to enhance transcription of the proximal EPB41L4A gene and additional genes in its two flanking topologically associated domains, and in trans by broadly regulating gene expression, including expression of snoRNAs, transcription of genes involved in nucleolar biology and the distribution of nucleolar proteins. These effects are phenocopied by the loss of SUB1, an interactor of EPB41L4A-AS1, and are observed following transient perturbations of EPB41L4A-AS1 that do not affect steady-state SNORA13 levels or the rRNA modification it helps install. Exogenous expression of the full-length EPB41L4A-AS1 locus but not SNORA13 expression can rescue the trans-acting transcriptional effects of its perturbation. The EPB41L4A-AS1 gene is thus a versatile locus producing RNA molecules acting on multiple levels for key cellular functions.

Defining the chromatin-associated protein landscapes on Trypanosoma brucei repetitive elements using synthetic TALE proteins

keith.matthews@ed.ac.uk (Christos Spanos) — Tue, 10 Mar 2026 00:00:00 +0000

Kinetoplastids, such as Trypanosoma brucei, are eukaryotes that likely separated from the main lineage at an exceptionally early point in evolution. Consequently, many aspects of kinetoplastid biology differ significantly from other eukaryotic model systems, including yeasts, plants, worms, flies, and mammals. As in many eukaryotes, the T. brucei genome contains repetitive elements at various chromosomal locations, including centromere- and telomere-associated repeats and interspersed retrotransposon elements. T. brucei also contains intermediate-sized and mini-chromosomes that harbour abundant 177 bp repeat arrays and 70 bp repeat elements implicated in Variable Surface Glycoprotein (VSG) gene switching. In many eukaryotes, repetitive elements are assembled in specialised chromatin such as heterochromatin; however, apart from centromere- and telomere-associated repeats, little is known about chromatin-associated proteins that decorate these and other repetitive elements in kinetoplastids. Here, we utilise affinity selection of synthetic TALE DNA binding proteins designed to target specific repeat elements to identify enriched proteins by proteomics. Validating the approach, a telomere repeat binding TelR-TALE identifies many proteins previously implicated in telomere function. Furthermore, the 70R-TALE designed to bind 70 bp repeats indicates that proteins involved in DNA repair are enriched on these elements that reside adjacent to VSG genes. Interestingly, the 177 bp repeat binding 177R-TALE enriches for many kinetochore proteins, suggesting that intermediate-sized and mini-chromosomes assemble kinetochores related in composition to those located on the main megabase chromosomes. This provides a first insight into the chromatin landscape of repetitive regions of the trypanosome genome with relevance for their mechanisms of chromosome integrity, immune evasion, and cell replication.

Acetylation of H3K115 is associated with fragile nucleosomes at CpG island promoters and active regulatory sites

yaten2020@gmail.com (Dipta Sengupta) — Wed, 04 Mar 2026 00:00:00 +0000

Acetylation of lysine residues in the tail domain of histone H3 is well characterised, but lysine residues in the histone globular domain are also acetylated. Histone modifications in the globular domain have regulatory potential because of their impact on nucleosome stability but remain poorly characterised. In this study, we report the genome-wide distribution of acetylated H3 lysine 115 (H3K115ac), a residue on the lateral surface at the nucleosome dyad, using chromatin immunoprecipitation. In mouse embryonic stem cells, we find that detectable H3K115ac is enriched at the transcription start site of active CpG island promoters, but also at polycomb-repressed promoters prior to their subsequent activation during differentiation. By contrast, at enhancers, H3K115ac enrichment is dynamic, changing in line with gene activation and chromatin accessibility during differentiation. Most strikingly, we show that H3K115ac is detected as enriched on ‘fragile’ nucleosomes within nucleosome-depleted regions at promoters and active enhancers, where it coincides with transcription factor binding, and at CTCF-bound sites. These unique features suggest that H3K115ac correlates with, and could contribute to, nucleosome destabilisation and that it might be a valuable marker for identifying functionally important regulatory elements in mammalian genomes.

Massively parallel reporter assay for mapping gene-specific regulatory regions at single-nucleotide resolution

cepko@genetics.med.harvard.edu (Alastair J Tulloch) — Wed, 25 Feb 2026 00:00:00 +0000

Precise gene regulation is essential for tissue development and function, yet mapping cis-regulatory modules (CRMs) at high resolution and in specific cell types remains challenging. We introduce two complementary strategies—a locus-specific massively parallel reporter assay (LS-MPRA) and a degenerate MPRA (d-MPRA)—designed to overcome limitations in throughput, resolution, and prior knowledge requirements. LS-MPRA uses BAC-based libraries to densely sample genomic regions, enabling unbiased interrogation of millions of DNA fragments for CRM activity. D-MPRA applies systematic mutagenesis to resolve CRM architecture at single-nucleotide resolution, nominating essential bases that may function as TF binding sites or other regulatory elements. We applied these methods to retinal genes expressed in mature rods and bipolar interneurons using in vivo and ex vivo mouse (Mus musculus) tissue. LS-MPRA recapitulated known CRMs and identified previously uncharacterized CRMs, including those embedded in neighboring genes. Applied to Olig2, a dynamically expressed gene in retinal progenitors, LS-MPRA identified three CRM regions, which d-MPRA and motif analyses further dissected. CUT&RUN confirmed direct binding of candidate TFs. Extending LS-MPRA to chick (Gallus gallus) retina and spinal cord demonstrated cross-species and cross-tissue applicability. Together, these approaches provide a rapid, scalable, inexpensive, and accessible platform for CRM discovery that can be carried out without prior element annotation and with tunable (small) fragment sizes.

The chromatin remodeller CHD4 regulates transcription factor binding to both prevent activation of silent enhancers and maintain active regulatory elements

B.D.Hendrich@exeter.ac.uk (Andria Koulle) — Tue, 03 Feb 2026 00:00:00 +0000

Chromatin organisation and transcriptional regulation are tightly coordinated processes that are essential for maintaining cellular identity and function. ATP-dependent chromatin remodelling proteins play critical roles in control of genome structure and in regulating transcription across eukaryotes. Their essential nature, however, has made it difficult to define exactly how these functions are mediated. The chromatin remodeller CHD4 has been shown to be capable of sliding nucleosomes in vitro, and to regulate chromatin accessibility and gene expression in vivo. Using an inducible depletion system, here we identify a second mechanism of action for CHD4 in actively restricting the residence time of transcription factors (TFs) on chromatin. Together, these activities result in distinct, context-dependent outcomes: at highly accessible regulatory elements, CHD4 limits TF binding to maintain regulatory function, while at low-accessibility euchromatic regions, it prevents TF engagement and sustains chromatin compaction, thereby silencing cryptic enhancers. Collectively, these mechanisms enable CHD4 to reduce transcriptional noise while preserving the responsiveness of active regulatory networks.

ATAD2 mediates chromatin-bound histone chaperone turnover

faycal.boussouar@univ-grenoble-alpes.fr (André Verdel) — Tue, 20 Jan 2026 00:00:00 +0000

ATAD2, a conserved protein which is predominantly expressed in embryonic stem (ES) cells and spermatogenic cells, emerges as a crucial regulator of chromatin plasticity. Our previous parallel studies conducted in both ES cells and S. pombe highlighted the fundamental role of ATAD2 in facilitating chromatin-bound histone chaperone turnover. Focusing on mouse spermatogenesis, we demonstrate here that ATAD2 regulates the HIRA-dependent localization of H3.3 on the genome and influences H3.3-mediated gene transcription. Moreover, by modulating histone eviction and the assembly of protamines, ATAD2 ensures proper chromatin condensation and genome packaging in mature sperm. Disruption of Atad2 function in mice leads to abnormal genome organization in mature spermatozoa. Together, these findings establish a previously overlooked level of chromatin dynamic regulation, governed by ATAD2-controlled histone chaperones binding to chromatin, which defines the balance between histone deposition and removal.