High-resolution Hi-C analyses have traditionally leaned on hundreds of millions of sequenced read pairs, a convention driven more by precedent than by evidence. The problem is that no one had systematically established the minimum depth required for the different classes of features that researchers routinely compute from contact maps. Granovsky and Polovnikov tackled this by performing progressive random subsampling of twelve Hi-C libraries spanning multiple cell types and experimental protocols. By repeatedly downsampling each library to different depths, they could track how each derived metric fluctuated as data became sparser, effectively simulating dozens of cheaper experiments from every expensive one already in hand.

The crucial innovation lies in how the authors defined sufficiency. Rather than declaring a depth adequate when a metric reached some arbitrary fraction of its full-depth value, they introduced a biologically motivated criterion grounded in the variability between independent biological replicates. The logic is elegant: if the scatter in a metric caused by reducing sequencing depth falls below the scatter observed between genuine replicate experiments, then adding more reads cannot improve the estimate any further, because biological noise has become the dominant source of variation. The required depth for each metric, then, is the point at which subsampling-induced scatter first reaches that replicate-level floor.

Applying this criterion across their panel of libraries produced strikingly different thresholds for different metrics. The genome-average loop-density estimate, derived from the log-derivative of the contact probability function P(s), reached its biological floor at approximately seven million read pairs. The genome-average loop-size estimate required roughly twenty million. Insulation scores, which quantify the depletion of contacts at boundaries between chromatin domains, needed around eighty million read pairs. And compartment eigenvectors, the principal descriptors of active versus inactive chromatin organization, demanded approximately one hundred ten million read pairs when computed at one-hundred-kilobase resolution. Compartment, insulation, and boundary analyses were performed on a subset of nine libraries with comparable full-depth coverage, while loop inference was evaluated across all twelve.

These numbers carry real weight for experimental design. Loop density and loop size, which reflect the fingerprint of the loop-extrusion process that shapes chromatin into loops anchored by CTCF and cohesin, turn out to be remarkably cheap to measure accurately at the genome-wide level. A lab interested primarily in global extrusion characteristics can obtain biologically saturated estimates with a small fraction of the sequencing typically deployed. In contrast, compartment eigenvectors at kilobase resolution sit at the expensive end of the spectrum, approaching the very depths that have become standard practice in high-resolution studies. The hierarchy of requirements means that a one-size-fits-all sequencing budget inevitably overpays for some measurements and may still underdeliver on others.

Perhaps the most consequential finding is that for every metric examined, depth-induced variability fell below biological replicate variability well before full sequencing depth was reached. In other words, the marginal value of additional reads on a single library diminishes to zero long before the deepest libraries stop yielding information. Once the subsampling scatter hits the replicate floor, extra sequencing refines nothing that matters, because the remaining uncertainty reflects genuine biological variation between cell populations rather than statistical noise. This reframes the entire question of depth: the ceiling is set not by the sequencer but by the biology itself.

The authors are careful to delineate the scope of their conclusions. The thresholds they report apply to genome-averaged characteristics, not to local metrics at specific genomic loci. A researcher hunting for a boundary shift at a particular disease-associated locus, or validating a single enhancer-promoter loop, faces a different statistical regime in which rare contacts must be sampled densely enough to resolve one narrow window of the genome. Genome-wide averages benefit from the law of large numbers across millions of loci, which is precisely why they saturate at comparatively modest depths, while locus-specific calls cannot borrow strength from the rest of the map in the same way.

The practical upshot for the field is a reallocation argument. If global biophysical features are the goal, the analysis suggests that increasing the number of biological replicates is likely to improve inference more effectively than sequencing individual libraries to greater depth. Multiple shallower libraries sample the true biological heterogeneity of a cell type, whereas one ultra-deep library samples a single biological state with precision that quickly becomes redundant. For groups planning Hi-C campaigns, this means the sequencing budget conversation should begin with the specific metrics to be computed, not with a blanket target of hundreds of millions of read pairs per sample.

The study also carries methodological significance for how the community benchmarks genomics pipelines. Subsampling-based calibration against replicate variability offers a general template that could be extended to other contact-map assays, such as Micro-C, and to other classes of derived quantities beyond those examined here. Because the work relied entirely on publicly available sequencing datasets, with no new human or animal data generated, it demonstrates how reanalysis of existing archives can yield design guidance of immediate value to the field. The research was supported by the Russian Science Foundation, with additional support to Polovnikov from the Alexander von Humboldt Foundation.

As Hi-C and its descendants become routine tools in chromatin biology, genome assembly, and disease genomics, the economics of sequencing decisions increasingly shape what questions laboratories can afford to ask. Granovsky and Polovnikov have converted a folk assumption about depth into a quantified, metric-by-metric benchmark, and in doing so they have handed experimentalists a map of where each additional million read pairs stops buying accuracy and starts buying only cost. For a field that has often equated more data with better science, the message is a bracing one: sometimes the deepest insight comes from knowing exactly when to stop sequencing.

Subject of Research: Sequencing depth requirements for Hi-C-derived chromatin metrics

Article Title: Systematic assessment of sequencing depth requirements for Hi-C-derived metrics

Article References: Granovsky, A., & Polovnikov, K. (2026). Systematic assessment of sequencing depth requirements for Hi-C-derived metrics. BMC Genomics, 27(1), Article 860. https://doi.org/10.1186/s12864-026-13455-7

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13455-7

Keywords: Hi-C, sequencing depth, chromatin organization, A/B compartments, insulation score, loop extrusion, contact probability, biological replicates, benchmark, genomics, next-generation sequencing, chromatin structure

Telomere-to-telomere, or T2T, assembly represents the gold standard of modern genomics. Conventional genome assemblies leave behind hundreds or even thousands of unresolved stretches known as gaps, typically in regions crammed with repetitive DNA such as centromeres, ribosomal RNA gene clusters and other satellite-rich zones. These gaps matter. They hide genes, obscure structural variation and make it impossible to say exactly how many chromosomes a species truly carries. A T2T assembly eliminates those holes entirely, reading each chromosome as one continuous thread from the telomere at one end to the telomere at the other. Achieving this for a plant genome is notoriously difficult, since plant genomes are often loaded with repetitive transposable elements that confound sequencing algorithms.

The team overcame the challenge by combining three complementary sequencing strategies. Pacific Biosciences HiFi reads provided highly accurate long stretches of DNA, Oxford Nanopore ultra-long reads extended those stretches across the most stubborn repetitive regions, and Hi-C, a technique that captures the physical contacts between chromosomes inside the cell nucleus, was used to order and orient the sequences into full-length chromosomes. The final assembly spans roughly 890.50 megabase pairs and achieves a scaffold N50 of 44.26 megabases, meaning half of the assembled sequence resides in chromosomes longer than 44 million bases. The entire genome was organized into exactly 20 gap-free chromosomes, each capped by telomeres, with 40 telomeres and 20 putative centromere regions identified in total.

One of the most consequential results is the definitive resolution of the chromosome number of I. cornuta. Botanists have debated the basic chromosome number of this species for years, with conflicting counts circulating in the cytological literature. The new gap-free assembly shows unambiguously that the species carries 20 pairs of chromosomes, a diploid complement written as 2n = 2x = 40. Crucially, the team did not rely on sequencing data alone. An independent karyotype analysis of root-tip cells, in which chromosomes are stained and counted directly under the microscope, confirmed the genomic count. Agreement between the molecular assembly and classical cytogenetics settles a long-standing controversy and provides a firm foundation for evolutionary comparisons across the Aquifoliaceae family.

Genome quality was assessed with the metrics that the assembly community takes most seriously. The annotation, recovered using RNA sequencing data, predicted 35,437 protein-coding genes. Benchmarking with BUSCO, a test that searches for a set of universally conserved single-copy genes, returned a completeness score of 98.33 percent, indicating that almost the entire expected gene repertoire is present. The base-level accuracy of the assembly, expressed as a quality value or QV, reached 48.97, which corresponds to an error rate of roughly one mistake in every 79,000 bases. These figures place the Chinese holly assembly among the most accurate plant genomes published to date and demonstrate that the integration of HiFi, ultra-long and Hi-C data can deliver reference-grade sequences even for species with no prior genomic resources.

With the reference in hand, the researchers turned to evolutionary history. Comparative genomics against related plant species revealed two whole-genome duplication events in the ancestry of I. cornuta, ancient episodes in which the entire genetic complement was doubled and subsequently pared down through gene loss and divergence. Whole-genome duplications are a major engine of plant evolution, because duplicate genes are free to accumulate mutations and take on new functions. The analysis also uncovered lineage-specific expansions of gene families, with a pronounced enrichment in pathways governing secondary metabolism, the biochemical arsenal plants use to synthesize defensive compounds, pigments and medicinal molecules.

These expanded gene families are particularly intriguing given what the Chinese holly is known for. The species thrives under stresses that would defeat many ornamentals, and its tissues contain bioactive compounds of pharmaceutical interest. The authors are careful, however, to frame these expansions as candidate gene sets that could potentially contribute to environmental adaptability. Genomic evidence of expansion does not by itself prove function. Verifying the biological roles of these genes will require transcriptomic profiling across stress conditions and direct functional assays, such as gene knockout or overexpression experiments, work that the T2T reference now makes dramatically easier to design and interpret.

The significance of the assembly extends well beyond a single species. Ilex is a large and ecologically diverse genus of more than 400 evergreen and deciduous trees and shrubs distributed across temperate and tropical regions, and it had never before had a gap-free reference genome of any kind. The I. cornuta assembly therefore serves as a genomic anchor for the entire genus, enabling researchers to map other holly species against a complete, contiguous blueprint, reconstruct chromosome evolution within the family, and pinpoint the genetic basis of traits ranging from sex determination, a famously thorny question in hollies, to fruit color and cold hardiness.

For breeders, the practical implications are immediate. Ornamental holly breeding has traditionally depended on slow phenotypic selection and conventional crossing, approaches that are poorly suited to traits controlled by many genes or by structural variants buried in repetitive DNA. A telomere-to-telomere reference changes that calculus. It allows genome-wide association studies, marker-assisted selection and eventually genome editing to be pursued with confidence that the underlying sequence is complete and correctly ordered. Genes involved in stress tolerance, secondary metabolite production and ornamental quality can now be located precisely, tracked in breeding populations and, in time, targeted directly.

The work also stands as a showcase of how far genome science has traveled. Little more than a decade ago, completing a plant chromosome end to end was deemed unreachable; today, a coordinated pipeline of three sequencing technologies can deliver a gap-free reference for a species whose genome had never been assembled before. The Chinese holly now joins the small but growing roster of plants with fully finished genomes, and the resource, deposited and openly described by the Nanjing-led team, is freely available to researchers worldwide. For a genus that decorates gardens, supports traditional medicine and endures some of the toughest growing conditions in its native range, the holly has finally been given a complete instruction manual, and scientists are only beginning to read it.

Subject of Research: Telomere-to-telomere gap-free genome assembly of the Chinese holly, Ilex cornuta

Article Title: Telomere-to-telomere, gap-free genome assembly of the Chinese holly, Ilex cornuta

Article References: Zhou, P., Xu, K., Gong, W., Li, F., Sun, H., Zhang, Q., & Zhang, M. (2026). Telomere-to-telomere, gap-free genome assembly of the Chinese holly, Ilex cornuta. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10103-w

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10103-w

Keywords: Ilex cornuta, Chinese holly, telomere-to-telomere, genome assembly, centromere, chromosome number, whole-genome duplication, comparative genomics, PacBio HiFi, Oxford Nanopore, Hi-C, molecular breeding

The team attacked the problem with an unusually rich experimental resource: bulk Hi-C contact maps generated from the ventral hippocampus, a brain region central to memory and emotion, of eight inbred mouse strains. Five of these were classical laboratory strains and three were wild-derived, capturing a meaningful slice of the genetic diversity within a single species. The resulting dataset comprises 14.55 billion valid contact pairs, an enormous catalog of who-touches-whom information across the mouse genome. By comparing chromatin architecture across strains that differ at millions of sequence positions, the researchers could ask which features of genome folding are stable enough to survive both genetic drift and deliberate breeding, and which are labile.

Before any evolutionary claims could be made, however, the authors confronted a stubborn methodological problem. The most popular analytical tools treat TADs as discrete objects: an algorithm scans a Hi-C contact map and draws sharp lines where it detects a boundary. When the researchers applied such discrete calling across replicate samples, they found poor concordance between replicates, meaning the same biological sample analyzed twice could yield noticeably different sets of called domains. That instability makes discrete TAD calls a shaky foundation for cross-strain or cross-species comparison, because apparent differences might reflect algorithmic noise rather than genuine biology.

The solution was to abandon the binary view. Instead of asking whether a boundary exists at a given position, the team measured separation scores, a continuous metric that quantifies how strongly a genomic location insulates the regions on either side. Rather than a yes-or-no verdict, every point along the genome receives a graded insulation value, and weakly insulated boundaries can be distinguished from strongly insulated ones. This shift from categorical to continuous measurement substantially improved reproducibility between replicates, giving the researchers a reliable yardstick for comparing chromatin architecture across strains and, ultimately, across species. The lesson is one the field is likely to absorb broadly: when a biological property varies in degree, measuring it as a degree rather than a category can dissolve apparent irreproducibility.

Armed with this framework, the researchers identified 2,857 highly insulated genomic regions whose insulation is conserved across all eight mouse strains. These are places where the folding of the chromosome creates a particularly impermeable wall, and where that wall appears in every strain examined, from the most domesticated laboratory line to the wildest-derived strain. The consistency across such genetically diverse backgrounds suggests that these boundaries are not accidents of recent breeding history but features actively maintained by the cell.

What kind of genomic real estate hosts these conserved, strongly insulated boundaries? The team found they are markedly enriched for multiple binding sites of CTCF, the architectural protein best known for stitching DNA loops together and anchoring the boundaries of chromatin domains. They also sit in accessible, open chromatin, lie near super-enhancers, the dense clusters of regulatory elements that drive expression of genes defining cell identity, and are found close to housekeeping genes, the essential maintenance machinery every cell must keep running. In other words, the most insulated boundaries are not scattered randomly through the genome; they cluster around the regulatory and structural elements that biologists already consider among the most functionally consequential.

The evolutionary signal extends below the level of chromatin folding into the DNA sequence itself. The conserved highly insulated regions show greater DNA sequence conservation across placental mammals than would be expected by chance, indicating that the nucleotide letters underlying these boundaries have been constrained by purifying selection over deep evolutionary time. They also display more stable DNA methylation patterns, suggesting that their epigenetic state is buffered against the genetic perturbations that distinguish one mouse strain from another. Together, these converging lines of evidence, from protein binding, chromatin accessibility, sequence conservation and epigenetic stability, paint the strongly insulated boundary as a multiply protected feature of the mammalian genome.

The most striking result emerges when the comparison is pushed beyond mice. In a cross-species analysis, the researchers measured how often insulated regions occupy the same genomic positions in human and mouse, two lineages separated by roughly 90 million years of evolution. They found that the positional overlap between the two species increases steadily with insulation strength, climbing to 80.7 percent for the most highly insulated regions. That figure is remarkable: it means that for the strongest boundaries, the vast majority of locations are shared between human and mouse, implying that the folding architecture at these sites has been maintained essentially intact since the common ancestor of the two species. Weakly insulated boundaries, by contrast, overlap far less, consistent with the idea that only the most robust architectural walls are under strong selective constraint.

Not every difference in chromatin conformation between strains carries deep meaning, however. The study also examined structural genomic rearrangements, deletions, duplications and inversions of DNA segments that differ among mouse strains, and found that these are the primary driver of chromatin conformation differences between strains. Yet when the team searched for functional consequences of those conformational differences, they found only limited evidence that they matter for gene regulation or phenotype. This is a subtle but important nuance: much of the strain-to-strain variation in genome folding may be passive, a mechanical consequence of DNA sequence rearrangement rather than a cause of biological divergence. The functional story, the data suggest, lives in the conserved, strongly insulated boundaries rather than in the variable ones.

The practical payoff of the study is a prioritization tool. Because insulation strength is continuous, reproducible and predictive of conservation, researchers screening a disease-associated genomic variant can now consult the insulation landscape to judge whether a nearby TAD boundary is likely to be an ancient, functionally critical architectural element or a labile, probably inconsequential one. Given that disruptions of TAD boundaries have been implicated in developmental disorders and cancers, for example when structural variants allow an enhancer to reach a gene it was never meant to touch, a quantitative framework for ranking boundary importance could sharpen the interpretation of human genomic data. The work also reframes the long-running TAD debate: rather than asking whether TADs as a class are functionally meaningful, the field can now ask which specific boundaries carry the strongest evidence of constraint, with insulation strength serving as the guide. Funded in part by the National Institutes of Health and the Howard Hughes Medical Institute’s Hanna H. Gray Fellows Program, the study demonstrates that a careful rethinking of measurement, from discrete calls to continuous scores, can turn a contested question in genome biology into a tractable, quantitative one.

Subject of Research: Evolutionary conservation of topologically associating domain boundaries measured by Hi-C derived insulation strength in mouse and human genomes

Article Title: Hi-C derived insulation strength predicts evolutionary conservation of topologically associating domains

Article References: Soto, D. C., Chen, P. B., Huang, H., Galasso, J., Chen, Z. J., Luo, C., Ernst, J., & Flint, J. (2026). Hi-C derived insulation strength predicts evolutionary conservation of topologically associating domains. Genome Biology. https://doi.org/10.1186/s13059-026-04298-2

Image Credits: AI Generated

DOI: 10.1186/s13059-026-04298-2

Keywords: TADs, Hi-C, chromatin architecture, insulation strength, CTCF, genome evolution, mouse strains, separation scores, super-enhancers, DNA methylation, comparative genomics, Genome Biology

Position effect variegation is a phenomenon familiar to geneticists working on organisms from fruit flies to humans: when a gene is moved to a different chromosomal neighborhood, its activity can change dramatically depending on the local chromatin environment, the packaging of DNA and associated proteins that determines whether a region is transcriptionally accessible or silenced. In Drosophila, relocating the white gene near heterochromatin disrupts eye pigmentation; in yeast, inserting exogenous genes into silent chromatin regions represses their transcription. Yet despite its universality across eukaryotes, the position effect had never been systematically dissected in filamentous fungi such as P. rubens, largely because the tools for doing so simply did not exist.

The research team, led by Gang Liu of the Chinese Academy of Sciences, chose P. rubens for good reason. Formerly known as Penicillium chrysogenum, this industrial workhorse has produced the world’s penicillin for decades and harbors dozens of secondary metabolite biosynthetic gene clusters in its genome, most of which remain transcriptionally silent under standard laboratory conditions. That silence, the new study suggests, may partly reflect the chromatin contexts in which these clusters sit. Understanding where in the genome a foreign pathway will be welcomed, rather than silenced, could unlock the fungus’s full potential as a chassis for making pharmaceuticals and other valuable chemicals.

To build the screening platform, the researchers adapted the piggyBac transposon system, originally isolated from the cabbage looper moth Trichoplusia ni. PiggyBac is prized among genome engineers because it inserts precisely into TTAA DNA sequences without leaving genomic scars, and in organisms such as fission yeast and Pichia pastoris it distributes itself nearly randomly across the genome. The team engineered a strain in which a piggyBac cassette carrying a selectable marker was embedded within a G418 resistance gene, then supplied a hyperactive, codon-optimized transposase called hyPBase, decorated with nuclear localization signals, on an autonomously replicating plasmid. When the transposase was expressed, the cassette excised cleanly and reinserted elsewhere, restoring G418 resistance only in cells where transposition had occurred.

Quantitative PCR confirmed that every resistant colony carried exactly one copy of the transposon, meaning that any subsequent differences in gene expression could be attributed to insertion site rather than copy number. The researchers also swapped the constitutive promoter driving the transposase for a starch-inducible promoter from P. rubens itself, giving them temporal control over transposition and stabilizing the engineered strains. Transposition efficiency peaked at around eight percent on day four of induction, a level the authors describe as sufficient for generating large-scale insertion libraries in this multicellular organism, which is far harder to manipulate than single-celled yeasts.

High-throughput sequencing of more than 13,000 independent insertions revealed that piggyBac behaves differently in P. rubens than in previously studied hosts. Only about 40 percent of insertions landed at TTAA sites, and the surrounding nucleotide preferences diverged sharply from those observed in yeast, insects, mice and Candida. The transposon showed a mild tendency toward local hopping near the donor locus but otherwise spread broadly across all chromosomes, hitting all 59 secondary metabolite gene clusters annotated in the genome and disrupting the core biosynthetic genes of 43 of them, evidence that these clusters are dispensable for growth and candidates for future genome minimization. Notably, insertion density was highest within open reading frames, a preference that should aid gene function discovery.

With the mutagenesis platform validated, the team turned it into an expression-mapping tool. They constructed a strain carrying a green fluorescent protein reporter driven by a moderately strong promoter on the mobile cassette, induced transposition, and then converted fungal mycelia into single-cell protoplasts for analysis by fluorescence-activated cell sorting. Two populations were collected: P4, comprising 14 percent of cells with fluorescence indistinguishable from untransformed controls, representing genomic loci where the reporter was silenced, and P7, the top 1.88 percent of cells with the highest fluorescence, representing hotspots of expression. Clones from both groups remained stable over five generations, confirming that the differences reflected genuine chromosomal position effects rather than transient physiological states.

To understand why some loci were hot and others cold, the researchers turned to Hi-C, a sequencing method that captures the three-dimensional folding of chromosomes. The analysis revealed that P. rubens chromosomes are organized into A and B compartments, mirroring the architecture of mammalian genomes, where compartment A corresponds to active, accessible chromatin and compartment B to compacted, inactive regions. Insertion sites from the P7 high-expression library were significantly enriched in compartment A, while P4 low-expression sites showed no particular enrichment in either compartment, hinting that silencing involves additional local features beyond gross compartmentalization. Neither P4 nor P7 sites clustered near the boundaries of topologically associating domains, suggesting that large-scale chromatin compartmentalization, rather than local boundary structure, is the primary determinant of expression potential. Genes near P7 sites were also enriched in primary metabolic pathways such as amino acid biosynthesis, consistent with those regions being constitutively active.

The decisive test came with physcion, an anthraquinone pigment produced by medicinal herbs such as Rheum and Polygonum. Physcion, the 6-O-methylated derivative of emodin, is registered as a plant-derived fungicide effective against powdery mildew, downy mildew, grey mould and anthrax, with low toxicity to humans and animals. Its biosynthetic pathway, fully elucidated in recent years, begins with malonyl-CoA condensed by a thioesterase-deficient non-reducing polyketide synthase, proceeds through atrochrysone and emodin intermediates, and ends with a critical O-methylation step catalysed by an O-methyltransferase using S-adenosylmethionine. The team first disrupted pcbAB, the gene responsible for penicillin biosynthesis, to eliminate competition from the fungus’s native flagship product, then inserted the synthetic physcion gene cluster, carried on two DNA fragments encoding the polyketide synthase, thioesterase, decarboxylase and methyltransferase, into five loci each from the P4 and P7 libraries.

The results were striking. Junction PCR and quantitative PCR confirmed correct, single-copy integration at every site, yet strains carrying the cluster at P4 loci produced an average of only 0.95 milligrams per liter of physcion, with most samples below 1.2 milligrams per liter. Strains with the identical cluster at P7 loci produced between 27 and 83 milligrams per liter, averaging 56.0 milligrams per liter, a roughly fiftyfold improvement attributable purely to chromosomal address. Quantitative RT-PCR showed markedly elevated transcription of the polyketide synthase gene AnPKS in several P7 strains, correlating with higher yields, although one P4 strain bucked the trend, indicating that transcript abundance alone does not fully explain pathway output and that further regulatory layers tied to chromosomal position remain to be explored.

The implications extend well beyond a single compound. The authors argue that P. rubens should now be regarded as a chromatin-optimized chassis for secondary metabolite production, and that rational integration-site selection, guided by expression hotspot maps, should become standard practice in fungal metabolic engineering. The methodology, from inducible piggyBac mutagenesis through FACS-based expression sorting to Hi-C-informed interpretation, offers a blueprint transferable to other industrially significant filamentous fungi, where heterologous gene silencing has long frustrated efforts to express foreign biosynthetic pathways. As synthetic biology moves into the era of three-dimensional genomics, this study makes a compelling case that in the design of microbial cell factories, the most important question may not be what you insert, but where.

Subject of Research: Position effect variegation and heterologous biosynthesis of physcion in Penicillium rubens

Article Title: Position Effect Variegation of Penicillium rubens Genome and Application in Heterologous Production of the Plant‐Derived Anthraquinone Physcion

Article References: Wu, M., Fu, Y., Tang, L., Yao, Y., Pan, Y., & Liu, G. (2026). Position Effect Variegation of Penicillium rubens Genome and Application in Heterologous Production of the Plant‐Derived Anthraquinone Physcion. Microbial Biotechnology, 19(10), Article e70449. https://doi.org/10.1111/1751-7915.70449

Image Credits: AI Generated

DOI: 10.1111/1751-7915.70449

Keywords: Penicillium rubens, position effect variegation, piggyBac transposon, physcion, heterologous expression, chromatin, Hi-C, secondary metabolites, biosynthetic gene cluster, metabolic engineering, FACS, filamentous fungi

A new study published in BMC Genomics provides the first three-dimensional answer, and the result is remarkable. A team led by Martha Valiadi and Adam Monier at the University of Exeter, working with colleagues in Greece and at the Institut Pasteur in Paris, applied high-throughput chromosome conformation capture, or Hi-C, to O. tauri and mapped how its genome folds in space. Hi-C works by chemically cross-linking DNA segments that sit close together in the nucleus, fragmenting the genome, and then sequencing the paired fragments to build a contact map of physical interactions. When the team analysed these maps, they found that the low-GC regions of the two outlier chromosomes fold into self-contained loops of interaction that resemble the topologically associating domains, or TADs, familiar from animal and plant genomes. These TAD-like structures have sharp boundaries that effectively wall the outlier regions off from the rest of their own chromosomes and from the rest of the genome.

The finding is significant because spatial insulation was never previously demonstrated in organisms with genomes this compact. O. tauri holds the record as the smallest known free-living eukaryote, and its genome of roughly thirteen million base pairs is squeezed into a nucleus that occupies only a fraction of the cell. In larger eukaryotes, TADs are thought to help regulate gene expression by limiting the reach of regulatory elements such as enhancers, so that signals from one part of the genome do not bleed into another. Finding TAD-like architecture in an organism with so little genomic real estate suggests that the physical compartmentalisation of DNA is not a luxury of complex life but a fundamental feature that even the most streamlined eukaryotic genomes maintain.

The outlier regions themselves carry a distinctive chromatin fingerprint that the researchers uncovered by mining existing transcriptomic, methylation and nucleosome positioning data across the Mamiellales. Compared with the rest of the genome, the outlier regions are hypomethylated, meaning they carry fewer chemical methyl tags on their cytosine bases, and they are transcriptionally hyperactive, with genes inside them being expressed at unusually high levels. Nucleosome spacing across these regions is also altered, indicating that the DNA is packaged differently around the histone proteins around which it is wound. Taken together, these features describe a chromatin environment that is open, active and chemically distinct from the surrounding genome, and this profile is conserved across the species of the order that the team examined.

Yet the two outlier chromosomes are not identical twins in behaviour. The team found that transcription is consistently elevated in the low-GC region of the big outlier chromosome, which they designate BOC1, in every Mamiellales species they analysed. The small outlier chromosome tells a different story: transcriptional activity in its low-GC region, SOC1, varies from species to species. This asymmetry hints that the two outlier chromosomes may have different evolutionary origins or different functional roles, even though both share the same hallmark sequence composition and the same spatial sequestration within the nucleus. Understanding why one outlier region is universally busy while the other is more permissive remains an open question for future work.

One long-standing hypothesis about outlier chromosomes has been that they serve as repositories for transposable elements, the mobile genetic sequences that can copy and paste themselves around the genome. Because transposable elements often carry different GC compositions than host DNA and can be silenced by methylation, sequestering them in a separate chromatin compartment would be a sensible genome-defence strategy. The new study confirms that outlier regions are indeed enriched in transposable elements in most Mamiellales species, including a variety of element types such as Mutator-like elements and long terminal repeat retrotransposons. But the correlation is not universal, and this is where the study delivers its most conceptually important twist.

In two species, Bathycoccus prasinos and Ostreococcus lucimarinus, the researchers found no substantial enrichment of transposable elements in the outlier regions, yet those regions still retain their characteristic low-GC composition and their elevated BOC1 transcription, and the Hi-C data show that they remain spatially insulated. This decoupling demonstrates that the three-dimensional isolation of outlier chromosomes is not simply a by-product of harbouring mobile DNA. Spatial insulation, in other words, is a property that tracks the chromatin state of these regions rather than their transposable element content alone. It adds a genuine three-dimensional criterion to the list of sequence-based features, such as GC content and gene density, by which outlier chromosomes have traditionally been defined.

To test whether this compartmentalisation extends beyond the Mamiellales, the team reanalysed published Hi-C data from two other compact marine eukaryotes: Mantoniella tinhauana, a close relative within the same order, and Pelagomonas calceolata, a stramenopile alga only distantly related to the green lineage. In both cases, the data revealed consistent patterns of insulation around the outlier regions, suggesting that the spatial sequestration of compositionally distinct chromosome segments may be a recurrent feature of compact picoeukaryotic genomes more broadly. If this holds, it would imply that the physical organisation of the nucleus in these tiny cells follows rules that have evolved independently multiple times, converging on the same solution of walling off unusual DNA into its own neighbourhood.

The technical achievement behind these conclusions should not be understated. Hi-C experiments typically require millions of cells, and working with an organism as small as O. tauri demanded careful optimisation of cross-linking, digestion and ligation steps to recover enough informative contacts from such a tiny genome. The team also used iterative correction to normalise their contact matrices, removing biases introduced by restriction site distribution and read depth, before calling the boundaries of the TAD-like domains. The resulting maps provide the first glimpse of how a eukaryotic genome at the extreme lower limit of cellular complexity arranges itself in three dimensions, and they demonstrate that even a nucleus barely larger than a bacterial cell can sustain organised chromosomal architecture.

For the broader field of genome biology, the study reframes outlier chromosomes from curiosities of sequence composition into bona fide spatial compartments with conserved epigenetic identities. It raises new questions about how their boundaries are established and maintained in an organism that lacks many of the architectural proteins found in larger eukaryotes, and about what functional advantages insulation confers, whether in protecting the core genome from transposable elements, regulating the expression of specialised gene families, or managing the conflicts between different chromatin states within a single nucleus. As sequencing and chromosome conformation capture technologies continue to reach ever smaller and stranger organisms, the humble O. tauri, long a model of genomic minimalism, now also stands as a model of how much organised structure can be packed into the smallest of packages.

Subject of Research: Three-dimensional genome organisation and spatial insulation of outlier chromosomes in the picoeukaryote Ostreococcus tauri

Article Title: Three-dimensional genome organisation reveals spatially insulated outlier chromosomes in Ostreococcus tauri

Article References: Valiadi, M., Harrison, K., Loe-Mie, Y., Williams, B., Ankrett, D., Smirnoff, N., & Monier, A. (2026). Three-dimensional genome organisation reveals spatially insulated outlier chromosomes in Ostreococcus tauri. BMC Genomics. https://doi.org/10.1186/s12864-026-13416-0

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13416-0

Keywords: Ostreococcus tauri, Hi-C, chromosome conformation capture, topologically associating domains, outlier chromosomes, picoeukaryotes, Mamiellales, genome organisation, transposable elements, chromatin, DNA methylation, BMC Genomics

DNA methylation is one of biology’s most conserved regulatory mechanisms. Enzymes called DNA methyltransferases attach methyl groups to cytosine bases, producing 5-methylcytosine, a mark that can be faithfully copied through cell division yet remains responsive to developmental and environmental cues. In humans and classic model organisms, methylation has been studied intensively, underpinning phenomena as diverse as X-chromosome inactivation, genomic imprinting, and the maintenance of cellular identity. In domestic animals, however, the picture has remained fragmentary. Most livestock studies have relied on differential methylation analysis — simply comparing methylated regions between groups of animals with different phenotypes. Such comparisons identify correlations but cannot say which epigenetic differences are actually driven by genetic variation, nor which ones matter for traits.

To close that gap, a team led by Yiting Wang, Zitao Chen, and Qishan Wang generated and integrated an extraordinary dataset from pig skeletal muscle: 185 whole-genome bisulfite sequencing samples, 148 RNA-seq transcriptomes, and 8 three-dimensional chromatin interaction maps, alongside whole-genome sequencing data. Fifty-six of the bisulfite samples were newly generated for the study, drawn from Jinhua pigs raised in Zhejiang Province and Duroc pigs raised in Anhui Province, while 129 came from public repositories. The bisulfite approach converts unmethylated cytosines to uracil during library preparation, allowing methylation levels to be read out base by base. The team sequenced on Illumina NovaSeq 6000 instruments, verified conversion efficiency with unmethylated lambda DNA spike-in controls, and ultimately profiled more than 5.25 million individual CpG sites and roughly 1.25 million regional CpG blocks across the porcine genome.

A crucial methodological hurdle was obtaining reliable genotypes for samples that had only bisulfite data. The researchers called SNPs directly from the bisulfite reads using Bis-SNP, then imputed them to sequence-level density with Beagle, using the PHARP v3 pig haplotype reference panel. Benchmarking against matched whole-genome sequencing data from twenty Duroc animals showed that, at a stringent dosage R-squared threshold above 0.9, imputed genotypes achieved nearly 90 percent precision and over 93 percent overall concordance with the reference calls. After merging with WGS-derived variants, the final dataset contained more than 12.2 million high-quality SNPs — a foundation robust enough for quantitative trait locus mapping at scale.

The centerpiece of the analysis is a block-resolved methylation quantitative trait locus, or meQTL, atlas. Rather than treating each CpG site in isolation, the team used the wgbstools toolkit to segment the genome into co-methylated blocks — typically a few hundred base pairs long — and mapped genetic variants within one megabundle of each block’s start position using tensorQTL. The results were striking. Independent cis-meQTL signals numbered 49,801 at single CpG sites and 448,028 at blocks, implicating 67,333 genetically regulated CpGs and 436,630 blocks. Cross-cohort validation on chromosome 1 showed that roughly 85 to 87 percent of associations detected in the newly generated cohort replicated in the public cohort with concordant effect directions. Allele-specific methylation analysis provided independent confirmation: block pairs showing recurrent allele-specific methylation were more than twice as likely to harbor meQTLs, with 98.5 percent of overlapping signals agreeing in allelic direction.

Heritability estimates underscored the biological meaning of these signals. CpGs with detectable meQTLs showed a mean cis-heritability of about 0.41, and regulated blocks about 0.25, whereas unregulated sites and blocks hovered near 0.04 to 0.05. Regions governed by multiple independent genetic variants were progressively more heritable, and rarer alleles tended to show larger effects on methylation. Enrichment analyses placed meQTL variants preferentially in promoters, enhancers, and transcription start site-proximal regions, and sequence-based prediction models suggested the strongest effects on chromatin accessibility in skeletal and cardiac muscle cells. Roughly 19 percent of meQTL variants were predicted to alter transcription factor binding motifs — including motifs for ARNT::HIF1A, HAND2, ZEB1, and SOX10 — although the authors caution that these remain computational predictions rather than experimentally validated binding changes.

Connecting methylation to gene expression, the team performed expression quantitative trait methylation mapping, pairing each gene with CpGs and blocks within one megabase of its transcription start site. They identified 14,726 significant site-gene pairs and 4,637 block-gene pairs, with effects distributed almost evenly between positive and negative directions — a pattern the authors attribute to the mixture of promoter, gene body, enhancer, and distal contexts captured by the analysis rather than a contradiction of the canonical repressive role of promoter methylation. To establish that these methylation-expression links share a genetic basis, the researchers deployed colocalization, HyPrColoc, and summary-data-based Mendelian randomization. Together these approaches supported 15,602 site-gene and 86,318 block-gene relationships, with thousands backed by multiple methods. Hi-C chromatin contact maps then supplied spatial context: some distal CpG-gene pairs shared the same topologically associating domain, and a handful — such as a CpG more than 500 kilobases from SIPA1L2 — were physically connected by the same chromatin loop.

The leap to complex traits came from integrating the meQTL atlas with genome-wide association statistics for 91 growth, carcass, and meat quality traits from PigBiobank. Complementary methods — colocalization, SMR, epigenome-wide association studies using genetically predicted methylation, and HyPrColoc — yielded 73,580 methylation-trait associations, with 8,097 supported by at least two approaches. Several loci stood out. A CpG in the promoter of NEDD4L, a gene implicated in muscle development and insulin resistance, associated strongly with average daily gain and backfat thickness. A block within the HSPB3 promoter, spanning three enhancers, associated with lean cut percentage, loin muscle area, and loin depth. At the MPC2 locus, a single variant, rs346166613, drove both site-level and block-level methylation signals linked to backfat thickness, with MPC2 highly expressed in adipocytes. The team also built methylation-based genetic scores from genotype-predicted methylation; these achieved moderate out-of-sample prediction, peaking for backfat thickness in Duroc pigs at a correlation of 0.353 and about 6 percent predictive variance, and combining them with conventional polygenic scores improved prediction beyond either alone.

Perhaps the most compelling result is a full regulatory cascade validated at the bench. Multi-trait colocalization and mediation Mendelian randomization identified 12,171 CpG-gene-trait triplets, with 2,702 showing significant mediation — often with more than 90 percent of the genetic effect on the trait apparently transmitted through methylation and expression. One prioritized pathway centered on a CpG block near SOCS3, a modulator of JAK-STAT signaling previously linked to myogenic differentiation. The block, its methylation, SOCS3 expression, and average daily gain all shared genetic signals, and Hi-C data placed the block and the gene in the same TAD. Using CRISPR-dCas9-TET1 targeted demethylation in immortalized porcine muscle satellite cells, the researchers reduced methylation at the block, observed increased SOCS3 protein by western blot, and measured enhanced cell proliferation and altered cell-cycle distribution — proof of concept that the computationally prioritized locus has real regulatory teeth.

The authors are careful about scope. The study covers only skeletal muscle; colocalization and Mendelian randomization establish convergent genetic evidence, not definitive causality; and functional editing validated just one locus among thousands of candidates. Methylation-based scores, being genotype-derived, capture genetic rather than environmental epigenetic information. Still, the resource — publicly queryable through the PSMEA database — and the prioritization framework, which distilled 6,894 candidate genes across 84 traits into Gold, Silver, and Bronze tiers, offer breeders and biologists a powerful new lens. By showing that a genetically predictable slice of the methylome carries trait-relevant, mechanistically interpretable information, the study transforms DNA methylation from a correlational curiosity into a working layer of livestock genomics, with implications that could eventually ripple from the pig pen to human medicine.

Subject of Research: Genetic regulation of DNA methylation and its role in complex trait variation in pig skeletal muscle

Article Title: Integrative genomics decoding DNA methylation-mediated genetic control of complex traits in pigs

Article References: Wang, Y., Chen, Z., Fan, M., Liu, C., Gu, J., Yu, P., Jin, W., Zhang, Z., Xie, S., Yang, T., Jiang, J., Zhang, Z., Wang, Z., ZHang, Z., Fang, L., Pan, Y., & Wang, Q. (2026). Integrative genomics decoding DNA methylation-mediated genetic control of complex traits in pigs. Journal of Advanced Research. https://doi.org/10.1016/j.jare.2026.10.001

Image Credits: AI Generated

DOI: 10.1016/j.jare.2026.10.001

Keywords: DNA methylation, meQTL, pig genomics, epigenomics, skeletal muscle, Mendelian randomization, colocalization, Hi-C, CRISPR-dCas9-TET1, methylation-based genetic scores, livestock breeding, SOCS3

ASFV belongs to the nucleocytoplasmic large DNA viruses, a group of complex viruses whose genomes encode many of their own replication machinery. Although the virus assembles and replicates in cytoplasmic factories, its entry into a macrophage sets off a cascade of events that reaches into the nucleus. Previous work had suggested that ASFV can modulate host gene expression epigenetically, but the scale and dynamics of the nuclear changes during early infection had never been systematically mapped. The new study addresses that gap by tracking the infection at 2, 4, and 6 hours post-infection, a window in which the virus establishes itself before progeny virions begin to spread.

To capture this pivotal phase, the researchers deployed an unusually comprehensive multi-omics strategy. Time-course RNA sequencing charted the host transcriptome as it changed hour by hour. CUT&Tag profiling measured where RNA polymerase II, the enzyme that transcribes protein-coding genes, was sitting across the genome. Immunoprecipitation followed by mass spectrometry identified the proteins that associate with the polymerase during infection. Finally, Hi-C, a method that captures the physical contacts between distant stretches of chromatin, revealed how the genome folds in three-dimensional space. Crucially, the team ran this entire pipeline using both a virulent ASFV strain and its live-attenuated derivative, allowing them to distinguish changes caused by the virus in general from those attributable to specific virulence factors.

The most striking finding concerns the global organization of the genome. In healthy cells, chromatin is partitioned into compartments of active euchromatin and silent heterochromatin, and further subdivided into topologically associating domains, or TADs, which are self-contacting genomic neighborhoods that help regulate which genes talk to which regulatory elements. The study found that early ASFV infection significantly increased heterochromatinization across the host genome, shifted the positions of TAD boundaries, and altered both the intensity of chromatin interactions and the spatial distances between interacting genomic regions. In other words, within hours of the virus entering the cell, the host genome was physically refolded.

One of the most intriguing observations is that viral DNA itself appears to participate in this reorganization. The Hi-C data revealed interactions between viral genomes and host chromatin, suggesting that incoming ASFV DNA does not simply float in the nucleus as an inert passenger. Instead, it seems to make physical contact with host chromosomes, raising the possibility that the virus actively recruits host genomic regions to viral replication compartments or sequesters immune genes into repressive nuclear environments. The authors suggest that this chromatin reorganization may contribute to ASFV immune evasion, though the precise mechanisms linking physical genome folding to immune suppression remain to be worked out.

The transcriptional consequences of this remodeling were equally revealing. The host antiviral transcriptional program, the set of interferon-stimulated genes and inflammatory pathways that normally swing into action when a macrophage detects a pathogen, turned out to be indistinguishable between cells infected with the virulent strain and cells infected with the attenuated virus. This was a surprise, because the attenuated strain lacks MGF-encoded factors, a family of viral genes long associated with virulence and immune modulation. The result demonstrates that the earliest wave of innate immune activation is independent of these virulence determinants, implying that whatever the MGF proteins do to tip the balance toward lethal disease must happen later, or through mechanisms other than silencing the initial antiviral transcriptional response.

RNA polymerase II itself became a focal point of the analysis. The researchers found that the total protein levels of the polymerase did not change appreciably during early infection, yet its behavior was profoundly altered. Its genomic occupancy was redistributed across the genome, and its interactome, the network of proteins it physically associates with, was remodeled. Core polymerase subunits and components of the Mediator complex, a large coactivator that bridges transcription factors to the polymerase, became enriched in the polymerase-associated fraction. In contrast, sequence-specific transcription factors, the proteins that normally direct the polymerase to particular genes in response to signals, were depleted from that fraction.

This shift in the polymerase’s social circle is mechanistically significant. A polymerase that is increasingly bound to Mediator and core subunits but less associated with gene-specific transcription factors suggests a reprogramming of transcriptional control away from signal-responsive gene activation and toward a more basal or virally steered mode. Combined with the increased heterochromatinization and the repositioning of TAD boundaries, the picture that emerges is one of coordinated change: the physical folding of the genome, the distribution of the transcription machinery, and the resulting transcriptome are all being reshaped together during the first six hours of infection.

The implications extend beyond basic virology. African swine fever continues to inflict substantial economic losses on the global swine industry, and the absence of a widely available safe and effective vaccine has made understanding the virus’s evasion strategies a research priority. By showing that ASFV manipulates host chromatin architecture so early and so extensively, the study opens a new dimension for antiviral intervention. If specific host factors mediate the observed heterochromatinization or the redistribution of polymerase occupancy, those factors could become drug targets. Similarly, the finding that early innate immune activation proceeds normally even without MGF-encoded virulence factors suggests that the critical battle between host defense and viral evasion is fought on a chromatin landscape, not merely at the level of individual signaling pathways.

The work also adds ASFV to a growing list of viruses known to touch the three-dimensional genome, but with a distinctive twist. Because ASFV is a cytoplasmic replicator, its nuclear influence must be exerted through delivered proteins, RNAs, or the viral DNA itself, rather than through a nuclear replication cycle. Disentangling which viral components drive the chromatin remodeling, and determining whether blocking that remodeling restores full antiviral gene expression, are the natural next questions. For now, the study provides the most systematic view yet of how a lethal agricultural pathogen rewrites the physical grammar of the host genome in its opening hours, and it suggests that the nucleus, long viewed as a bystander in ASFV infection, is in fact an active battlefield.

Subject of Research: Chromatin architecture remodeling in host cells during early African swine fever virus infection

Article Title: ASFV early infection dynamically remodels host chromatin architecture to evade immune responses

Article References: Xu, Y., Yang, B., Zhu, J., Sunkang, Y., Xing, L., Wang, C., Yang, J., Xu, W., Dai, J., Zhang, J., Cao, G., & Xiao, K. (2026). ASFV early infection dynamically remodels host chromatin architecture to evade immune responses. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06467-9

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06467-9

Keywords: African swine fever virus, ASFV, chromatin architecture, Hi-C, RNA polymerase II, macrophages, immune evasion, topologically associating domains, heterochromatin, innate immunity, virus-host interactions, multi-omics

The team, led by Jun Gao and Lingwei Sun of the Institute of Animal Science and Veterinary Medicine at the Shanghai Academy of Agricultural Sciences, with corresponding authors Caifeng Wu and Jianjun Dai, built the assembly from scratch using PacBio third-generation long-read sequencing combined with Hi-C technology. Long reads capture stretches of DNA far longer than older short-read methods, which is essential for resolving repetitive regions, structural variants, and other difficult stretches of the genome. Hi-C then provides the crucial scaffolding step: by chemically fixing chromosomes inside the cell nucleus and sequencing the physically adjacent DNA fragments, Hi-C data reveal which sequences belong to the same chromosome and how they are ordered, allowing the contigs to be anchored into complete chromosome-scale assemblies.

After a de-redundancy step to remove duplicated sequences, the assembled Pudong White Pig genome totaled approximately 2.68 gigabases, in line with the expected size of the pig genome. The quality metrics that matter most to genome builders told a favorable story: the new assembly showed higher sequence integrity and fewer genomic gaps than Sscrofa11.1, the Duroc-derived reference genome that is currently the most widely used standard in pig genomics. That comparison is significant because reference genomes shape nearly every downstream analysis, from mapping reads in association studies to calling variants; gaps and missing segments in a reference can hide biologically important variation, particularly for breeds that diverged from the reference animal’s lineage long ago.

A reference genome for a single breed, however, only tells part of the story. To capture the fuller spectrum of pig genetic diversity, the researchers constructed a pan-genome from 30 domestic and international pig breeds. A pan-genome represents the union of all genomic content across a set of individuals, distinguishing the core sequences shared by all breeds from the accessory sequences present in only some. In this analysis, the team identified a total of 135,859 non-reference sequences, or NRSs, stretches of DNA that simply do not exist in the standard reference genome. These absent sequences can include functional genes, regulatory elements, and structural variants that influence traits ranging from disease resistance to reproduction, which is precisely why they are invisible to studies that rely on a single reference.

Stringent quality control filtering validated 65,193 of those non-reference sequences, with a combined length of 108.33 megabases, roughly 4 percent of the pig genome’s total size. That is a substantial amount of genetic material that the standard reference fails to capture. Within this validated set, the researchers identified 594 non-reference sequences specific to Shanghai’s indigenous pig breeds, found across the four Shanghai native pig populations included in the pan-genome analysis. These breed-specific insertions represent a molecular signature of local genetic heritage, shaped by centuries of adaptation to the climate, feeding practices, and selection pressures of the Yangtze River delta region.

Some of these sequences land in scientifically intriguing places. One insertion site, specific to Shanghai indigenous pigs, sits at the BMPR1B gene, which is linked to reproductive performance in Taihu lake pigs, a famous group of Chinese native breeds renowned for their exceptional fecundity. The presence of a breed-specific insertion at a locus associated with such an economically vital trait suggests that local breeds may harbor unique regulatory or coding variation affecting litter size, variation that commercial reference genomes cannot reveal. For breeders working to conserve and improve native stocks, pinpointing such loci is a first step toward understanding and potentially exploiting the genetic basis of traits that make indigenous pigs valuable.

The analysis also revealed a striking pattern in the distribution of non-reference sequences: a hotspot region concentrated in the central portion of chromosome X, spanning roughly the 59 to 89 megabase interval. Hotspots of this kind can indicate regions of unusual structural dynamism, perhaps shaped by selection, recombination patterns, or the history of the X chromosome in domestication. Because the X chromosome plays a distinctive role in inheritance and can carry loci affecting fertility and other traits, a concentrated reservoir of reference-missing sequence there is a finding that will likely prompt closer examination in future studies of pig genetics and breeding.

Beyond the pan-genome work, the assembly allowed the team to confirm a previously reported splice mutation at position 41,486,012 on chromosome 8 of the Sscrofa11.1 reference genome. This locus is central to the genetics of coat color: at this site, both the Pudong White Pig and the Shanghai White breed carry the same dominant white genotype characteristics seen in the Landrace and Yorkshire breeds, the two dominant commercial white pig lines worldwide. The confirmation ties the Pudong White Pig’s defining visual trait to a known molecular mechanism and demonstrates that the new assembly can be used to validate and contextualize variants originally discovered on the older reference, an important cross-check for the genomics community.

The broader significance of the work lies in what it provides for conservation and breeding. The Pudong White Pig is endangered, and endangered livestock breeds face a double risk: the loss of the animals themselves and the loss of the genetic adaptations they carry, adaptations that may prove valuable as agriculture confronts disease pressure, climate change, and shifting market demands. A chromosome-level reference genome is a permanent, digital record of that heritage. It enables accurate genotyping of remaining herds, supports marker-assisted selection and genomic selection programs aimed at rebuilding population numbers without losing genetic diversity, and allows researchers to detect inbreeding and manage breeding pairs to maximize retained variation.

The study also adds to a growing recognition that global livestock genomics needs more than one reference per species. As this work shows, a single Duroc-based reference leaves out more than 100 megabases of real, validated sequence found across just 30 breeds, including hundreds of sequences unique to Shanghai’s native pigs. The Pudong White Pig assembly, funded by China’s National Key R&D Program and the Shanghai Agricultural Science and Technology Innovation Project, and conducted with ethics approval from the Shanghai Academy of Agricultural Sciences, offers a template for similar efforts: combine long reads with Hi-C, anchor every chromosome, then embed the result in a multi-breed pan-genome to reveal what single references miss. For a white-coated survivor of Shanghai’s agricultural landscape, the new genome is both a scientific milestone and a lifeline.

Subject of Research: Chromosome-level de novo genome assembly and pan-genome analysis of the endangered Shanghai Pudong White Pig

Article Title: Chromosome-level genome assembly of the Shanghai Pudong White Pig (Sus scrofa domesticus‌)

Article References: Gao, J., Sun, L., Cao, M., Tu, W., Zhang, H., Zhang, S., Xu, J., He, M., Zhang, D., Wu, C., & Dai, J. (2026). Chromosome-level genome assembly of the Shanghai Pudong White Pig (Sus scrofa domesticus‌). BMC Genomics. https://doi.org/10.1186/s12864-026-13407-1

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13407-1

Keywords: Pudong White Pig, genome assembly, PacBio sequencing, Hi-C, pan-genome, non-reference sequences, Sus scrofa domesticus, endangered livestock breed, BMPR1B, dominant white coat color, chromosome X, BMC Genomics

The tool, called Spatial ChIP, or ChIPSP, is an R package developed by Tianyi Zhou, Kevin Song, Hui Huang, and Qin Feng of the Center for Nuclear Receptors and Cell Signaling and the Department of Biology and Biochemistry at the University of Houston, together with Ning Lyu of the Division of Pharmacoepidemiology and Pharmacoeconomics and the Harvard-MIT Center for Regulatory Science at Harvard Medical School. Described in the journal BMC Genomics, ChIPSP integrates standard ChIP-seq data with chromatin loop interactions derived from Hi-C, a genome-wide chromosome conformation capture assay that detects which DNA segments physically come into contact inside the nucleus. By overlaying transcription factor binding peaks onto Hi-C maps, the package prioritizes candidate transcription factor-associated genes that lie within the same three-dimensional regulatory neighborhoods, even when they are far apart in linear genomic distance.

The technical logic behind ChIPSP is straightforward but powerful. Hi-C experiments, often performed at resolutions ranging from tens of kilobases down to a few kilobases, yield millions of pairwise chromatin interactions that can be filtered into significant loop calls. ChIPSP takes these loop anchors and asks which of them overlap transcription factor binding sites identified by ChIP-seq, and which of the opposing anchors contain gene promoters. A gene becomes a candidate target when its promoter participates in a loop whose other anchor is occupied by the transcription factor of interest. This spatial framework captures enhancer-promoter communication that crosses chromatin loop boundaries, situations in which the nearest-gene heuristic would either pick the wrong gene or miss the true regulatory relationship entirely. The result is a ranked table of candidate genes accompanied by the supporting loop and peak evidence, allowing researchers to inspect the underlying interactions for any locus of interest.

To evaluate the package, the team applied it to one of the most clinically consequential transcription factors in oncology: the androgen receptor, or AR, the nuclear hormone receptor that drives prostate cancer growth and is the principal target of modern androgen receptor pathway inhibitors such as darolutamide. Using ChIP-seq data from LNCaP prostate cancer cells, a widely used model of androgen-dependent prostate cancer, ChIPSP identified 1,499 candidate AR-associated genes. Strikingly, 658 of these genes, more than forty percent of the candidate list, were missed entirely by conventional linear annotation methods. In other words, more than four hundred additional potential AR targets emerged simply by asking how the genome folds rather than how it reads in a straight line.

The biological plausibility of these newly detected candidates was tested using RNA-seq. Many of the 658 spatially linked genes proved to be androgen-responsive, changing their expression in response to androgen signaling, which is exactly what would be expected of genuine AR targets. Pathway analysis of the expanded candidate set revealed enrichment in developmental transcriptional programs that were distinct from the pathways captured by standard ChIP-seq annotation alone. This suggests that conventional approaches have not merely been undercounting targets but may have been systematically skewing the perceived biology of androgen receptor signaling toward a subset of programs that happen to sit near AR binding sites in linear space.

Among the individual loci highlighted by the analysis, the genes KRT8, which encodes the keratin 8 intermediate filament protein, and MAF, which encodes a transcription factor implicated in cancer biology, stood out as consistent with candidate long-range AR-associated regulation across chromatin loop boundaries. At these loci, AR-bound distal regions are brought into contact with the gene promoters by looping, providing a structural explanation for how the receptor could control genes that appear distant or unrelated in linear genomic coordinates. Supplementary analyses further supported the spatial linkages, showing androgen receptor occupancy and H3K27ac enhancer marks, a histone modification associated with active regulatory elements, at the relevant genomic regions, and connecting distal AR-bound sites to additional targets including NDRG1 and ERRFI1.

To demonstrate that the approach is not specific to a single receptor or cell type, the researchers applied ChIPSP to the glucocorticoid receptor, or GR, another nuclear hormone receptor, using ChIP-seq data from A549 lung cancer cells. In this second system, the package nominated candidate gene targets including IRS2, UBL3, and FOXO1. The team then showed, using RT-qPCR validation experiments, that these genes were responsive to dexamethasone, a synthetic glucocorticoid that activates the receptor. The confirmation that spatially nominated candidates respond experimentally to receptor activation provides a second, independent nuclear-receptor example and strengthens the case that the method captures genuine regulatory relationships rather than genomic coincidences.

The implications for cancer research are considerable. Nuclear receptors such as AR and GR are among the most heavily drugged classes of transcription factors, and identifying their full repertoire of target genes is essential for understanding both therapeutic response and resistance. Genes that regulate cell identity, survival, and lineage plasticity often sit far from the binding sites that control them, and a nearest-gene annotation strategy can leave entire regulatory programs unexamined. By bridging protein-DNA binding data with chromatin interaction maps, ChIPSP extends ChIP-seq annotation into a spatial framework and complements rather than replaces conventional peak-to-gene annotation. Because it is distributed as an R package, the tool is accessible to the same bioinformatics community that already processes ChIP-seq data, requiring only ChIP-seq peak calls and publicly available Hi-C loop datasets as input.

The work also arrives at a moment when three-dimensional genome organization has moved from a specialist interest to a mainstream concern in genomics. Hi-C and related chromatin conformation capture technologies have matured, and public repositories now hold chromatin loop maps for many of the same cancer cell lines routinely used in ChIP-seq studies. Tools like ChIPSP make it practical to combine these data layers routinely rather than treating 3D context as an afterthought reserved for detailed single-locus case studies. The authors note that Hi-C loop detection is resolution-dependent, an important technical caveat for users, since loops called at coarse resolution may merge distinct regulatory contacts, while very high resolution data may be needed to resolve fine-scale enhancer-promoter architecture.

For researchers studying transcription factors in cancer and beyond, ChIPSP offers a practical route to regulatory targets that are invisible to conventional approaches. The package’s developers, who received no external funding for the research, have made the tool available as an open-access contribution, with the underlying study published in BMC Genomics and accompanied by ranked candidate gene lists, pathway analyses across KEGG, Gene Ontology, Hallmark, and Reactome databases, and experimental validation data. As spatially aware annotation becomes part of the standard ChIP-seq analysis pipeline, the field may find that the genomes of cancer cells have been holding far more regulatory information in their folds than linear analysis ever revealed.

Subject of Research: An R package integrating ChIP-seq with Hi-C chromatin loop data to characterize spatial transcription factor gene regulation in three-dimensional genomic context.

Article Title: Spatial ChIP (ChIPSP), an R package for characterizing spatial gene regulation

Article References: Zhou, T., Song, K., Huang, H., Lyu, N., & Feng, Q. (2026). Spatial ChIP (ChIPSP), an R package for characterizing spatial gene regulation. BMC Genomics. https://doi.org/10.1186/s12864-026-13371-w

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13371-w

Keywords: ChIPSP, ChIP-seq, Hi-C, three-dimensional genome organization, chromatin looping, transcription factor, androgen receptor, glucocorticoid receptor, prostate cancer, enhancer-promoter interactions, R package, gene regulation

To appreciate why this matters, it helps to consider the two measurements the method combines. The first, chromatin accessibility, is conventionally assayed by a technique known as ATAC-seq, or assay for transposase-accessible chromatin using sequencing. ATAC-seq exploits an engineered enzyme, a hyperactive Tn5 transposase, that preferentially inserts sequencing adapters into regions of the genome that are physically open. Because open chromatin tends to correspond to active promoters, enhancers, and other regulatory elements, the density of transposase insertion at any given locus serves as a proxy for that locus’s regulatory activity. ATAC-seq has become one of the most widely used assays in genomics precisely because it is comparatively simple, requires modest amounts of material, and can be applied to single cells, yet on its own it says nothing about where in a tissue the measured cell resides or how its DNA is folded.

The second measurement, chromatin organization, is conventionally assayed by Hi-C, a method that captures the three-dimensional contacts between genomic regions. In Hi-C, DNA that is physically close together in the nucleus, even if far apart along the linear chromosome, is crosslinked, cut, ligated together, and then sequenced; the resulting contact maps reveal loops, topologically associating domains, and larger-scale compartments that shape which enhancers can reach which genes. Hi-C has been enormously influential in showing that genome folding is not a passive consequence of packaging but an active regulatory layer, with disruptions to loops and domains implicated in developmental disorders and cancer. Like ATAC-seq, however, conventional Hi-C is performed on populations of dissociated cells, which erases the spatial context in which those folding patterns occur.

Spatial-ATAC-Hi-C unites these two assays on the same thin tissue section. The general strategy follows the logic of spatial genomics platforms that have emerged over the past several years: a tissue section is placed on a substrate patterned with an array of indexed capture spots or otherwise barcoded in space, enzymatic reactions are carried out in situ, and the material released at each position is tagged with a spatial barcode before sequencing. In this case, the workflow is designed so that both the transposase-tagged fragments reporting on open chromatin and the ligation products reporting on long-range genomic contacts are captured and barcoded from the same tissue coordinates. After sequencing, computational processing separates the two modalities, producing for each spatial pixel or spot both an accessibility profile and a contact profile, alongside histological information from the underlying tissue.

The dual readout is what gives the method its distinctive power. Accessibility data alone can identify which regulatory elements are active in a given tissue region, and contact data alone can show how the genome is folded there, but the combination allows researchers to ask whether the two are coupled at the same location. A region of open chromatin sitting in a tissue microenvironment where the relevant loop is present may behave very differently from an identically open region where the loop is absent. Conversely, cell types that look similar in their accessibility profiles may differ in their higher-order folding, or vice versa. Having both measurements from the same spatial coordinates makes it possible to detect such discordances rather than inferring them by overlaying separately generated datasets, each with its own artifacts and alignment uncertainties.

The authors demonstrated the technique on mouse brain, a tissue whose elaborate cellular architecture makes it a natural testing ground for spatial genomics. The brain contains densely intermingled populations of excitatory and inhibitory neurons, astrocytes, oligodendrocytes, microglia, and other cell types arranged in layered and regionally specialized structures. Applying Spatial-ATAC-Hi-C to mouse brain sections allowed the researchers to profile chromatin accessibility and organization in a spatially resolved manner across these structures, illustrating how the method can recover spatially patterned regulatory information from a complex mammalian organ. The demonstration establishes that the chemistry and barcoding steps are compatible with intact, structurally preserved tissue rather than dissociated cells, which is the central technical hurdle for any spatial assay of this kind.

The second demonstration moved from normal tissue to disease, with applications to human glioblastoma and astrocytoma samples. Gliomas are tumors arising from glial or glial progenitor lineages, and glioblastoma in particular is among the most aggressive of human cancers, characterized by infiltrative growth, pronounced intratumoral heterogeneity, and poor responses to therapy. Spatially resolved measurements are especially valuable in this setting because the microenvironment of a tumor varies dramatically from one region to another: proliferative cores, invasive edges, hypoxic niches, and regions infiltrated by immune cells can all coexist within a single section. By co-profiling accessibility and chromatin architecture across such regions, the method offers a way to examine whether regulatory programs and genome folding differ between tumor compartments, and how tumor cells’ epigenetic states relate to their immediate surroundings.

For cancer biology, the ability to measure chromatin architecture in situ is particularly intriguing. Genome folding alterations, including changes in topologically associating domains and enhancer-promoter loops, have been implicated in oncogene activation and tumor suppressor silencing, but these observations have mostly come from cell lines or dissociated tumors, where the relationship between folding and tissue context is lost. A spatial assay that preserves histological landmarks means that contact maps can be interpreted alongside the pathology visible in the section, connecting molecular events to morphological features such as tumor margins or regions of distinct grade. The same logic applies to accessibility: enhancer activity in an invasive edge cell can be compared directly with that in a cell at the tumor core, without the confounding of bulk averaging.

Like all emerging technologies, Spatial-ATAC-Hi-C comes with considerations that will shape its adoption. Spatial assays of chromatin generally trade off resolution, throughput, and sensitivity: capturing both short fragments from transposase tagmentation and long-range ligation products from the same section is chemically demanding, and the amount of informative data recoverable per spatial unit depends on the size of the spots, the thickness of the section, and the depth of sequencing. Computational analysis is also more involved than for either assay alone, since contact maps are sparse at single-spot resolution and typically require aggregation, imputation, or specialized visualization to interpret. The authors’ demonstrations on mouse brain and human glioma samples suggest these challenges are tractable, but as with any new platform, broader adoption will depend on reproducibility across laboratories, tissue types, and sample qualities, including archived or clinically derived specimens.

Even so, the arrival of a method that co-profiles chromatin accessibility and spatial genome organization marks a meaningful expansion of the spatial genomics toolkit. Until now, researchers studying gene regulation in tissues have had to choose between knowing where a regulatory state occurs and knowing how the genome is physically configured, or else stitch together separate experiments. Spatial-ATAC-Hi-C closes that gap, opening the possibility of systematically mapping how the folded genome varies across development, physiology, and disease within the native architecture of organs. For fields ranging from neurobiology to cancer research, where epigenetic state and tissue context are inseparable determinants of cell behavior, the ability to read both layers of regulation from the same tissue section is likely to find rapid and wide application.

Subject of Research: Spatially resolved co-profiling of chromatin accessibility and three-dimensional chromatin architecture in mammalian tissues

Article Title: Spatial chromatin architecture and accessibility co-profiling of mammalian tissues

Article References: Wang, P., Wang, J., Wang, Q., Youngblood, M. W., Cheng, Y., Tao, B., Wong, J. H.-Y., Luan, Y., Yu, S., Swaroop, A., Nandoliya, K. R., Najem, H., Fu, Y., McCortney, K., Zhang, D., Diao, Y., Heimberger, A. B., Sonabend, A. M., Horbinski, C. M., … Yue, F. (2026). Spatial chromatin architecture and accessibility co-profiling of mammalian tissues. Nature Methods. https://doi.org/10.1038/s41592-026-03217-4

Image Credits: AI Generated

DOI: 10.1038/s41592-026-03217-4

Keywords: Spatial-ATAC-Hi-C, chromatin accessibility, chromatin architecture, spatial genomics, ATAC-seq, Hi-C, mouse brain, glioblastoma, astrocytoma, epigenomics, gene regulation, Nature Methods