The research team, led by Nirmalya Ganguli with Srimoyee Koner as first author, isolated and characterized promoter regions from three mouse genes that are known to be active specifically in testicular germ cells: Ldhc, Tex13c, and Dazl. Each of these genes represents a different aspect of sperm biology. Ldhc encodes the testis-specific form of lactate dehydrogenase, an enzyme that has been described as the ultimate testis-specific gene because of its tightly restricted expression pattern. Tex13c belongs to a family of intronless genes expressed after meiosis, and its protein product is thought to be involved in transcriptional repression within the nucleus of developing germ cells. Dazl, meanwhile, is a master translational regulator of spermatogenesis, essential for the proper development of male germ cells.

Why does such a comparative study matter? Promoters are the workhorses of functional genomics. When researchers introduce a gene, a reporter, a short hairpin RNA, or a genome-editing tool into cells, the promoter determines whether the construct will be active in the right cells at the right time. Ubiquitous promoters, such as the widely used CMV promoter, can drive expression broadly, but that lack of specificity is a liability in a tissue as complex as the testis, which contains germ cells at many different stages of development as well as somatic support cells such as Sertoli and Leydig cells. A promoter that fires only in germ cells gives researchers a way to manipulate those cells while leaving their neighbors untouched. Yet, as the authors note in their background section, no prior comparative analysis had established which promoters are most robust for driving expression at various stages of mammalian germ cell development.

Specificity and strength of promoter activity depend on transcription factors that bind to the promoter region. The regulatory logic of germ cell genes is distinctive: many late-stage genes lack canonical TATA boxes and instead rely on cAMP-responsive elements and other motifs that respond to the hormonal and developmental cues of spermatogenesis. Because each promoter recruits its own combination of transcription factors, two promoters from genes expressed in the same tissue can behave very differently when linked to a transgene. That variability is exactly what the Hyderabad team set out to quantify by placing candidate promoters head to head under identical experimental conditions.

The methodological centerpiece of the study is testicular electroporation, a non-surgical technique that delivers DNA constructs directly into the cells of the mouse testis. Rather than creating transgenic mouse lines, which can take months and considerable resources, electroporation allows researchers to inject a plasmid into the seminiferous tubules and apply short electrical pulses that open transient pores in cell membranes, letting the DNA enter. The technique has been established in earlier work as a rapid way to test promoter activity in the native environment of the testis, where germ cells experience the full complement of their normal transcription factors. The team used this approach to validate the expression of marker proteins driven by each candidate promoter in vivo.

When the results were compared, a clear hierarchy emerged. The Ldhc promoter exhibited the strongest germ cell-specific expression of the three, followed by the Dazl promoter. The Tex13c promoter, consistent with its more restricted post-meiotic role, showed a narrower activity profile. For researchers planning gene-function studies, this ranking is immediately actionable: the Ldhc promoter emerges as the preferred driver when maximum expression in germ cells is needed, while the Dazl promoter offers a strong alternative with its own pattern of activity across germ cell stages.

To demonstrate that the winning promoter could do more than glow in a reporter assay, the team carried out a proof-of-concept functional experiment. They placed a short hairpin RNA targeting the c-Kit gene under the control of the Ldhc promoter and delivered it to the mouse testis. The c-Kit gene encodes a receptor tyrosine kinase that plays a well-documented role in the survival and proliferation of differentiating germ cells, and its manipulation is a standard test of whether a delivery system can actually change gene expression in the testis. The result: expression of the shRNA downregulated c-Kit in the mouse testis, showing that the Ldhc promoter is not merely active but capable of driving biologically meaningful silencing constructs in germ cells.

The implications extend beyond mouse genetics. Spermatogenesis is a frequent casualty of human infertility, and disorders of sperm production remain difficult to diagnose at the genetic level despite decades of routine clinical testing. Robust germ cell-specific promoters give researchers a precise instrument for probing gene function during sperm development, potentially illuminating which genes, when disrupted, underlie male infertility. The authors also point to a second, broader application: these promoters can be used to express genome-editing tools specifically in germ cells. Restricting editing machinery to the germ cell population would be a prerequisite for any strategy that seeks to modify the germline, whether for research into developmental genetics or for longer-term applications in animal biotechnology, where engineered lines of livestock or laboratory animals are produced through the germline.

There are also technical nuances worth appreciating. Earlier work by members of the same group had isolated and characterized promoters from the mouse genome to drive post-meiotic germ cell-specific expression, and the present study builds on that foundation by expanding the comparison and validating activity in vivo. The use of an optimized microRNA-like backbone for the shRNA construct reflects current best practice in RNA interference, designed to achieve effective silencing from a single copy of the construct. Quantitative real-time PCR with the comparative threshold method provided the readout of gene expression changes, and the animal experiments were conducted with approval from the Institutional Animal Ethics Committee of the National Institute of Animal Biotechnology, underscoring the regulatory rigor behind the work.

For the field of reproductive biology, the study fills a practical gap that has persisted for years. Single promoters have been validated before, including the classic demonstration in transgenic mice that the LDHC promoter drives testis-specific expression, and germ cell reporters based on Dazl have been generated previously. What was missing was a side-by-side evaluation conducted under the same conditions, in the same tissue, with the same delivery method, so that researchers could make an informed choice among candidates. By ranking Ldhc above Dazl and establishing both as reliable drivers during spermatogenesis, the Hyderabad team has handed gene-function researchers a validated toolbox. As genome editing moves deeper into studies of development and fertility, the humble promoter, the oldest tool in the transgenic toolkit, is proving once again that the success of any genetic experiment begins with choosing the right switch.

Subject of Research: Comparative characterization of germ cell-specific promoters for driving transgene expression during mouse spermatogenesis

Article Title: Comparative evaluation of germ cell-specific promoters identifies robust drivers of transgene expression during mouse spermatogenesis

Article References: Koner, S., Das, A., Chatterjee, S., & Ganguli, N. (2026). Comparative evaluation of germ cell-specific promoters identifies robust drivers of transgene expression during mouse spermatogenesis. Molecular Biology Reports, 53(1), Article 1648. https://doi.org/10.1007/s11033-026-12822-7

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12822-7

Keywords: promoters, spermatogenesis, germ cells, Ldhc, Dazl, Tex13c, transgene expression, testicular electroporation, c-Kit, male fertility, functional genomics, gene regulation

The research team, led by Imad Abugessaisa of the RIKEN Center for Integrative Medical Sciences together with colleagues including Wendy van de Sande of Erasmus MC, built their atlas using a technique called LQ-ssCAGE, or low-quantity single-strand cap analysis of gene expression. CAGE-based methods capture the 5-prime capped ends of RNA molecules, which mark the exact positions where RNA polymerase II initiated transcription. Because the method both identifies these transcription start sites and quantifies how abundantly each one is used, it provides a two-in-one view of promoter activity and gene expression. The researchers prepared 72 LQ-ssCAGE libraries and, in parallel, 72 standard RNA-seq libraries from the same samples, using the RNA-seq data as an independent quality check on the CAGE measurements.

The experimental design was carefully constructed to mimic a real infection and its treatment. Half of the larvae were infected with Madurella mycetomatis, a high-priority fungal pathogen on the World Health Organization’s fungal priority list and the most common cause of eumycetoma, a neglected tropical disease characterized by tumorous lesions in subcutaneous tissue. The infected and uninfected larvae were then divided into three groups: one treated with solvent only, one with the antifungal drug itraconazole, and one with ravuconazole, which was recently assessed in the first clinical trial for eumycetoma. Samples were collected at four time points, 6, 30, 72, and 168 hours, with three biological replicates per condition, producing a rich time-course dataset of promoter activity during infection and therapy.

Sequencing yielded roughly 53.3 million cap-trapped 5-prime reads, which the team mapped to the current reference genome assembly, CSIRO_AGI_GalMel_v1, a high-quality assembly scoring 99.3 percent on the lepidoptera BUSCO benchmark and containing 15,138 genes and pseudogenes. Quality control was thorough. Gene body coverage showed the expected strong 5-prime enrichment for CAGE libraries and uniform coverage for RNA-seq libraries, sequencing depth was consistent across samples with no outlier batches, and pairwise correlation analysis between normalized RNA-seq and CAGE counts showed strong positive Spearman correlations, confirming that the two independent assays produced reproducible expression profiles.

From the raw data, the researchers identified 1,219,124 individual CAGE transcription start sites, which they filtered down to 332,601 biologically relevant positions. Clustering these signals produced 40,200 tag clusters, from which they derived two tiers of transcription start site peaks: a permissive set of 27,942 peaks detected in at least four samples, and a robust set of 5,437 peaks detected in all samples. They also identified 728 bidirectional clusters, the molecular signature of active enhancers, which after filtering to non-exonic intronic and intergenic locations yielded 141 candidate active enhancers. Users of the atlas can therefore choose between the permissive and robust annotations depending on how conservative their analyses need to be.

The validation evidence for these calls is strong. Among all CAGE peaks, 32.1 percent overlapped annotated 5-prime untranslated regions, and more strikingly, 80.27 percent of all CAGE tags fell within 5-prime UTRs, exactly where active promoters should sit. Genome-wide enrichment analysis showed that the identified peaks were strongly and specifically enriched at annotated promoters of both protein-coding and non-coding RNA genes, while intronic and intergenic regions were depleted. Dinucleotide frequency analysis at the dominant start sites revealed the characteristic initiator sequence bias expected at insect RNA polymerase II promoters, and the atlas-derived annotations displayed a markedly stronger core promoter signature, including TATA box and GC patch signals, than the existing NCBI reference annotation, demonstrating a genuine improvement in transcription start site resolution.

The atlas also revealed the two canonical shapes of animal promoters. Using the interquartile range of tag clustering as a measure of initiation precision, the team classified 4,746 broad promoters, where transcription starts at many scattered positions, and 1,679 sharp promoters, where initiation is concentrated at a single precise site. As expected from earlier CAGE studies in mammals and flies, sharp promoters carried a stronger TATA box positioned roughly 30 bases upstream of the start site, while broad promoters were GC-rich, consistent with CpG island-associated architecture. Most genes, about 6,899, used a single dominant start site, whereas roughly 2,300 genes used multiple sites, and 71.1 percent of promoter-associated genes were linked to more than one tag cluster, echoing the widespread alternative promoter usage documented in Drosophila.

Perhaps the most clinically intriguing result concerns what did and did not change during infection. RNA-seq confirmed differentially expressed genes between infected and healthy larvae, consistent with the team’s earlier work on iron regulatory pathways during M. mycetomatis grain development. However, no differentially expressed genes were detected between infected larvae and infected larvae treated with itraconazole or ravuconazole. The authors note that this mirrors recent clinical observations in which neither drug showed measurable activity during the first six months of eumycetoma treatment, with persistent fungal viability and no reduction in lesion size or plasma beta-1,3-D-glucan levels. Differential transcription start site usage analysis did flag a handful of genes, including a cystinosin homolog and a peroxidase-like isoform, whose promoter selection shifted between healthy and infected samples, hinting that infection may add a layer of transcriptional regulation beyond simple changes in gene abundance.

Using expression correlations between nearby elements, the team predicted physical interactions between enhancers and promoters, finding that 682 of 37,320 transcription start sites interacted with 156 of 218 active enhancers. Interacting start sites were expressed at significantly higher levels than non-interacting ones, and the analysis also uncovered super-enhancers, clusters of closely spaced bidirectional enhancers. De novo motif discovery on the top 5 percent of expressed start sites pointed to RFX family transcription factors, with the RFX1 ortholog showing 7.1-fold enrichment, a factor conserved across animals and known in flies as a key regulator of ciliary and sensory neuron genes. The authors are candid about limitations: only about 700,000 reads per sample were generated, and saturation analysis suggests the depth was sufficient for highly expressed promoters but likely underestimated weak enhancers. Even so, with all data deposited in NCBI GEO and Mendeley Data, and the annotations browsable in the ZENBU genome browser, this first draft start site atlas gives the growing G. mellonella research community a foundation for gene regulation studies comparable to the reference resources long available for human and mouse.

Subject of Research: Genome-wide mapping of transcription start sites in Galleria mellonella larvae during fungal infection and antifungal treatment

Article Title: Genome-wide mapping of the Galleria mellonella transcription start sites during fungal infection and treatment

Article References: Abugessaisa, I., Konings, M., Manabe, R.-I., Tagami, M., Severin, J., Hasegawa, A., Kawashima, T., Kinoshita, H., Noma, S., Takahashi, C., Verbon, A., Okazaki, Y., van de Sande, W. W., & Kasukawa, T. (2026). Genome-wide mapping of the Galleria mellonella transcription start sites during fungal infection and treatment. iScience, 29(10), Article 117675. https://doi.org/10.1016/j.isci.2026.117675

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117675

Keywords: Galleria mellonella, transcription start sites, CAGE, Madurella mycetomatis, eumycetoma, promoters, enhancers, gene regulation, itraconazole, ravuconazole, genome annotation, infection model

The technical hurdle the team faced is one that has haunted genome biologists for years: the three-dimensional arrangement of enhancers and promoters at the scale of individual nucleosomes—the fundamental spools around which DNA wraps—has remained essentially invisible. Popular chromosome conformation capture methods such as Hi-C average over millions of cells and blur out anything smaller than tens of kilobases. Super-resolution imaging can reach the nanoscale, but only for a handful of selected loci at a time. To bridge the gap, the researchers developed a simulation framework that leverages region-capture Micro-C contact maps, an assay that zooms in on megabase-scale windows with nucleosome-level resolution, to infer full conformational ensembles of genomic regions.

A central innovation of the work is a data-processing step the authors call neighbor balancing. Standard Micro-C analysis assumes that contact density is uniform across the genome, an assumption that quietly erases one of the most biologically meaningful signals: the fact that some stretches of DNA are simply more densely packed, and therefore more frequently in contact, than others. The new balancing strategy identifies variation in contact density instead of normalizing it away. When the team applied the method, sharp dips in contact density appeared precisely at active promoters and enhancers—places where the chromatin fiber opens up and nucleosomes thin out. These dips had been smoothed over in coarser, genome-wide datasets, but region-capture data preserved them, and neighbor balancing made them interpretable.

With the corrected contact maps in hand, the researchers turned to a maximum-entropy inversion approach, a technique rooted in statistical mechanics that finds the least-biased set of polymer structures consistent with the experimental constraints. The resulting simulated ensembles were then put through a battery of validation tests. The simulated structures reproduced pairwise distance distributions measured independently by chromatin tracing, a super-resolution imaging method that follows the positions of dozens of genomic loci in single cells. The simulations also contained packing domains and nucleosome clutches—discrete clusters of neighboring nucleosomes—matching structures that electron microscopy and super-resolution imaging studies had previously observed directly in cells. In other words, the model did not merely fit the data it was trained on; it recovered structural features seen by entirely different experimental techniques.

The most striking finding emerged when the team examined what those structures actually look like. Far from being uniformly open, euchromatin generally forms condensed domains with packing densities comparable to those of heterochromatin. The difference lies in scale: euchromatic domains are smaller than their heterochromatic counterparts, but the chromatin fiber inside them is nearly as tightly packed. Local nucleosome concentrations inside domain interiors frequently exceeded 400 micromolar, an extraordinarily crowded environment for DNA. This result aligns with a growing body of imaging evidence suggesting that so-called open chromatin is condensed but liquid-like in living cells, and it forces a rethinking of what the light and dark bands of classical cytology actually represent.

If euchromatin is mostly dense, how do genes get read? The answer, according to the simulations, lies in the geometry of the domain surfaces. Kilobase-scale regions at promoters and enhancers often protrude from the condensed domains, extending outward into the surrounding nuclear space where they become highly accessible. The team found that protrusion probability, the positions of clutch boundaries, and local contact density all correlate closely with ATAC-seq coverage, a genome-wide measure of chromatin accessibility. Where the fiber pokes out of a domain, transcription factors can find their binding sites; where it is buried inside, it cannot. Accessibility, in this picture, is not a bulk property of open chromatin but a surface phenomenon.

This arrangement effectively compartmentalizes regulatory elements from the surrounding chromatin, and the authors argue that the geometry serves a functional purpose. By lifting enhancers and promoters onto domain surfaces, the cell facilitates protein binding and enhancer–promoter communication, the long-range conversations between distant regulatory elements that switch genes on. The condensed cores may act as scaffolds that keep related regulatory elements in physical proximity, while the exposed protrusions provide the docking sites. The study also found that both packing domains and clutches tend to be smaller around ATAC-seq peaks, suggesting that local accessibility reshapes chromatin organization at multiple scales simultaneously.

The hierarchical picture that emerges runs from nucleosomes to clutches to domains, and it holds across species and cell types. The team validated their framework in mouse embryonic stem cells and in several human cell lines, finding condensed domains throughout. Even when the researchers examined data from cells depleted of cohesin, the ring-shaped protein complex that extrudes DNA loops, the local structure was largely maintained: chromatin expanded at large scales, but clutch-scale organization and the correlation between protrusions and accessibility persisted. This indicates that the condensed-domain architecture of euchromatin is not simply a byproduct of loop extrusion but reflects more fundamental physicochemical interactions within the chromatin fiber itself.

The implications reach well beyond structural biology. Misregulated enhancer–promoter communication underlies many developmental disorders and cancers, and drugs that target chromatin regulators, such as bromodomain inhibitors, are already in clinical use. A model in which accessibility depends on whether a regulatory element sits on a domain surface offers a concrete structural hypothesis for how such drugs work and why their effects are so context-dependent. It also reframes a long-standing question in the field: rather than asking how euchromatin stays open, biologists may now ask how specific regulatory elements are actively extruded or maintained on domain surfaces, and what molecular machinery performs that positioning.

The study’s computational framework, along with its simulation trajectories and analysis code, has been made publicly available, allowing other groups to apply the approach to their own region-capture datasets. As region-capture Micro-C spreads through the genomics community, the MIT team’s method could become a standard tool for converting contact maps into physically realistic, nucleosome-resolution structures. What began as a technical exercise in matrix balancing has delivered a conceptual shift: the active genome is not a loose tangle waiting to be read, but a dense, well-organized material whose most important working parts hang, deliberately and accessibly, on the outside.

Subject of Research: Three-dimensional organization of euchromatin and regulatory elements at nucleosome resolution

Article Title: Euchromatin forms condensed domains with short active regions on the surface

Article References: Paggi, J. M., Long, L. Y., & Zhang, B. (2026). Euchromatin forms condensed domains with short active regions on the surface. Nature Genetics. https://doi.org/10.1038/s41588-026-02775-9

Image Credits: AI Generated

DOI: 10.1038/s41588-026-02775-9

Keywords: chromatin, euchromatin, heterochromatin, nucleosomes, Micro-C, enhancers, promoters, gene regulation, 3D genome organization, polymer simulations, chromatin accessibility, super-resolution imaging