The investigators used co-immunoprecipitation assays to identify BRD4 as an FBXO28-associated protein, then examined how the two molecules regulate transcription. FBXO28 mediated ubiquitination of BRD4 at lysine 395 through a lysine 63-linked conjugation model. Unlike ubiquitination processes that direct a substrate for destruction, this modification did not cause BRD4 degradation. Instead, it enhanced BRD4 activity as a transcriptional coactivator: changing FBXO28 expression altered the abundance of BRD4-target genes and affected BRD4 binding to the promoters of those genes.

The proposed downstream mechanism involves P-TEFb, a complex associated with transcriptional elongation. FBXO28-dependent ubiquitination of BRD4 facilitated activation of P-TEFb and subsequent phosphorylation of the serine 2 residue in the C-terminal domain of the largest subunit of RNA polymerase II. This phosphorylation was linked by the researchers to the initiation of transcription. The study therefore connects a ubiquitin-signaling event to changes in transcriptional programs that support the proliferation and movement of lung cancer cells.

The authors also described a positive feedback loop. BRD4 bound to the FBXO28 promoter, and increased FBXO28 transcription further strengthened the relationship between the two proteins. The experimentally supported model is that FBXO28 ubiquitinates BRD4, activated BRD4 increases FBXO28 expression, and elevated FBXO28 can reinforce BRD4 modification and activity. Overall, the work implicates the FBXO28–BRD4 feedback loop as a contributor to non-small cell lung cancer malignancy. Because the evidence comes from patient-sample associations and functional cellular experiments, the results indicate a promising research pathway rather than demonstrating an effective intervention or clinical benefit in people with lung cancer.

Subject of Research: FBXO28-mediated BRD4 ubiquitination and transcriptional activation in non-small cell lung cancer.

Article Title: FBXO28 promotes the malignancy of non-small cell lung cancer through ubiquitination-mediated activation of BRD4

Article References: Liang, Y., Jiao, Q., Zhang, F., Bai, W., Yang, J., Luo, X., Zhang, J., Tang, X., Tang, J., Zhang, Z., Yan, J., Lai, F., Sun, L., Cao, L., Zhang, W., & Lv, X.-B. (2026). FBXO28 promotes the malignancy of non-small cell lung cancer through ubiquitination-mediated activation of BRD4. Cell Death Discovery. https://doi.org/10.1038/s41420-026-03364-9

Image Credits: AI Generated

DOI: 10.1038/s41420-026-03364-9

Keywords: FBXO28, BRD4, non-small cell lung cancer, ubiquitination, transcription, P-TEFb, RNA polymerase II, cancer progression, positive feedback loop, promotes, malignancy, non-small

Zygotic genome activation, commonly abbreviated ZGA, is the moment when a newly fertilized egg stops relying on the maternal RNA stockpiled inside the oocyte and begins transcribing its own genes at scale. In mice, this transition culminates during the 2-cell stage, with what researchers call major ZGA occurring in late 2-cell embryos. It is a famously fragile period. A large fraction of embryos cultured in vitro stall at exactly this point, failing to complete the switch from maternal to embryonic control. That developmental arrest is one of the central bottlenecks for assisted reproductive technologies, or ART, and any intervention that eases the passage through ZGA has direct clinical relevance.

The researchers began by comparing mouse embryos cultured under hypoxic conditions with those grown in normal atmospheric oxygen, and with embryos developing in vivo. The results were clear: hypoxic culture improved blastocyst formation and reduced developmental arrest during the ZGA window. When the team profiled gene expression in late 2-cell embryos, they found that the zygotic transcriptome of hypoxia-cultured embryos closely mimicked the patterns seen in physiologically developing, in vivo embryos during major ZGA. In other words, low oxygen did not merely push embryos along; it steered their gene expression toward a more natural trajectory, blunting the artificial signature that in vitro culture typically imprints on the embryonic genome.

To understand how oxygen levels could exert such an influence, the investigators turned their attention to histone modifications, the chemical tags that decorate the protein spools around which DNA is wound. Their focus fell on H3K4me3, the trimethylation of histone H3 at lysine 4, a mark long associated with active and poised promoters. Using CUT&Tag, a sensitive technique for mapping chromatin marks in scarce samples, the team compared H3K4me3 landscapes between hypoxia- and normoxia-cultured late 2-cell embryos. The comparison revealed a striking redistribution. Under hypoxia, H3K4me3 became specifically enriched at the promoters of zygotic genes, while it decreased at transposable elements and at oocyte-specific genomic domains.

That redistribution matters because it tracks precisely with what an embryo needs at this stage. Zygotic genes must be switched on, while the transcription of transposable elements and lingering oocyte programs must be restrained. The elevated H3K4me3 at zygotic promoters under hypoxia was not an isolated observation; the team showed that higher expression deciles of genes corresponded to progressively higher H3K4me3 levels, confirming the expected positive correlation between this mark and transcriptional output. Genes that were upregulated under hypoxia showed enhanced H3K4me3 around their transcription start sites, and genome browser views at representative loci such as Kank2 and Grb2 illustrated the pattern directly.

The mechanistic story deepened when the researchers examined how H3K4me3 related to chromatin accessibility and the machinery of transcription itself. Regions of the genome that were chromatin-accessible, regions enriched for RNA polymerase II, and regions enriched for the Ser2-phosphorylated form of RNA polymerase II, known as Ser2P, all exhibited higher H3K4me3 enrichment in hypoxia-cultured late 2-cell embryos. Ser2 phosphorylation is a critical modification of the polymerase’s C-terminal domain that marks the elongation phase of transcription, when RNA synthesis is actively proceeding along the gene body. The co-localization of the histone mark with these polymerase states suggested that H3K4me3 under hypoxia was not just a passive correlate of activity but was functionally entwined with both the initiation and the elongation of zygotic transcripts.

Direct measurements supported that interpretation. Global levels of RNA polymerase II and of its Ser2-phosphorylated form were both increased under hypoxia, and the polymerase was recruited to the very regions that had gained H3K4me3, initiating transcription and driving elongation there. Immunofluorescence imaging of 5-EU incorporation, which labels newly synthesized RNA, showed that transcriptional activity in hypoxia-cultured embryos matched that of in vivo embryos at the late 2-cell stage, reinforcing the physiological fidelity of the hypoxic culture condition. At key ZGA loci including Obox3, Klf17, Ep300 and L3mbtl2, the team observed the H3K4me3 and polymerase signals converging on the genes that define the embryonic program.

Perhaps the most compelling piece of evidence came from a pharmacological experiment. The researchers treated embryos with CPI-455, an inhibitor of the lysine demethylases that normally erase H3K4me3. Blocking demethylation raised H3K4me3 levels, and, crucially, Pol II and Ser2P signals increased as a result. That causality test ties the epigenetic mark to the transcriptional machinery in a directional way: when H3K4me3 is preserved, the polymerase arrives in greater numbers and proceeds more vigorously into elongation. It also implicates enzymes such as KDM5B, a lysine demethylase targeted by this class of inhibitors, as potential regulators of the ZGA landscape that hypoxia appears to counterbalance.

Not every consequence of hypoxia pointed toward activation. Consistent with the reduced H3K4me3 at repetitive elements, the team found lower LINE1 transcription in hypoxia-cultured embryos, while ERV1 and ERVK retrotransposon transcripts were significantly upregulated under normoxia in keeping with their elevated H3K4me3 marks; LINE2, ERVL, B2 and B4 elements showed no significant differences. This pattern suggests that hypoxia helps the embryo silence potentially destabilizing repeat transcription while sharpening the activation of genuine zygotic genes, a double benefit at a developmental stage when genome integrity and precise timing are both at a premium.

The broader significance of the study lies in the bridge it builds between clinical practice and basic developmental biology. Low-oxygen culture has been a mainstay of assisted reproduction largely on empirical grounds, adopted because embryos simply did better. With this work, the field gains a molecular rationale: hypoxia raises promoter H3K4me3, promotes chromatin accessibility, and orchestrates acute zygotic transcription by fueling both polymerase recruitment and productive elongation. The authors frame their findings as a theoretical foundation for optimizing ART protocols and improving clinical success rates. If the same H3K4me3-dependent mechanism operates in human embryos, it could inform how culture conditions are tuned, how embryo quality is assessed, and potentially how pharmacological agents that modulate histone methylation might be used to rescue embryos that would otherwise arrest. For now, the mouse data provide a vivid picture of an embryo’s first genetic awakening being tuned, quite literally, by the air it breathes, or rather by the carefully reduced oxygen that mimics the environment of the oviduct where early development naturally unfolds.

Subject of Research: Epigenetic regulation of zygotic genome activation under hypoxic embryo culture

Article Title: H3K4me3-dependent transcript elongation drives hypoxia-enhanced zygotic genome activation

Article References: Chen, B., Sun, X., Miao, Y., Sun, S., Deng, M., & Liu, H. (2026). H3K4me3-dependent transcript elongation drives hypoxia-enhanced zygotic genome activation. BMC Biology. https://doi.org/10.1186/s12915-026-02762-w

Image Credits: AI Generated

DOI: 10.1186/s12915-026-02762-w

Keywords: zygotic genome activation, hypoxia, H3K4me3, RNA polymerase II, transcript elongation, embryo culture, assisted reproductive technologies, chromatin, epigenetics, mouse embryo, BMC Biology, fertility

The research team, led by investigators at Harbin Medical University and Northeast Agricultural University, used two complementary models of cell fate change: the reprogramming of mouse embryonic fibroblasts into induced pluripotent stem cells and the differentiation of embryonic stem cells into neuroectoderm. In both systems, disrupting SEC with KL-2, a peptidomimetic compound that selectively interferes with the interaction between the SEC scaffold protein AFF4 and the P-TEFb subunit Cyclin T1, dramatically impaired the transition. Crucially, the compound had no effect on the survival or proliferation of fibroblasts or stem cells under steady-state conditions, indicating that SEC is dispensable for maintaining a cell’s existing identity but indispensable for building a new one.

Timing proved to be everything. When the researchers inhibited SEC during the early days of reprogramming, the efficiency of induced pluripotent stem cell colony formation collapsed, and cells remained trapped in a mesenchymal state, never acquiring the epithelial morphology that marks successful reprogramming. RNA sequencing at day four revealed that more than half of the differentially expressed genes were downregulated, including core reprogramming factors such as Klf4 and Myc, along with cell cycle genes like Orc1 and pluripotency-associated genes such as Sall1. The downstream consequences were severe: cells accumulated in the G0/G1 phase, spindle structures became multipolar and abnormal, and karyotype analysis showed widespread chromosome number abnormalities in daughter cells.

To understand how SEC controls these genes, the team turned to the biophysics of transcription. After RNA polymerase II initiates transcription, it typically pauses 20 to 100 nucleotides downstream of the transcription start site, awaiting a release signal before productive elongation begins. Using published RNA polymerase II ChIP-seq datasets, the researchers quantified pause dynamics with two complementary metrics: the Traveling Ratio, which compares promoter-proximal to gene-body polymerase occupancy, and a two-dimensional Traveling Matrix method that simultaneously maps changes at both regions. Their analysis showed that early-activated genes like Klf4 and Myc carry pre-established paused polymerase even before reprogramming begins, and that reprogramming triggers their rapid release. In contrast, Oct4 and Sox2 lack pre-paused polymerase and instead acquire pause and release in a coupled, sequential manner during the mid-late stage.

CUT&Tag experiments confirmed that early SEC inhibition caused polymerase to pile up at the promoters of Klf4 and Myc while depleting from their gene bodies, a signature of failed pause release. ChIP-qPCR further showed reduced Ser2 phosphorylation of the polymerase C-terminal domain, the hallmark of productive elongation, and elevated pause indices at both loci. Ethynyl uridine incorporation assays demonstrated a global collapse in nascent RNA synthesis after SEC disruption, while overexpression of the SEC component ELL boosted it. The picture that emerged is one in which SEC acts as the trigger for the first transcriptional wave of reprogramming by liberating polymerase that was already parked at the most critical genes.

The most striking discovery, however, lay beyond the canonical mechanism. SEC is classically understood to promote pause release through P-TEFb-mediated phosphorylation of the polymerase C-terminal domain and the pause factors DSIF and NELF. But the researchers noticed that more than 40 percent of SEC target transcripts carry N6-methyladenosine, or m6A, the most abundant internal modification on eukaryotic messenger RNA. In Drosophila, m6A had already been shown to promote pause release, but whether the same held true in mammals was unknown. When the team inhibited SEC early in reprogramming, global m6A levels dropped sharply, as measured by RNA dot blot, ELISA, and the antibody-independent GLORI-seq method, which converts unmodified adenosines to inosines for single-nucleotide quantification. More than 23,000 m6A sites showed decreased modification, and the classic DRACH motif was enriched among the affected peaks, confirming their identity as bona fide m6A marks.

Using metabolic labeling with 4-thiouridine to isolate newly transcribed RNA, the researchers showed that SEC regulates m6A deposition co-transcriptionally. The single-base SELECT assay revealed that SEC inhibition significantly reduced m6A at specific sites in nascent Klf4 and Myc transcripts, including the promoter-proximal A701 site and gene-body A855 site of Klf4, while leaving non-m6A control sites untouched. Molecular docking predicted a close interaction between the SEC component ELL and the m6A methyltransferase METTL3, and this was validated by co-immunoprecipitation and immunofluorescence co-localization. ChIP-qPCR demonstrated that METTL3 and ELL co-occupy the promoters of Klf4 and Myc, and that SEC inhibition evicts both from these loci without changing total METTL3 protein levels. More than half of the transcripts losing m6A after SEC inhibition overlapped with those affected by METTL3 knockout, pointing to a shared regulatory pathway.

Functional experiments sealed the argument. Inhibiting METTL3 with the selective inhibitor STM2457 during the early stage of reprogramming reproduced the effects of SEC disruption: Klf4 and Myc expression fell, polymerase accumulated at their promoters, Ser2 phosphorylation declined, and reprogramming efficiency collapsed. Intriguingly, the study also uncovered a stage-specific duality: while early METTL3 inhibition was catastrophic for reprogramming, inhibition during the mid-late stage actually enhanced colony formation, suggesting that m6A plays opposing roles at different phases of the process. This may explain why previous studies have reported conflicting results about METTL3’s role in pluripotency. Most decisively, combined inhibition of SEC and METTL3 produced no additional increase in polymerase pausing beyond either treatment alone, and METTL3 overexpression could not rescue pause release when SEC was disrupted — evidence that the two operate in a single pathway, with SEC providing the scaffold that recruits METTL3 to specific loci.

The team then asked whether the same logic governs normal development. During retinoic acid-induced differentiation of embryonic stem cells, SEC inhibition selectively impaired the neuroectoderm lineage while leaving mesoderm and endoderm differentiation largely intact. Neural genes such as Nes and Tubb3 showed increased promoter-proximal pausing and reduced Ser2 phosphorylation upon SEC disruption, and global m6A levels again declined. STM2457 treatment similarly blocked neural marker acquisition, and reanalysis of published NET-seq data from METTL3 knockout cells revealed stalled nascent transcripts at the promoters of Nes and Tubb3. The authors propose that the functional divergence between SEC and the related elongation factor BRD4, which is known to drive mesendoderm specification, may act as a molecular determinant of lineage choice.

The implications extend well beyond the culture dish. Because SEC is dispensable for cell survival under steady-state conditions but essential whenever a cell must adopt a new identity, the findings suggest it may be a common driver of cell fate transitions throughout life, including embryonic development, tissue regeneration, and the initiation and progression of diseases such as cancer. The discovery that a transcription elongation complex doubles as a scaffold for RNA methylation adds a new layer to the growing intersection between the epitranscriptome and the transcriptional machinery, and hints that m6A deposited through the SEC-METTL3 pathway may act on R-loops to resolve paused polymerase. If confirmed, targeting this pathway could open new therapeutic avenues for conditions in which cell identity goes awry, from tumorigenesis to infertility.

Subject of Research: Role of the Super Elongation Complex and METTL3-mediated m6A modification in regulating RNA polymerase II pause release during cell identity transitions

Article Title: SEC Mediates m6A Deposition and Transcription Pause Release to Drive Cell Identity Transition

Article References: Zhang, Z., Xu, J., Wu, Y., Qu, Y., Hu, X., Sun, Y., Wu, Y., Liu, Z., Wang, J., & Lei, L. (2026). SEC Mediates m 6 A Deposition and Transcription Pause Release to Drive Cell Identity Transition. Advanced Science, Article e78131. https://doi.org/10.1002/advs.78131

Image Credits: AI Generated

DOI: 10.1002/advs.78131

Keywords: Super Elongation Complex, m6A, METTL3, RNA polymerase II, transcription pause release, iPSC reprogramming, embryonic stem cells, neuroectoderm differentiation, Klf4, Myc, cell fate transition, epitranscriptomics

The authors begin from a striking statistic: only about one percent of the 3.2 billion base pairs of the human genome codes for functional proteins, yet roughly 80.4 percent of the genome participates in non-protein-coding regulation of gene expression in at least one cell type. This enormous regulatory investment underscores how vital epigenetic gene regulation is at every stage of single- and multicellular life. Epigenetic modifications, chemical changes to DNA and the histone proteins around which it is wrapped, fold the genome into permissive and restrictive chromatin structures and nuclear arrangements. When these systems go awry, they contribute to the initiation and maintenance of diseased states. Yet while whole-genome mapping has revealed the landscape of these marks, the dynamics and variability of their interactions at the level of individual cells remain far less understood.

That variability is what biologists call transcriptional noise, the cell-to-cell variance in messenger RNA content, often quantified by the coefficient of variation, a mean-normalised standard deviation. Noise comes in two flavours. Extrinsic noise arises from subtle differences in the cellular microenvironment that affect processes such as growth rate and cell volume, and it tends to influence all gene expression within a given cell uniformly. Intrinsic noise, by contrast, stems from the randomness of biochemical reactions involving small molecule numbers, such as transcription factors binding to their cognate motifs, and it affects each gene uniquely. The distinction was famously established in bacteria by the Elowitz laboratory, which used two reporter genes expressing yellow and cyan fluorescent proteins under the same promoter: fluctuations in the combined signal reflected extrinsic noise, while divergence between the two colours within a single cell revealed intrinsic noise. The greatest source of intrinsic noise, the review emphasises, is the stochastic switching of gene promoters between active and inactive states.

The consequence of this switching is that messenger RNA is produced in short discrete bursts interspersed with longer periods of relative inactivity, a process occurring on timescales ranging from minutes to hours. Cell-to-cell variability results from the burst size, the number of RNA molecules produced during the active state; the burst frequency, how often transitions to the active state occur; and the degradation rate of the resulting transcripts. Remarkably, recent work has shown that fluctuations in gene expression can persist for several cell divisions, in line with the idea that bursting may drive phenotypic divergence of otherwise identical cells. On shorter timescales, bursting provides a way for cells to respond rapidly to stimuli. Understanding its regulation matters greatly for fields such as oncology, where heterogeneity in gene expression is associated with poor prognosis and drug resistance.

Here the review makes its most provocative conceptual move. The traditional two-state model, in which promoters flip between a single ON and a single OFF state, is increasingly challenged by evidence for more than two promoter states. The authors propose, in agreement with others, that bursting should be understood as a multi-scale phenomenon: during a macroscopic ON state, multiple small microbursts occur, corresponding to convoys of RNA polymerase II traversing the gene, separated by short and shallow OFF states lasting roughly 1.5 minutes, while deep OFF states extend far longer, around 9 to 34 minutes. The accumulation of these microbursts within each ON state produces a larger macroburst. This distinction matters because different experimental techniques operate at different scales. Single-cell transcriptomics generally captures the macroscale, lacking time resolution and fitting pre-determined models to distribution data, whereas live-cell imaging using MS2 or PP7 bacteriophage coat protein reporter systems offers very high temporal resolution and therefore probes the microscale. Even gene length matters: imaging a long gene captures macroburst dynamics as multiple polymerase convoys progress simultaneously, while a short gene reveals individual microbursts.

Armed with this framework, the authors turn to the epigenetic machinery. DNA methylation, long viewed as a stable, repressive mark, turns out to be far more dynamic than generally appreciated. It responds rapidly to extracellular stimuli, as seen in immune cells following bacterial infection, primarily at distal enhancers, and in post-mitotic neurons, where 1.4 percent of assessed CpG sites rapidly underwent demethylation or de novo methylation after stimulation. Intriguingly, 87 percent of differentially methylated CpG sites showed decreased methylation after an associated increase in transcription factor binding, suggesting that changes in DNA methylation are often a consequence rather than a cause of altered gene expression. Multiple transcription factors can recruit the methyltransferases DNMT3A or DNMT3B to trigger promoter hypermethylation, and methyl-CpG-binding proteins such as MeCP2 recruit repressive complexes, although MeCP2 has also been shown to activate genes and induce extensive chromatin unfolding in models of Rett syndrome.

The genomic location of methylation determines its effect on noise. Promoter methylation has been implicated with higher levels of transcriptional noise, apparently acting as a trigger that forces ON-to-OFF state transitions and increases the burstiness of genes, while unmethylated promoters can remain active and quieter. Gene body methylation, by contrast, is associated with reduced noise, a finding replicated in Arabidopsis thaliana and the sea anemone Exaiptasia pallida, and may work by suppressing transcription from cryptic intragenic promoters or by inhibiting deposition of the histone variant H2A.Z, which is itself linked to increased noise. In mouse embryonic stem cells, DNA methylation was identified as a key regulator of stochastic phenotypic state switching, although studies of Polycomb-targeted active genes found no such effect, underscoring the context dependency of these mechanisms.

Histone variants and nucleosome density add another layer. The H3.3 variant has a turnover time of approximately two hours, strikingly similar to the interval between transcription bursts for hormone-responsive genes, suggesting that H3.3 occupancy could act as a form of transcriptional memory; indeed, H3.3 and its methylatable fourth lysine residue are essential for retaining memory of gene activity states after nuclei transplantation. The chaperone HIRA deposits H3.3 at decondensed, transcriptionally active promoters and enhancers, while DAXX deposits it in condensed inactive chromatin, recruiting histone deacetylase II to maintain repression. The variant macroH2A, when paired with the transcription factor NRF-1, reduces noise by maintaining the ON state, whereas macroH2A nucleosomes lacking NRF-1 may promote stochastic ON-OFF switching. Independently of variant identity, a higher density of promoter-proximal nucleosomes was found to lower burst frequency and increase expression noise, slowing the rate of OFF-to-ON transitions.

Histone post-translational modifications show equally nuanced effects. Broadly, histone acetylation marks active genes, and evidence is accumulating that it mainly regulates burst size: the HDAC inhibitors trichostatin and SAHA increased the burst size of latent HIV production and, in a PP7-tagged cell line, predominantly affected burst size. Yet site-specific epigenetic rewriting using CRISPR-based dead Cas9 fusion proteins has revealed that targeted promoter acetylation can predominantly influence burst frequency, as at the circadian Bmal1 promoter, while enhancer acetylation increased burst duration of the FOS gene in neurons, mediated by the reader protein BRD4 and the release of paused RNA polymerase II. Histone methylation appears more tied to transcriptional memory: H3K4 methylation persists along gene bodies in yeast for over an hour after transcription ceases, transmits burst frequency between mother and daughter Dictyostelium cells, and in breast cancer cells, broad H3K4me3 peaks were associated with more homogeneous expression, while narrow peaks accompanied heterogeneous expression and drug resistance via the demethylase KDM5B.

Finally, the three-dimensional organisation of the genome and its enhancer networks constrain these dynamics. Macro-level features such as A and B compartments and topologically associated domains appear tied to stable, lineage-specific expression rather than acute bursting, although TAD disruption can cause developmental malformations by rewiring enhancer contacts. Enhancer-promoter loops, which can span up to three megabases, are far more dynamic: deleting specific CTCF sites in T helper 2 cells increased gene expression noise by destabilising loops, while forced looping and stronger enhancers in Drosophila increased burst frequency without changing burst size. Genome-wide analyses confirm that enhancers primarily modulate burst frequency, and the mediator complex drives the rapid succession of initiation events that produces a burst of polymerase activity. The authors conclude that resolving the true genome-wide effects of epigenetics on bursting will require high-throughput imaging of thousands of loci under epigenetic perturbation, but with such techniques on the horizon and single-cell transcriptomics exploding, the field stands on the verge of finally connecting the epigenetic code to the pulsating rhythm of gene activity.

Subject of Research: Epigenetic regulation of transcriptional bursting and gene expression noise in eukaryotic cells

Article Title: Transcription bursting and epigenetic plasticity: an updated view

Article References: Beckman, W. F., Jiménez, M. Á. L., & Verschure, P. J. (2021). Transcription bursting and epigenetic plasticity: an updated view. Epigenetics Communications, 1(1), Article 6. https://doi.org/10.1186/s43682-021-00007-1

Image Credits: AI Generated

DOI: 10.1186/s43682-021-00007-1

Keywords: transcription bursting, epigenetics, gene expression noise, DNA methylation, histone variants, histone modifications, chromatin architecture, enhancers, RNA polymerase II, topologically associated domains, transcriptional memory, single-cell imaging

Interstrand crosslinks, or ICLs, are among the most cytotoxic forms of DNA damage because they covalently tether the two strands of the double helix to one another, blocking both DNA replication and transcription. Cisplatin, a platinum-based drug, induces these crosslinks as part of its antitumor arsenal, and the resulting suppression of transcription contributes significantly to the drug’s ability to kill rapidly dividing cancer cells. However, cells possess a dedicated pathway known as transcription-coupled repair that can remove such lesions from actively transcribed genes, and this repair capacity can mitigate cisplatin’s effects, contributing to treatment resistance in some tumors. Understanding exactly how the crosslink stalls the polymerase, and how that stall is recognized by the repair machinery, is therefore of direct clinical relevance.

The Zhejiang team assembled defined RNA polymerase II elongation complexes on DNA templates containing a site-specific cisplatin-induced interstrand crosslink, positioning the lesion at various distances from the enzyme’s active site. By vitrifying these complexes and imaging them with cryo-electron microscopy, the researchers were able to reconstruct a molecular movie of sorts, following the polymerase as it advanced toward the crosslink and ultimately ground to a halt. The eight captured states span the journey from the initial approach of the lesion through the final stalled configuration, offering a step-by-step structural narrative that has never before been available for this class of DNA damage.

The most striking discovery emerged when the polymerase reached the crosslink itself. Rather than simply blocking progress like a physical roadblock, the crosslinked guanine bases underwent an unexpected conformational transformation. The enzyme actively remodeled the crosslink, prying the damaged guanines into a novel configuration positioned above the bridge helix, a structural element of the polymerase that plays a central role in translocating DNA and RNA through the enzyme. This rearrangement had not been observed for any other type of transcription-blocking lesion and reveals RNA polymerase II in an unanticipated role: that of a structural remodeler of the very damage it encounters.

The consequence of this remodeling is functionally decisive. By relocating the crosslinked base above the bridge helix, the polymerase prevents the damaged guanine from entering the active site, the catalytic chamber where templated nucleotide addition normally occurs. With the lesion physically excluded from the position where it would need to reside to serve as a template, transcription cannot proceed in the conventional manner. The structural data show that the remodeled crosslink creates an empty nucleotide-binding site poised in an abnormal configuration, setting the stage for a secondary failure mode that the researchers characterized in detail.

That secondary failure is ATP misincorporation followed by backtracking. Because the active site is left in an aberrant state, the polymerase can misinsert adenosine triphosphate opposite the position where the crosslinked guanine should have been read. Following this misincorporation event, the enzyme undergoes backtracking, a process in which the polymerase slides backward along the DNA and RNA, extruding the freshly synthesized 3′ end of the RNA transcript from the active site. Backtracking is a well-known mechanism of transcriptional arrest, and the new structures demonstrate how the remodeled crosslink actively drives the enzyme into this arrested configuration rather than merely obstructing forward motion.

The study also examined oxaliplatin, another clinically important platinum chemotherapy drug, and found that its interstrand crosslinks stall polymerase II through a conserved mechanism. Notably, the structural analyses revealed that when the platinum crosslink sits several nucleotides downstream of the active site, the polymerase drives the platinum adduct from the minor groove of the DNA helix into the major groove, and the crosslinked guanine on the non-template strand migrates toward the upstream side of the complex as the lesion approaches the catalytic center. These observations, together with experiments using alternative DNA sequences, demonstrated that the remodeling behavior is not an artifact of a particular template but a general property of how polymerase II handles platinum crosslinks.

A central question in transcription-coupled repair concerns what happens after the polymerase stalls. Cockayne syndrome protein B, or CSB, is an ATP-dependent translocase that is recruited to stalled polymerase II and is considered the initiating factor of transcription-coupled repair. One might expect CSB to either help the polymerase bypass the lesion or actively displace the stalled enzyme to clear the way for repair enzymes. The new data show that CSB does neither. In the reconstituted system, CSB neither promoted bypass of the cisplatin crosslink nor displaced the stalled polymerase, and the transcription factor TFIIS, which can stimulate RNA cleavage and restart arrested polymerases, likewise failed to rescue transcription through the lesion. This failure to resolve the blockage effectively commits the crosslinked site to the transcription-coupled repair pathway, ensuring that the damage is flagged for processing rather than erroneously bypassed.

The structural comparisons revealed additional mechanistic elegance. The fork loop-2 region of the polymerase was observed to separate the template and non-template DNA strands at the downstream fork of the transcription bubble, and this separation appears to facilitate the migration of the crosslinked non-template guanine as the lesion is remodeled. Furthermore, alignment of the cisplatin-stalled complexes with previously determined structures of polymerase II bound to CSB homologs, including yeast Rad26 and the human CSB-CSA-UVSSA complex, showed that the crosslink-stalled enzyme adopts a conformation competent for recruiting these repair factors, mirroring the way polymerase II arrested by bulky lesions on the template strand presents itself to the repair machinery.

The implications of this work extend from basic molecular biology to the clinic. By identifying RNA polymerase II as an active remodeler of cisplatin-induced interstrand crosslinks and defining the structural basis of the resulting stall, the study fills a long-standing gap in our understanding of how platinum drugs exert their transcription-blocking effects at atomic resolution. Because the efficiency of transcription-coupled repair influences cellular sensitivity to cisplatin, with repair-deficient cells such as certain testis tumor cells showing heightened drug sensitivity, the molecular details uncovered here could inform strategies to modulate repair pathways and improve therapeutic outcomes. The cryo-electron microscopy maps and atomic models have been deposited in public databases, ensuring that the structural framework established by the Zhejiang team will serve as a foundation for future studies of crosslink repair, polymerase stalling, and the cellular response to platinum-based chemotherapy.

Subject of Research: Structural mechanism of transcription stalling by cisplatin-induced DNA interstrand crosslinks

Article Title: Transcription remodeling and stalling by cisplatin-induced interstrand crosslinks

Article References: Zhu, L., Zhao, W., Ye, L., Liu, Y., Deng, W., Qian, B., Yang, X., & Xu, J. (2026). Transcription remodeling and stalling by cisplatin-induced interstrand crosslinks. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02338-3

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02338-3

Keywords: cisplatin, interstrand crosslink, RNA polymerase II, transcription-coupled repair, cryo-electron microscopy, DNA damage, Cockayne syndrome protein B, oxaliplatin, backtracking, platinum chemotherapy, bridge helix, DNA repair

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 story begins in 1969, when Robert Roeder and William Rutter first separated multiple forms of DNA-dependent RNA polymerase from eukaryotic cells. Over the following decades, biochemists painstakingly identified the general transcription factors, TFIID, TFIIB, TFIIE, TFIIF, and TFIIH, that guide polymerase II to promoters and position it precisely at the transcription start site. The TATA box, discovered in histone genes in the late 1970s, became the archetypal promoter element, and the crystal structure of the TATA-box-binding protein bound to DNA in 1993 revealed how this saddle-shaped factor bends the double helix nearly ninety degrees. But these early pictures were static snapshots of isolated parts. What the machinery does as a whole, and how it moves, remained opaque.

Cryo-electron microscopy changed everything. Where X-ray crystallography demanded rigid, homogenous crystals that giant, flexible assemblies rarely form, cryo-EM can capture heterogeneous, dynamic complexes frozen in vitreous ice, and computational classification can sort millions of particle images into distinct conformational states. Structures of the human pre-initiation complex, or PIC, the mega-assembly of polymerase II and general transcription factors on promoter DNA, progressed from low-resolution models to structures resolved at near-atomic detail. In 2021, researchers resolved the polymerase II pre-initiation complex at 2.9 angstroms, revealing the initial stages of DNA opening, and subsequent work captured the PIC in complex with the Mediator co-activator, with TFIIH, and even with the first nucleosome downstream of the promoter.

At the heart of the new understanding is TFIID, a massive 1.2-megadalton complex of the TATA-box-binding protein and thirteen TBP-associated factors, or TAFs. TFIID serves as the primary scaffold for promoter recognition. In its free state, it adopts a compact, autoinhibited conformation in which the lobes that will grip promoter DNA are folded inward. Binding of TBP to the TATA box triggers a dramatic rearrangement, and recent structures show that TFIID engages not only the TATA box but also downstream promoter elements, including the downstream core promoter region, or DPR, discovered through machine-learning analysis of human promoter sequences. Because the majority of human promoters lack a TATA box altogether, these TAF-mediated contacts with downstream DNA are essential for accurate initiation across most of the genome, and different TAF subunits are now known to dictate promoter selectivity and to integrate signals from sequence-specific activator proteins.

Superimposed on this scaffold is Mediator, the thirty-subunit co-activator that physically bridges enhancer-bound transcription factors to the promoter-bound machinery. Structural studies of the complete Mediator-PIC complex, including landmark human structures published in 2021, revealed how Mediator’s head, middle, and tail modules wrap around the polymerase and how its dissociable kinase module can regulate the interaction. The Pol II C-terminal domain, an intrinsically disordered tail of heptad repeats extending from the largest polymerase subunit, threads into the PIC-Mediator assembly, and its phosphorylation by the TFIIH-associated kinase module, through cyclin-dependent kinase 7, marks a critical checkpoint. Serine 5 phosphorylation of the CTD not only helps trigger promoter escape but also recruits the mRNA capping enzyme, coupling the earliest steps of transcription to RNA processing.

Perhaps the most striking theme to emerge from recent structural work is the role of chromatin itself. The +1 nucleosome, the first nucleosome downstream of the transcription start site, is no longer viewed as a mere obstacle. Structures of the +1 nucleosome-bound PIC-Mediator complex show that this histone package acts as a position-dependent regulator, physically orienting the pre-initiation complex, influencing transcription start-site selection, and even enforcing directionality. Epigenetic marks matter here: trimethylation of histone H3 lysine 4 anchors TFIID through the bromodomain of TAF3, while acetylation marks recruit BET-family factors that bridge TFIID and nucleosomes. The nucleosome, in other words, transmits epigenetic information directly into the transcription machinery, helping the PIC integrate genetic sequence and chromatin state into a single decision about where and in which direction to initiate.

The transition from initiation to elongation emerges as a choreographed, programmed process rather than a simple handoff. Before productive synthesis begins, polymerase II must melt the promoter DNA, a task performed primarily by the XPB translocase subunit of TFIIH, which pulls double-stranded DNA into the polymerase cleft to generate torsional stress. Early attempts at synthesis are often abortive: the enzyme repeatedly synthesizes and releases short RNAs of fewer than five nucleotides because the nascent transcript is thermodynamically unstable in the complex. During this phase, the enzyme scrunches downstream DNA into its active site, storing mechanical energy. Structural and single-molecule studies now delineate a three-step mechanism of promoter escape, in which steric pressure from accumulated template DNA and forward translocation of the RNA-DNA hybrid rupture the narrow channel formed by Pol II and TFIIB, causing general transcription factors to dissociate, the transcription bubble to collapse from roughly twenty-one to a stable eleven nucleotides, and the polymerase to commit to elongation, typically after transcribing about eight to twenty-three nucleotides.

Crucially, the review frames transcription initiation as a process governed by surface competition. Each stage of initiation involves factors that bind overlapping or competing surfaces on polymerase II. As CTD phosphorylation proceeds, inhibitory factors are released and regulatory factors are progressively exchanged: Mediator dissociates, DSIF and NELF bind to impose promoter-proximal pausing, and elongation factors await release signals. This model explains how the same binding surfaces can recruit different partners at different times, and how re-initiation, in which scaffold factors remain promoter-bound to launch successive rounds of polymerase loading, can generate the transcriptional bursting observed in single cells. It also rationalizes why paused polymerase can inhibit new initiation, creating feedback loops that shape gene expression dynamics.

The medical implications are substantial. TAF subunits such as TAF1 and TAF10 are required during development but dispensable in certain adult tissues, and PROTAC-mediated degradation of TAF1 has been shown to induce apoptosis in acute myeloid leukemia cells, making components of the initiation machinery emerging drug targets. As cryo-EM continues to push toward imaging these assemblies inside cells, and as single-molecule and nascent-RNA sequencing methods add temporal resolution, the field is converging on a unified view: transcription initiation is not a static lock-and-key event but a dynamic, chromatin-informed, programmed pipeline, and seeing it in atomic detail is finally telling us how life reads its own blueprint.

Subject of Research: Structural and mechanistic basis of transcription initiation by RNA polymerase II in eukaryotes

Article Title: The molecular basis of transcription initiation by RNA polymerase II

Article References: Chen, X., & Xu, Y. (2026). The molecular basis of transcription initiation by RNA polymerase II. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/s41580-026-01019-2

Image Credits: AI Generated

DOI: 10.1038/s41580-026-01019-2

Keywords: RNA polymerase II, transcription initiation, pre-initiation complex, TFIID, Mediator, cryo-electron microscopy, TATA box, promoter escape, plus-one nucleosome, CTD phosphorylation, TFIIH, gene regulation