Molecular Cell

Human quinone reductase 2 (QR2, NQO2) is a cytosolic flavoprotein involved in cell physiology and metabolism, and implicated in several diseases. However, the mechanisms that govern its oligomeric assembly and diverse functional outcomes remain incompletely understood. Here, we employ native mass spectrometry to directly resolve the dynamic oligomeric landscape of recombinant human QR2 expressed in Escherichia coli, preserving non-covalent interactions and enabling analysis of assembly behavior under native conditions. QR2 is predominantly observed as a dimer stabilized by multiple non-covalently bound ligands, giving rise to discrete species. Top-down native mass spectrometry reveals a single intact proteoform, excluding covalent modification or covalently bound flavins as drivers of oligomerization. Binding of flavin adenine dinucleotide (FAD) robustly stabilizes the dimer, while unexpectedly, flavin mononucleotide (FMN) also promotes dimer formation. As FMN and FAD differ structurally by the presence of an adenine dinucleotide moiety, we hypothesized that purine nucleotide binding itself may modulate QR2 assembly. Consistent with this, we identify a new concentration-dependent effect of guanosine-triphosphate (GTP) on QR2 dimerization. Functional reductase assays show that flavin-stabilized dimers exhibit the highest catalytic activity, whereas GTP-induced dimers retain reduced activity. Binding of the inhibitor YB537 abolishes activity despite promoting dimer formation. Together, these findings reveal a ligand-dependent structural plasticity in QR2 oligomerization that is decoupled from reductase function, suggesting that QR2 dimerization serves a wider regulatory role beyond simply supporting reductase catalysis.

Neuronal maturation is associated with extensive changes in gene expression and chromatin organization. However, the molecular mechanisms that control the epigenetic landscape in terminally differentiated neurons remain poorly understood. Here, we show that maturing cerebellar granule cells undergo a striking and specific increase in the levels of the repressive histone modification H3K27me3 across different genomic regions, including individual genes, broad intergenic regions, and gene clusters. The accumulation of H3K27me3 coincides with a developmental switch from EZH2 to EZH1 and colocalizes with H3K36me2 and DNA non-CpG methylation. Using mice with a conditional deletion in the catalytic domain of EZH1, we demonstrate that the maintenance of H3K27me3 in mature neurons depends on EZH1. Unexpectedly, an almost complete loss of H3K27me3 in postmitotic GCs induces minimal changes in gene expression and chromatin accessibility at 7 months of age. Using single-nucleus RNA sequencing (snRNAseq) from the mouse neocortex, we show that, similarly to GCs, the loss of EZH1-mediated H3K27me3 also has a minimal impact on cortical neuron gene expression. The amino acid composition of EZH1 suggests reduced sensitivity to H3K36 methylation, providing a potential basis for its activity in chromatin contexts that are not permissive for EZH2. Together, our results show that a postmitotic switch from EZH2 to EZH1 establishes novel chromatin domains in neurons with a minimal role in transcriptional maintenance.

Stress granules (SGs) are dynamic, membrane-less assemblies that form in the cytoplasm in response to cellular stress. The ordered recruitment of proteins into SGs is fundamental to condensate composition and function, yet the molecular determinants of this ordered client recruitment remain incompletely understood. Using proximity photo-crosslinking proteomics, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a TIA1-proximal protein preferentially enriched in SGs under arsenite stress. Knockdown of hnRNPA2B1 preferentially delayed TIA1 enrichment in G3BP1-marked SGs at 20 min without affecting G3BP1 or the overall SG-positive cell fraction, and this phenotype showed directional rescue upon re-expression. In vitro droplet reconstitution assays with purified proteins revealed that hnRNPA2B1 and RNA cooperatively increased TIA1 incorporation capacity into G3BP1 condensates, an effect not attributable to changes in droplet size. Kinetic fitting identified hnRNPA2B1 + RNA as uniquely increasing the plateau amplitude of TIA1 recruitment (Cohens d = 1.62 versus RNA-alone condition). Coarse-grained simulations support an inside-out assembly model in which hnRNPA2B1 stabilizes the condensate core through homotypic interactions while RNA-bound TIA1 accumulates at the periphery. Together, these findings identify hnRNPA2B1 as a capacity-determining modulator of early TIA1 recruitment and provide a framework for understanding ordered protein assembly within stress granules.

Epigenetic regulation is tightly linked to cellular metabolism through chromatin-modifying enzymes that depend on central metabolites as co-substrates. Methionine is an essential amino acid that is directly converted by methionine adenosyltransferase 2A (MAT2A) into S-adenosylmethionine (SAM), the universal methyl donor required for histone and DNA methylation. Although methionine restriction/depletion can alter the chromatin methylation landscape and improve physiological outcomes in diverse biological systems, it remains unclear whether these effects arise from loss of methionine itself or from secondary depletion of SAM. Here, we show that methionine depletion induces nuclear accumulation of MAT2A together with redistribution of H3K9 methylation, derepression of transposable elements, activation of stress-response pathways, and broad transcriptional reprogramming. Surprisingly, pharmacologic inhibition reduced intracellular SAM to levels comparable to methionine depletion but failed to reproduce these major epigenetic or transcriptional responses. Furthermore, depletion of the SAM-sensor SAMTOR and inhibition of KDM4 histone demethylases did not prevent methionine-dependent chromatin remodeling, indicating that canonical SAM-sensing pathways are not required for this adaptation. Instead, methionine depletion uniquely induced innate immune and integrated stress-response programs consistent with a viral mimicry-like state. These findings demonstrate that methionine availability, rather than SAM abundance, functions as a primary metabolic signal regulating epigenetic adaptation to nutrient stress. Our data support a model in which methionine is sensed independently of SAM abundance and acts upstream of stress signaling pathways that secondarily remodel chromatin.

RAF kinases interpret signals from the three major RAS isoforms to initiate MAPK pathway activation, yet the molecular logic that governs isoform-specific RAS recruitment and the early events that relieve RAF autoinhibition are not yet fully understood. In particular, how the modular N-terminal regulatory architecture of CRAF and ARAF, anchored by the multifunctional cysteine-rich domain (CRD), discriminates among HRAS, KRAS, and NRAS has remained a central unresolved question. Here, we combine quantitative biophysical measurements with structural and dynamic analyses to define how RAS isoform identity and CRD engagement shape the earliest steps of RAF activation. These studies reveal unexpectedly divergent modes of RAS recognition between CRAF and ARAF and expose previously unappreciated functions of the CRD in modulating RAS affinity and intramolecular regulatory contacts. We further identify a direct link between RAS binding and destabilization of RAF autoinhibition, providing mechanistic insight into how RAS initiates the transition from an inactive monomer to an activation-competent assembly. Finally, we show that emerging KRAS inhibitors variably perturb KRAS-CRAF interactions, offering insight into how these therapeutics influence early RAS-RAF signaling events. Together, this work uncovers distinct biophysical principles that govern RAS-RAF selectivity and reveals a regulatory role for the CRD that reframes our understanding of RAF activation and its dysregulation in RAS-driven cancers. SignificanceProteins in the RAS-RAF signaling pathway control cell growth and are frequently mutated in cancer. Despite their importance, how different RAS proteins selectively recruit RAF kinases has remained incompletely understood. This study reveals that the cysteine-rich regulatory region of RAF plays a central role in distinguishing RAS isoforms and controlling RAF activation. These insights clarify early steps in MAPK signaling and may guide the development of improved therapies targeting RAS-driven cancers.

Post-transcriptional regulation is critical for mammalian embryogenesis yet has been underexplored. We previously showed that RNA-binding Pumilio proteins (Pum1/2) are essential for early mouse embryogenesis and embryonic stem cell (ESC) functions. Here, using acute protein degradation systems combined with time-resolved RNA-seq and eCLIP, we delineate a two-phase regulatory hierarchy modulated by Pum1/2 in mouse ESCs. The first phase, occurring within 10 hours of Pum1/2 depletion, is predominantly the stabilization of over 100 Pum1/2-target mRNAs, while the second phase, occurring in subsequent 66 hours, propagates to over 1,000 mRNAs mostly through indirect regulatory effects. Functionally, Pum1/2 depletion delays transition from naive to formative pluripotency, impairs neuroectoderm differentiation, and enhances germline specification. Mechanistically, Pum1/2 directly repress mRNAs encoding PRC2 subunits, including Suz12, thereby constraining H3K27me3 deposition at neuroectodermal gene loci. These findings establish Pum1/2 as biphasic post-transcriptional regulators of pluripotency and lineage balance and link RNA stability control to chromatin-mediated silencing. HIGHLIGHTSO_LIAcute Pum1/2 degradation provides temporal resolution for profiling post-transcriptional regulation. C_LIO_LIPum1/2 destabilize more than 100 direct targets and modulate a biphasic network of over 1,000 mRNAs. C_LIO_LIPum1/2 loss delays pluripotency transition, suppresses neuroectoderm, and promotes germline fate. C_LIO_LIPum1/2 directly regulate Suz12 mRNA decay to modulate PRC2-mediated repression. C_LI

Abstract/SummaryPrecise regulation of enzyme recruitment during Okazaki fragment maturation (OFM) is essential for faithful and efficient lagging-strand DNA synthesis. Emerging evidence suggests that PARP1 contributes to OFM yet its specific functions remain unclear. Here, we define context-dependent functions of PARP1 during OFM. Under physiological conditions, PARP1 co-localizes with PCNA in early S phase and restrains Pol {delta}-PCNA- mediated strand-displacement DNA synthesis, thereby preventing the formation of long 5' flaps, which is refractory to FEN1 cleavage. On the other hand, in LIG1-deficient cells, in which DNA nicks and unexpectedly long 5' flaps accumulate, PARP1 promotes the recruitment of LIG3 to catalyze OF ligation and DNA2 to facilitate long 5' flap processing. Collectively, our findings uncover previously unrecognized roles of PARP1 in regulating 5' flap dynamics to ensure efficient OFM and cell viability. HighlightsO_LIPARP1 plays context-dependent regulatory functions in Okazaki fragment maturation (OFM). C_LIO_LIPARP1 controls strand displacement DNA synthesis by the PCNA-Pol{delta} complex to dictate generation of short over long RNA-DNA flaps during canonic OFM. C_LIO_LIPARP1 senses unligated Okazaki fragments in DNA Ligase 1 deficient cells and suppresses unwanted conversion of DNA nicks into 5 flaps. C_LIO_LIProcessing of unligated nicks or flaps by DNA ligase 3 or DNA2, respectively in LIG1 deficient cells depends on PARP1. C_LIO_LIPARP1 inhibitors induce synthetic lethality with DNA ligase 1 or DNA2 inhibition. C_LI

The electron shuttle and coenzyme nicotinamide adenine nucleotide (NAD) is essential for cellular metabolism and homeostasis. NAD levels significantly fluctuate in cells, whilst several age-related diseases are associated with depletion of this metabolite. However, how NAD changes are monitored by nutrient/energy sensing signalling pathways remains poorly understood. We found that at physiological concentrations NAD controls the activity of the AMP-activated protein kinase (AMPK) in vitro and in human cells. Mechanistically, NAD binds gamma subunit of AMPK, and mutagenesis of the putative binding site renders the holoenzyme insensitive to NAD inhibition. Hyperactivation of AMPK in response to NAD depletion suppresses metabolic pathways including mammalian Target of Rapamycin Complex I (mTORC1) and autophagy. These results demonstrate that in addition to monitoring cellular energy levels AMPK functions as a NAD sensor, providing novel insight into how cells and tissues detect and respond to metabolic fluctuations with implications for stress resistance and ageing.

Post-translational modifications (PTMs) of chromatin remodelers are abundant but functionally understudied. Here we investigate the role of asymmetric dimethylation of arginine 1064 (BAF155me2a) on the SWI/SNF core subunit BAF155, a mark deposited by CARM1/PRMT4 that has been linked to tumor progression but whose molecular function remains unclear. Using immunoprecipitation-mass spectrometry with a dimethyl-specific antibody, we found that R1064me2 selectively enhances BAF155 interactions with RNA processing factors, including the anti-termination protein SCAF4, splicing factors, and the transcription factor RFX5. CUT&RUN profiling showed that BAF155me2a, SCAF4, and RFX5 co-occupy promoter regions, and reciprocal immunoprecipitations confirmed that the SCAF4-BAF155 interaction depends on R1064 methylation. To test the functional consequences of this modification, we generated cells expressing either wild-type BAF155 or a methylation-deficient BAF155-R1064K mutant. Loss of methylation did not alter chromatin accessibility, BAF155 genomic occupancy, or SCAF4 recruitment. However, nascent transcription measured by TT-seq revealed a coordinated reduction in 5' sense transcripts and upstream antisense transcripts (PROMPTs) at BAF155-bound promoters, with a quantitatively larger decrease in PROMPTs at SCAF4 co-bound sites. The effect was restricted to the promoter-proximal region and resolved toward the gene end, consistent with a defect in productive elongation downstream of RNA polymerase II recruitment. These data support a model in which BAF155 dimethylation provides a co-transcriptional interface coupling SWI/SNF to RNA processing machinery, and identify regulation of nascent transcription as a non-canonical function of SWI/SNF PTMs.

Regulation of protein synthesis is essential for maintaining cellular homeostasis during stress. The integrated stress response (ISR) is a conserved signaling pathway that modulates global mRNA translation through four eIF2 kinases--GCN2, PKR, PERK, and HRI. However, how these kinases are selectively activated and tuned to distinct stress signals to direct appropriate cell fate decisions remains poorly understood. Here, we employ ultra-deep mutagenesis screens to systematically map regulators of protein synthesis across diverse stress perturbations in human cells. This comparative approach identifies stress-specific translational control factors, including a previously unrecognized role for the E3 ubiquitin ligase RNF25 in selectively sustaining translation following UV irradiation and other RNA-damaging treatments. In this context, we demonstrate that RNF25 operates independently of its partner RNF14, and that its ubiquitin ligase activity, as well as its RWD-domain, is required to restrain excessive activation of the eIF2 kinase GCN2. Accordingly, loss of RNF25 results in hyperactivation of GCN2, exacerbated translation shutdown, and impaired cell proliferation following RNA damage--phenotypes that can be fully reversed by genetic or pharmacological inhibition of GCN2. Together, these findings uncover a previously unappreciated RNF25-GCN2 signaling axis and identify ISR-driven toxicity as a potential vulnerability in combination with RNA-damaging chemotherapeutics.

The TREX2 complex bridges transcription and RNA export. Its subunits show differences in expression, localization, and dynamics, suggesting distinct cellular roles. To understand the roles of individual TREX2 components, we characterized their interactomes. We identified novel, evolutionarily conserved SAC3(PCI-fold)-based subcomplexes with its PCID2 subunit. PCID2 acts as a scaffold for mutually exclusive yet structurally related subcomplexes with GANP, LENG8, and SAC3D1. These subcomplexes have alternative localization at the nuclear envelope, nuclear speckles and cytosol. LENG8 localizes in nuclear speckles and interacts extensively with the mRNA processing factors. LENG8 depletion alters mRNA processing and polyadenylation site usage. LENG8 thus acts upstream of the canonical TREX2 complex, in which PCID2 cooperates with GANP in mRNA export. Together, our findings reveal that TREX2 is not a uniform complex but a modular system, in which TREX2 subunits can assemble into functionally distinct subcomplexes through interacting partners that define their specificity and alternative functions. HIGHLIGHTSO_LITREX2 subunits play distinct roles in RNA retention and exhibit subunit-specific preference for interactions with different protein partners C_LIO_LIPCID2 forms mutually exclusive subcomplexes with GANP, LENG8, and SAC3D1 C_LIO_LIGANP, LENG8, and SAC3D1 alter PCID2 intracellular localization C_LIO_LILENG8 is a nuclear speckle protein that modulates alternative mRNA processing C_LI

The DNA damage response (DDR) is a complex network of interconnected pathways and sub-pathways that safeguards genome integrity. Deciphering the coordinated and complementary interactions among these pathways remains a major challenge. In this study, we employed CRISPR screening to systematically map the genetic interactions required for different sub-pathways of homologous recombination in human cells following PARP inhibitor treatment. Our approach recapitulated known interactions and uncovered several previously unrecognized connections. We identified RAD54L, in addition to ATRX, as a factor promoting the double Holliday junction (dHJ) pathway and demonstrated that RAD51AP1 and RAD54B function in synthesis-dependent strand annealing (SDSA). We provide evidence that loss of TOP3A induces a switch in HR sub-pathway usage from SDSA to the dHJ pathway. Furthermore, TOP3A deficiency abolishes the requirement for ATRX and the histone variant H3.3 in the dHJ pathway, while maintaining strict dependence on RAD54L. We further observed that H3.3 is involved in both HR sub-pathways, whereas its depositing chromatin remodelers HIRA and ATRX play pathway-specific roles in SDSA and dHJ, respectively. Together, our findings define the architecture underlying HR sub-pathway choice and reveal a key role for TOP3A in regulating pathway balance.

Selective autophagy restrains innate immune signalling to maintain tissue homeostasis, yet how this repression is rapidly relieved during infection remains unclear. Here, we show that under basal conditions the inhibitor of {kappa}B kinase {gamma} (IKK{gamma}) Kenny is sequestered at autophagosomes through Atg8 and the selective autophagy receptor Ref(2)P, thereby silencing Imd pathway activity. Bacterial infection disrupts this interaction, releasing the IKK complex to enable immune signalling. Mechanistically, we identify the initiator caspase Dredd as a direct interactor of the IKK{gamma} Kenny and show that Dredd binds and cleaves Kenny in a ubiquitination-dependent manner during infection. This cleavage removes an N-terminal LC3-interacting region, uncoupling the IKK complex from autophagosomal degradation. Dredd-mediated processing of Kenny stabilises the IKK complex and is required for activation of the NF-{kappa}B transcription factor Relish, robust antibacterial responses, and host survival following infection. Together, these findings uncover a mechanism by which caspase-mediated cleavage intersects with selective autophagy to dynamically control NF-{kappa}B signalling during bacterial infection. Short summaryBacterial infection activates NF-{kappa}B signalling by triggering caspase-dependent cleavage of the IKK subunit Kenny, releasing the IKK complex from autophagosomal repression to enable effective innate immune responses.

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein implicated in transcriptional control and oncogenesis, yet how its tandem bromodomains (BD1-2) contribute to nucleosomal engagement remains unresolved. Here we show that the tandem bromodomains of BRD4 cooperatively engage poly-acetylated histone H4 tails and nucleosomes in vitro and promote chromatin association in human cells. In stringent peptide pull-down and nucleosome-based biolayer interferometry assays, isolated BRD4 bromodomains bind weakly to poly-acetylated histone peptides and nucleosomes, whereas the tandem BD1-2 module binds much more robustly. These results closely mirror our observations in mammalian cells, where truncations lacking either bromodomain or pocket-disrupting mutations in either domain reduced chromatin association, with dual pocket disruption causing the strongest defect. In the BRD4 short isoform (BRD4-S), maximal chromatin association additionally required the region C-terminal to the BD2, which contains the basic residue-enriched interaction domain (BID) and extraterminal domain (ET), consistent with a multivalent chromatin engagement mechanism beyond the bromodomains alone. Functionally, dual pocket disruption attenuated BRD4-S-dependent breast cancer phenotypes, including impaired growth and reduced transwell migration. Together, these findings define how tandem bromodomains and adjacent BRD4-S regions cooperate to stabilize chromatin residence and inform therapeutic strategies aimed at more precisely disrupting BRD4 function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/719657v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@12d03beorg.highwire.dtl.DTLVardef@50cc45org.highwire.dtl.DTLVardef@92e3ecorg.highwire.dtl.DTLVardef@1b1a26d_HPS_FORMAT_FIGEXP M_FIG C_FIG

PDS5A, a regulatory subunit of the cohesin complex, and topoisomerase IIB (TOP2B), an enzyme resolving DNA topological problems, interact with CTCF and regulate transcription, chromatin loops, and genome organization. Yet, how PDS5A and TOP2B are recruited to chromatin to exert their function is not well-understood. Here, we studied the functional relationship between PDS5A and TOP2B and the resultant impact on genome organization and gene expression. Interestingly, TOP2B-PDS5A cooperate for their recruitment to CTCF-bound chromatin sites. The presence of catalytically active TOP2B increased PDS5A occupancy genome-wide. Notably, a novel PDS5A-CTCF interaction region in the CTCF N-terminal 95-116aa was required for CTCF-PDS5A-TOP2B interaction in vitro as well as for active TOP2B-mediated enrichment of PDS5A chromatin occupancy in vivo. The loss of CTCF(95-116aa) led to a reduced number of chromatin loops and dysregulated gene expression. In gliomas, PDS5A and TOP2B expression levels are variable and correlated, contributing to apparent heterogeneity in gene expression. Indeed, inducible knockdown of PDS5A led to reduced TOP2B occupancy and altered gene expression in the glioma genome. Importantly, PDS5A mediated sensitivity to TOP2 cancer drugs in glioma cells. This newly recognized functional interaction between PDS5A and TOP2B at chromatin boundaries clarifies the mechanisms fostering gene regulation through genome organization, with implications for glioma therapeutics.

DNA replication initiation requires activation of the CMG helicase to establish the replisome. This process involves the extrusion of single-stranded DNA (ssDNA) from the central channel of MCM double hexamers, allowing the two CMG helicases to pass each other; however, the factors that mediate this process in human cells remain unclear. We show that degron-mediated depletion of either MCM10 or RECQL4 alone causes only mild replication defects, whereas simultaneous depletion of both proteins completely blocks CMG activation. ChIP-seq analyses demonstrate that RECQL4 localises to replication initiation zones (IZs) independently of MCM10, whereas MCM10 recruitment to IZs is enhanced upon RECQL4 depletion, suggesting RECQL4 primarily functions in CMG activation, and MCM10 acts as a backup or supporting factor. Rescue experiments further indicate that RECQL4 cooperates with MCM10 through direct interaction, and that their ssDNA-binding activity underlies their functional overlap. We propose MCM10 and RECQL4 act cooperatively and redundantly to promote CMG activation.

Eggs of many species accumulate thousands of dormant mRNAs that are translated after fertilization at specific times and locations to direct development. However, how embryos coordinate translation of these mRNAs remains unclear. In this study, we identified sequential waves of translation critical for proper development progression. The first wave occurred within 1 h and included translation of ewsr1b mRNA that harbored a short 3' untranslated region (UTR) comprising 16 nucleotides. The resulting Ewsr1b protein triggered the second translation wave through binding cytoplasmic mRNAs, including pou5f3, which encodes a transcription factor promoting zygotic genome activation. In contrast, HuR and Syncrip repressed translation until the first and second waves, respectively. ewsr1b mRNA that had a long 3'UTR was translated in the second wave, and the 3'UTRs length determined protein localization and function. Overall, our findings reveal previously unknown molecular principles that coordinate translation timings and protein functions to drive long-term, multilayered processes.

The cytosolic iron-sulfur cluster assembly (CIA) pathway maturates essential nuclear and cytosolic Fe-S proteins required for genome maintenance and cellular metabolism. Nar1 (also called CIAO3 or IOP1) is a conserved Fe-S protein that connects the early and late steps of the CIA pathway, yet the molecular basis for its proposed function as a metallocluster carrier remains poorly defined. In particular, the interactions responsible for Nar1 recruitment to the CIA targeting complex (CTC) during cluster delivery remain unknown. Here, we define the molecular basis for Nar1 recruitment to the CTC using biochemical reconstitution, quantitative protein-protein interaction assays, and AlphaFold modeling. Our data reveal that Nar1 binds the CTC through two distinct interfaces. A primary interface comprises an electrostatic interaction that anchors Nar1 to a conserved acidic surface on the Cia1 subunit of the CTC and a secondary interface involves binding of Nar1s divergent targeting complex recognition peptide at the Cia1-Cia2 interface. Thus, Nar1 engages a conserved CTC surface that serves as a recruitment platform for multiple binding partners, including CIA clients. Computational structural models position the putative Fe-S cluster donor site of Nar1 adjacent to a proposed acceptor site on Cia2, suggesting that this bipartite binding mechanism positions Nar1 for transfer of an Fe-S cluster to the targeting complex. Together, these findings resolve conflicting models for Nar1 recruitment and establish a mechanistic framework for understanding how the CTC engages multiple binding partners during cytosolic iron-sulfur protein maturation.

During development, cellular identity is ultimately determined by transcriptional output: lineage-specific genes must be activated, while genes associated with alternative fates must be repressed. This process depends on the activity of chromatin remodelling complexes, which regulate the accessibility of transcription factors to chromatin regulatory elements. In addition, cellular identity is shaped by exposure to intercellular signals. Understanding the mechanisms by which extracellular signals are translated into changes in the transcriptional program is essential for understanding cell fate decisions during development, as well as in disease conditions such as cancer. Here we describe a rapid and widespread enhancer resetting event in response to ERK signalling in mouse ES cells. This process occurs in two distinct phases: an immediate, genome-wide alteration in transcription factor binding dynamics at regulatory regions which is dependent on the release of paused RNA Polymerase II, followed by the re-establishment of a context appropriate, stable chromatin state. We demonstrate that the chromatin remodelling complex NuRD is required for this reestablishment phase and for the appropriate transcriptional response to ERK signalling. We propose that enhancer resetting places genomic regulatory regions in a state which is permissive to the exchange of transcription factors in order to establish a new, stable enhancer topology enabling rapid yet precise transcriptional response to extracellular signals.

Stress granules (SG) are dynamic, membrane-less ribonucleoprotein assemblies that orchestrate cellular stress responses and rapidly disassemble upon stress relief. In Amyotrophic Lateral Sclerosis (ALS), mutations in RNA-binding proteins such as Fused in Sarcoma (FUS) impair SG dynamics, promoting the formation of aberrant and persistent granules. Although long non-coding RNAs (lncRNAs) are emerging as regulators of ribonucleoprotein organization, their mechanistic role in SG architecture and pathological remodeling remains largely unexplored. Here, we identify LINC00205 as a critical RNA regulator of pathological SG dynamics in FUSP525L-associated ALS. Using Neuroblastoma cells and human iPSC-derived Motor Neurons (MN), we show that LINC00205 is enriched in SG upon oxidative stress and directly interacts with mutant FUSP525L. Knock-out of LINC00205 selectively reduces the formation of FUSP525L-containing SG and restores physiological SG disassembly kinetics, without affecting normal SG or FUS expression levels. Mechanistically, LINC00205 acts as a multivalent RNA scaffold, directly binding mRNAs specifically enriched in pathological SG, such as PLCXD3 and PIK3CA, as well as the RNA helicase DHX36, which is preferentially associated with FUSP525L-containing SG. We demonstrate that LINC00205 promotes their specific recruitment into pathological SG, thereby contributing to the aberrant features of these assemblies. Together, our findings uncover an active lncRNA-driven mechanism that shapes the molecular composition of aberrant ALS-related SG and regulates their persistence, establishing lncRNAs as key organizers of RNA-protein assemblies under stress and providing a conceptual framework for modulating pathological condensates.