Molecular Cell

The human genome encodes several hundreds of ubiquitin E3 enzymes, most of which have poorly understood biological roles. In search for novel ubiquitin E3 enzymes involved in DNA damage tolerance, we identified RNF25 as a candidate to have a role in DNA replication stress tolerance. Under stress conditions, RNF25 translocates to the nucleus in a cGAS-dependent manner, and loss of RNF25 ubiquitin E3 activity leads to the accumulation of replication-dependent ssDNA gaps. Combining functional assays with with mass-spectrometry based proteomics approaches such as TULIP2, iPOND-MS and TurboID, we found that, mechanistically, RNF25 promotes the stability of the replication fork at Transcription-Replication Conflicts by the ubiquitin-mediated clearance of RAD18, mono-ubiquitinated PCNA, H2B-K120ub and RECQL, among others. Lack of RNF25 ubiquitin E3 activity promotes the occurrence of Transcription-Replication Conflicts and destabilizes reversed replication forks, enabling re-priming and accumulation of ssDNA gaps behind the replication fork, ultimately compromising genome integrity. Overall, RNF25 regulates DNA replication, transcription and translation in an ubiquitin-based goldilocks.

NF-{kappa}B must remain bound to chromatin long enough to activate innate immune genes, but what stabilizes this transcriptionally productive state is unknown. Here, we identify the Integrator complex as an essential determinant of NF-{kappa}B chromatin residence in mouse and human cells. Integrator associated with NF-{kappa}B and co-occupied its genomic targets. Single-molecule tracking revealed that Integrator markedly prolonged the chromatin residence of RELA molecules in a low-mobility state. Catalytically inactive INTS11-E203Q fully restored NF-{kappa}B-dependent transcription, separating this chromatin-retention function from RNA cleavage. Conditional Ints11 deletion abolished TLR4-dependent dendritic-cell activation in adult mice and impaired LPS-induced transcription in mouse embryonic fibroblasts. Loss of Integrator in human cancer cells similarly suppressed TNF-induced inflammatory gene expression. These findings uncover a noncatalytic function for Integrator that stabilizes NF-{kappa}B at enhancers, complementing its established catalytic control of RNA polymerase II pause release. By coupling signal recognition at enhancers to productive elongation, Integrator provides a molecular solution to a central challenge of multicellularity: converting transient extracellular cues into coherent gene-expression programs across complex chromatin landscapes.

The epigenetic inheritance of mammalian DNA methylation requires DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognition of hemi-methylated DNA and histone H3 N-terminal tails directs catalysis of H3K14, H3K18, and/or H3K23 mono-ubiquitination to recruit DNMT1. While it is appreciated that UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail and that DNMT1 recognizes this state through tandem ubiquitin interacting motifs, the mechanism that promotes successive ubiquitination and the biological function of multi-mono-ubiquitination are unknown. Here, we show that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD) to promote further ubiquitin deposition. Disruption of this ubiquitin reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss within late-replicating, CpG-sparse genomic regions that are characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those observed by disruption of UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading of H3 ubiquitination support CpG-sparse DNA methylation maintenance. Together, these findings establish a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable genomic regions of the mammalian methylome.

Faithful genome duplication requires coordination of DNA replication, chromatin assembly, and checkpoint signaling to prevent inappropriate mitotic progression. Using a targeted siRNA screen for factors modulating CHK1 inhibitor-induced DNA damage and premature mitotic entry, we identified replication-coupled chromatin assembly as a determinant of checkpoint dependence. Acute depletion of the CAF-1 subunit CHAF1A modestly delayed S-phase progression but caused little DNA damage, whereas inhibition of CHK1 or ATR triggered extensive DNA damage, premature mitotic entry, and loss of viability. Transcriptomic analyses revealed that CHK1 inhibition induced a transcriptional response enriched for B-MYB/Myb-MuvB (MMB) target genes in CHAF1A-deficient cells. Notably, induction of these genes occurred without corresponding increases in promoter accessibility, suggesting that enhanced MMB-dependent transcription is largely independent of widespread promoter opening. B-MYB depletion attenuated MMB target-gene induction, premature mitotic entry, and DNA damage. Depletion of ASF1 and NAP1L1 similarly enhanced checkpoint inhibitor sensitivity, indicating that increased dependence on ATR-CHK1 signaling is a broader consequence of impaired histone chaperone function. Together, our findings establish a functional link between replication-coupled chromatin assembly, ATR-CHK1 signaling, and MMB-dependent mitotic transcription.

The promoter-proximal pause (PPP), at most metazoan genes, is believed to prime transcriptional activation by release of paused RNAPII into the gene. We tested the pause-release model by abolishing the PPP and monitoring gene activation. An LLM-guided AI discovered that upstream G's and a G T/C dinucleotide robustly discriminates pauses from non-pauses. Insertion of G-less sequences into NDRG1 and HSP90AA1 abolished the PPP in an orientation-dependent way, but did not impair activation by hypoxia or heat-shock. Hence pause-release is dispensable for these responses. The 5' peak of RNAPII detected by ChIP persisted even when pausing, detected by NET-seq was absent. Instead of pausing, these promoter-proximal polymerases may undergo abortive initiation. RNAPII C-terminal domain (CTD) deletion suppresses the PPP and shifts it upstream without affecting sequence-dependent pausing, consistent with promoter-tethering by this IDR. We propose that PPP formation is initiated by G-dependent arrest, and stabilized by multiple factors including CTD-dependent tethering.

The cleavage of membrane-bound phosphoinositides produces inositol polyphosphates (IPs) and pyrophosphates (PP-IPs), small water-soluble signaling molecules that are highly conserved in eukaryotes and play a critical role in cell metabolism. Loss of the inositol pyrophosphate kinase (PP-IPk) Kcs1 and its mammalian ortholog IP6K1 was previously shown to cause hypersensitivity to genotoxic stress and reduced homologous recombination (HR) repair. To identify the mechanism linking PP-IPs to DNA double-strand break (DSB) repair, we combined quantitative proteomics with biochemical, genetic, and cell-cycle analyses in budding yeast. We found that loss of Kcs1, but not the PP-IPk Vip1, caused increased sensitivity to the radiomimetic drug zeocin. kcs1{Delta} cells displayed persistent Rad53 signaling and elevated {gamma}-H2A levels, remaining arrested in G1/S during treatment and accumulating in G2/M for hours after damage removal. Unexpectedly, Kcs1-dependent PP-IPs promoted DSB repair independently of the core HR machinery and of cell-cycle stage. Quantitative proteomics revealed increased levels of PHO pathway proteins in zeocin treated kcs{Delta} cells, and deletion of the CDK inhibitor PHO81 substantially rescued zeocin hypersensitivity, indicating that constitutive inhibition of the Pho80-Pho85 cyclin-dependent kinase underlies the DNA repair defect caused by Kcs1 loss. This function of Pho80-Pho85 was mechanistically distinct from its canonical role in phosphate homeostasis, and partially independent of its connections to Ku80 regulation. Together, our findings identify Kcs1-dependent PP-IP signaling as a new upstream input controlling Pho80-Pho85 activity during the DNA damage response, establishing an HR-independent axis for genome maintenance and linking PP-IP metabolism to the DNA damage response in eukaryotic cells.

Transcriptional memory is a phenomenon that allows cells to 'remember' prior stimulatory history, which can alter subsequent gene expression patterns and shape development, immunity, and disease. Although various epigenetic adaptations have been implicated, it remains unclear whether the mechanics of transcription alone are sufficient to install durable memory independently of complex activation and global phenotypic changes induced by inflammatory and environmental stimuli. Here, we employ CRISPR activation (CRISPRa) to uncouple gene activation from global signaling and test whether memory can be established selectively by a local transcriptional pulse in primary mesenchymal stromal cells (MSCs). Key stimulus-responsive genes (IDO1, CXCL11) remain sensitized for weeks after either IFN{gamma} exposure or locus-specific CRISPRa perturbations. Histone PTM profiling links this memory to epigenetic hysteresis involving persistent loss of promoter H3K27me3. Single-molecule chromatin mapping by DAF-seq showed that this adaptation does not coincide with gains in promoter accessibility, but suggests it supports increased transcription factor reacquisition in rechallenged cells. Finally, while H3K27me3 loss appears to be a key hallmark of strongly induced memory genes, multi-omic analyses showed that persistent H3K27me3 hysteresis also occurs at a small subset of non-memory genes. Coordination of H3K27me3 hysteresis with additional cis factors therefore likely shapes transcriptional memory in a locus-specific fashion, underscoring the utility of targeted CRISPRa perturbations for dissecting these responses.

DNA double strand breaks (DSBs) generated by genotoxic stress frequently contain chemically diverse termini that require end processing before ligation by non-homologous end joining (NHEJ). How end chemistry influences assembly and persistence of NHEJ synaptic complexes remains unclear. Using single-molecule imaging approaches in Xenopus egg extract, we measured formation and stability of the long-range (LRC) and short-range (SRC) complexes on nine DNA substrates spanning kinase-, polymerase- and nuclease-dependent processing requirements. LRC formation and lifetime were comparable across end types, and SRC formation and stability were largely insensitive to end chemistry. DNA hairpins, physiological intermediates of V(D)J recombination, were a notable exception that formed LRCs but inefficiently transitioned to SRCs. Depletion of cognate processing factors had modest effects on SRC formation and persistence, indicating that core NHEJ factors provide the principal structural support for synapsis across diverse termini. Although XLF was required for SRC formation on all substrates, reducing an XLF dimer from two intact tail/Ku-binding motifs to one selectively reduced productive SRC formation on ends requiring processing. Together, these results support a model in which partially redundant protein-protein interactions between core NHEJ factors maintain synapsis while end-specific processing reactions shape repair outcome.

Cells must prevent incompletely spliced transcripts from entering the cytoplasm, yet how splicing status is recognized and converted into selective nuclear retention remains unclear. Here, we identify a network of RNA-binding proteins that retain pre-mRNPs within nuclear speckles. Proteomic analysis of splicing intermediates accumulated in nuclear speckles identified a set of retention factors including BCLAF1, THRAP3 and SRSF7 that anchor pre-mRNPs to the SON scaffold and are required for pre-mRNP retention in speckles. Distinct RNA-binding patterns and transcript-specific retention factor requirements suggest that multiple mRNP features tune retention combinatorially. Super-resolution imaging revealed that the SON and SRRM2 speckle scaffolds form interdigitated networks with RNA enriched along networks and within interstitial speckle lumens. This architecture remodels in response to pre-mRNP load and is disrupted upon depletion of retention factors. Together, these findings define a pre-mRNP quality-control pathway that links splicing state to nuclear retention and the dynamic organization of nuclear speckles.

X chromosome inactivation in mammals is driven by the long non-coding RNA Xist, which localizes and spreads across the chromosome from which it is transcribed. Although several factors required for Xist localization have been identified, the underlying mechanism remains unclear. Here, we combine Nuclear-BRITE, a method for visualizing and quantifying the dynamics of individual Xist RNA molecules, with acute depletion of key localization factors. We find that Xist molecules remain constrained throughout interphase, consistent with continuous association with chromatin. Building on this observation, we identify a contact-mediated transfer mechanism in which 3D chromosome dynamics promote handover of Xist molecules between chromosomal sites. We further show that the Xist-binding proteins SPEN and HNRNPU play distinct roles in this process by promoting Xist - Xist and Xist - chromatin interactions, respectively. Together, our findings define a mechanism for Xist RNA spreading and provide a framework for understanding its unique chromosome-wide localization.

Signal-responsive enhancers must activate transcription and then return to a competent but inactive state, yet whether this transition is passive or actively driven has remained unresolved. We define this process as enhancer resetting and identify the androgen receptor (AR) as the factor that executes it as an endogenous response to estrogen signaling, independent of exogenous androgen. As estrogen receptor- (ER) undergoes ligand-induced proteasomal degradation during late-phase signaling, AR progressively accumulates in the nucleus and preferentially occupies highly active, persistent ER enhancers in a transcriptionally silent manner. This late-phase AR binding evicts the pioneer factor FOXA1 from chromatin while preserving baseline accessibility, a handoff mechanism that decouples chromatin openness from active transcription. Conversely, augmenting AR activity by DHT treatment or AR overexpression accelerates FOXA1 eviction, while disrupting AR chromatin binding, either by AR knockdown or a DNA-binding-deficient mutant, prevents it, leaving enhancers in an aberrantly permissive state that drives amplified ER rebinding and transcriptional hyperactivation upon subsequent estrogen stimulation. These findings establish active enhancer resetting as a mechanism safeguarding the fidelity of repeated transcriptional responses. Failure of this mechanism may underlie the transcriptional dysregulation that drives tumor progression and endocrine therapy resistance in ER-positive breast cancer, where estrogen signaling is chronic and cyclic.

Chromatin mobility, the exploration of 3D conformations by the chromatin fibre, is implicated in transcription and DNA repair, and is thought to decrease progressively as cells differentiate. Here, combining live-cell 3D single-nucleosome tracking with genomic approaches, we show that chromatin mobility instead changes in a cell-type-specific manner, increasing as pluripotent cells acquire posterior fates. This is accompanied by 3D genome reorganisation, including loss of Polycomb-associated interactions and emergence of CDX2-associated interactions. Withdrawal of posteriorising WNT/{beta}-catenin signalling rapidly reduces chromatin mobility and partially restores Polycomb-associated chromatin interactions, while Cdx1/2/4-null cells fail to acquire these WNT/{beta}-catenin-dependent changes. Together, our findings establish chromatin mobility as a regulated property of cell state, with WNT/{beta}-catenin signalling and CDX factors acting as mediators that fine-tune the sampling of 3D chromatin interactions during cell state transitions.

The DNA sensor cGAS is largely sequestered on chromatin in an inactive form, but how this state is regulated remains unclear. We show that cGAS preferentially binds nucleosomes containing the histone variant macroH2A1 (mH2A1) marked by H3K27me3 and H3K9me3. Cryo-EM reveals that cGAS selectively engages the mH2A1/H2B acidic patch within heterotypic nucleosomes. Depleting mH2A1.1 or blocking H3K27me3 deposition releases cGAS from chromatin and activates the cGAS-STING-IRF3 pathway. Genome-wide profiling reveals two cGAS-bound chromatin compartments: chromosome-arm sites co-occupied by mH2A1.1, methylated CpG islands, and CTCF, where cGAS and mH2A1.1 binding are interdependent; and centromeric regions (14% of peaks), which lack DNA methylation and mH2A1.1 dependence. These findings establish mH2A1 and H3K27me3 as determinants of cGAS chromatin tethering, linking nuclear cGAS regulation to facultative heterochromatin and opening new avenues for modulating cGAS-STING signaling.

High-resolution measurements of pathway activity make it possible to characterize how signaling responds to changing cellular conditions, to evaluate pathway-targeted inhibitors, and to dissect how sequence variation shapes signaling output. Here, we describe a sequencing-based assay that uses engineered, phospho-state-selective affinity reagents to quantify ERK activation state as a high-resolution, ratiometric readout of RAS/ERK MAPK pathway activity. Profiling ~1,600 BRAF variants at basal and elevated RAS-GTP levels allowed us to assign activity scores to somatic cancer and germline RASopathy variants and to distinguish residues that stabilize autoinhibition from those required for active dimer formation. We identified a large class of dominant negative variants that suppress signaling below endogenous levels through enhanced RAS engagement and an inability to form productive RAF dimers. Measuring the intracellular potency of the RAF inhibitor LXH254 against ~1,000 variants in parallel revealed hinge and activation-loop positions conferring resistance and showed that disrupting the BRAF-MEK interface is broadly sensitizing.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that coordinates energy production with biosynthetic demands in physiologically proliferating and cancer cells. PKM2 has also emerged as a non-canonical RNA-binding protein. Here, we combine targeted PKM2 mutagenesis with RNA-protein interaction assays, biochemical and biophysical analyses to define how RNA binding relates to PKM2 allosteric control and oligomeric state. We show that the allosteric activator fructose-1,6-bisphosphate (FBP) strongly reduces PKM2 binding to RNA. Mutants with impaired oligomerization show reduced RNA binding, and RNA association is favored by the tetrameric state. A positively charged surface patch in the FBP-binding region is essential for RNA binding and displays emergent properties. Single-residue variants further link RNA association to FBP-responsive tetrameric conformations. Our data integrate RNA binding with PKM2 allosteric regulation.

N-terminal acetylation of cellular proteins has been implicated in their proteasomal degradation through the Ac/N-degron pathway. Despite representing a major branch of the N-degron system, only a limited number of degrons and their motifs have been identified and characterized in this pathway, and the mechanism underpinning the recognition of N-terminal acetylation has remained largely elusive. Here, by interrogating FAM72A-mediated degradation of UNG2, we pinpointed the N-terminally acetylated sequence of UNG2 as a bona fide Ac/N-degron specifically recognized by the CTLHMKLN1-FAM72A E3 ubiquitin ligase. Rather than relying solely on a single acetylated N-terminal residue, this degron features a consensus Nt-AcM1-[ILV]2-G3 motif that governs both E3 targeting and the intracellular stability of the neo-substrates uncovered through proteomic screenings. Cryo-EM structure of the UNG2-FAM72A-MKLN1 complex further elucidated the molecular details supporting the high specificity of degron recognition. Notably, this degron pathway is subject to multilayered regulation, highlighting the characteristic conditionality of degron system. Our findings thus define a distinct AcM-[ILV]-G/N-degron pathway and provide mechanistic insights into degron recognition dictated by both N-terminal acetylation and sequence motif context.

Rapid and precise depletion of endogenous proteins is essential for dissecting gene function, yet existing degron technologies often require direct modification of endogenous coding sequences, which can compromise protein expression and limit their applicability. Here, we develop idTAG, an inducible degron platform that decouples protein expression from protein degradation by integrating Tet-On 3G-mediated transcriptional control with dTAG-mediated degradation. Systematic characterization reveals that conventional degron knock-in frequently causes unintended reduction of target protein expression at the mRNA level, representing an underappreciated limitation of current approaches. In contrast, idTAG enables efficient generation of degron cell lines while maintaining physiological protein abundance and allows programmable control of degradation kinetics through independent regulation of protein synthesis and degradation. Applying idTAG to RNA-binding proteins, we demonstrate that acute depletion of FUS has minimal effects on global RNA polymerase II transcription, whereas acute loss of SRSF5 selectively disrupts transcription termination at a subset of genes. Thus, idTAG provides a versatile platform for physiological and temporally controlled interrogation of endogenous protein functions.

Transcription occurs in the context of histone-based chromatin in eukaryotes and most archaea. The archaeal RNA polymerase and histones are ancestral to their eukaryotic counterparts, yet how RNA polymerase traverses histone-bound DNA in archaea remains poorly understood. Here, we reconstitute a nucleosome-associated transcription elongation complex (TEC) from Pyrococcus furiosus and capture its structure across multiple elongation states by cryo-electron microscopy (cryo-EM). High-resolution structures reveal that the RNA polymerase engages a three-dimer HPfB nucleosome positioned downstream. We identify direct physical interactions between HPfB and Rpo1N RNA polymerase subunit, establishing a defined polymerase-histone interface during transcription. Structural comparisons across defined elongation states demonstrate that RNA polymerase first translocates on DNA using the proximal histone dimer as an anchor, followed by destabilisation of the distal dimer. The DNA exiting the nucleosome is partially unwrapped and is redirected traversing the Rpo4/7 stalk. At extended transcript lengths nucleosome organisation is lost, indicating that transcription elongation ultimately disrupts histone-DNA interactions. This work provides the first structural insight into transcription through chromatin in archaea, revealing a mechanism in which the archaeal RNA polymerase actively remodels nucleosomes via DNA redirection and histone displacement, aided by direct polymerase-histone contacts.

The +1 nucleosome has emerged as a key regulator of eukaryotic transcription, but how it controls transcription initiation remains poorly understood. Here we reconstitute transcription through the +1 nucleosome using eleven purified yeast factors: RNA polymerase II (Pol II), the six general transcription factors (GTFs), TFIIS, the activator Pho4, and the SAGA and Mediator complexes. The system recapitulates key features of regulation observed in vivo. SAGA, acting with Pho4, directs pre-initiation complex (PIC) assembly to the correct position through its TBP-loading activity. Mediator stimulates transcription when the +1 nucleosome imposes a barrier to PIC formation, consistent with stabilization of productive TFIIH-DNA engagement. Contrary to the prevailing model, SAGA remains bound to the PIC after TBP loading and acetylates the +1 nucleosome within the assembled complex. The isolated PIC-Mediator-SAGA-nucleosome complex is transcriptionally active, and the repressive effect of the nucleosome is relieved by the DNA translocase activity of Ssl2, the TFIIH subunit that opens promoter DNA. TFIIH thus couples promoter melting to remodeling of the +1 nucleosome.

Ribonuclease Z enzymes remove 3' trailers from precursor tRNAs. In human cells, ELAC2/RNase ZL matures nuclear and mitochondrial pre-tRNAs, whereas the cytosolic short paralog ELAC1/RNase ZS repairs ANKZF1-cleaved tRNAs, but its broader substrate repertoire remains unclear. We investigated ELAC1 function using transcriptome profiling, small RNA sequencing, CLIP-seq, and biochemical reconstitution. ELAC1 was dispensable for canonical tRNA processing but associated with structured RNA polymerase III transcripts, prominently Y RNAs and vault RNAs (vtRNAs). Purified ELAC1 cleaved all four human vtRNAs near their 3' ends, with high-resolution mapping identifying a conserved cleavage position within the 3' extension. Efficient cleavage required structural features of the vtRNA terminal region. In contrast, ELAC2 showed little or no vtRNA cleavage under the conditions tested and displayed stricter substrate and cleavage-site selection on pre-tRNA. Loss and re-expression of ELAC1 altered structured ncRNA abundance and small-RNA end profiles. These findings identify vtRNAs as direct ELAC1 substrates and support functional specialization of human ELAC paralogs, with ELAC2 favoring precise pre-tRNA maturation and ELAC1 exhibiting broader RNA substrate specificity.