Opposing activities of conserved chromatin regulatory complexes, such as the Polycomb repressive complex 1 (PRC1) and the activating chromatin remodeler switch/sucrose non-fermentable (SWI/SNF), play critical roles in re...Opposing activities of conserved chromatin regulatory complexes, such as the Polycomb repressive complex 1 (PRC1) and the activating chromatin remodeler switch/sucrose non-fermentable (SWI/SNF), play critical roles in regulating gene expression during development and differentiation. The mechanisms by which these complexes compete to regulate chromatin states remain poorly understood. We combine single-molecule analysis and genomic approaches in cultured cells to demonstrate that the condensate-forming properties of PRC1 play an important role in excluding SWI/SNF from chromatin. Consistently, PRC1 compositions with higher condensate-forming propensity are more effective in preventing SWI/SNF binding. Conversely, SWI/SNF-bound chromatin significantly reduces PRC1 binding and subsequent condensate formation. Notably, SWI/SNF can suppress PRC1 condensate formation in an ATP-hydrolysis-independent manner. We propose that the condensate properties of different PRC1 compositions drive mutual PRC1-SWI/SNF antagonism to properly balance these competing regulatory activities during development.
Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartme...Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartments. Cohesin can also bind PDS5 proteins, which do not support loop extrusion but are required for the formation of CTCF boundaries. How PDS5 proteins perform this function is unknown. Here we show, by in vitro single-molecule imaging, that human PDS5 proteins stop loop extrusion by facilitating the dissociation of NIPBL from cohesin. Hi-C experiments suggest that this function is required for the establishment of CTCF boundaries in cells. In silico modeling indicates that PDS5 proteins enable the separation between compartments by limiting cohesin's velocity and chromatin residence time. The degree of this compartmentalization depends on the frequency with which chromatin is extruded relative to the time it takes for compartments to form. These results identify PDS5 proteins as key regulators of genome organization.
Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. Although condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, histone phosphorylation and deace...Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. Although condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, histone phosphorylation and deacetylation have been proposed to contribute to a further 2- to 4-fold reduction in mitotic chromatin volume. Here, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing highly synchronous mitotic entry. Our analysis reveals three temporally distinct programs of histone H3 phosphorylation on T3, S10, and S28 that could differentially regulate the association of readers with chromatin via methyl-phos switching. Mass spectrometry, quantitative chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, and immunofluorescence analyses reveal that H3 T3 phosphorylation is a mitosis-specific marker of heterochromatin, whose deposition requires H3K9me3. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, deacetylation does not drive mitotic chromosome formation. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.
Genome folding is not static but emerges from dynamic processes that control transcription, replication, recombination, and repair. DNA loop extrusion by cohesin is central to genome organization, yet it remains unclear...Genome folding is not static but emerges from dynamic processes that control transcription, replication, recombination, and repair. DNA loop extrusion by cohesin is central to genome organization, yet it remains unclear how cells can tune extrusion kinetics to achieve precise and functional chromosome folding patterns. Here, we show that extrusion rate acts as a tunable biophysical parameter in cells, quantitatively dialed by the respective dosage of the cohesin cofactors NIPBL and PDS5. Modulation of extrusion rate can offset changes in cohesin lifetime to buffer steady-state chromosome structure and transcriptional states, even in the face of abnormal extrusion dynamics. These findings provide a long-sought mechanistic basis for the genetic interactions between cohesin cofactors and for the molecular origin of haploinsufficiency in cohesinopathies, such as Cornelia de Lange syndrome.
Zaratiana C, M Y, Lee YA
… +19 more, Ong ABL, Liu TZY, Low SMC, Chang SMS, Tan S, Mustafa DNA, Ganesh A, Chang X, Koh XQ, Tay SH, Lee WJJ, Yuan JM, Khor CC, Koh WP, Dorajoo R, Li YE, Kasahara K, Wuestefeld T, Chen PB
Cis-regulatory elements (CREs) are central to dynamic gene regulation in hepatocytes, yet most functional annotations derive from in vitro models that poorly capture physiological regulation. We systematically profiled 1...Cis-regulatory elements (CREs) are central to dynamic gene regulation in hepatocytes, yet most functional annotations derive from in vitro models that poorly capture physiological regulation. We systematically profiled 109,386 human liver-derived CREs using massively parallel reporter assays in hepatocytes under matched in vitro and in vivo conditions. In vivo-active functional CREs (fCREs) were enriched for H3K27ac and chromatin accessibility and were regulated by diverse transcription factors in the human liver. We further demonstrate that gut microbiota-derived signals modulate fCRE activity and target gene expression in vivo, in part via the KEAP1/NFE2L2 antioxidant pathway. Specific microbial metabolites directly altered the activity of selected fCREs, and genetic variation within fCREs modified their responsiveness to microbial signals. Together, these findings reveal microbiota-dependent regulation of hepatic CREs and highlight condition-specific gene regulatory mechanisms in vivo.
The encounters between transcription and DNA replication may remodel replication dynamics, yet the coordination of these two essential processes remains elusive. Here, we developed a replication-associated Micro-C (Repli...The encounters between transcription and DNA replication may remodel replication dynamics, yet the coordination of these two essential processes remains elusive. Here, we developed a replication-associated Micro-C (Repli-MiC) method to map replication fountains, which are dynamic chromatin-interaction structures induced by coupled replication forks, at nucleosome resolution in mammalian cells. We implemented a reinforcement-learning-based computational framework to enable unbiased and quantitative characterization of replication fountains, thereby allowing precise assessment of how transcription influences sister-fork elongation. With this integrated platform, we found that co-directional transcription induces a bias in the speed of sister replication forks toward the transcriptional orientation without compromising fork coupling, which is further enhanced upon depletion of DNA topoisomerase I (TOP1). Conversely, head-on transcription potentially impairs fork elongation to weaken replication fountains. This study provides a comprehensive assay for profiling the entire DNA-replication elongation process and sheds light on the dual roles of transcription in modulating fork elongation.
Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI), yet the mechanisms driving elicitor-induced alkalinization...Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI), yet the mechanisms driving elicitor-induced alkalinization and its role in plant signaling remain unclear. Here, we demonstrate that inhibition of autoinhibited H-ATPases (AHAs) is required for elicitor-induced extracellular alkalinization. This alkalinization is essential for immune and cell-wall damage signaling mediated by diverse plasma membrane-localized receptor kinases (RKs), likely through modulation of ligand-receptor interactions. Mechanistically, RKs transduce elicitor-triggered signaling via the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1), which inhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF) interactions through a conserved phosphorylation event. This phosphorylation-driven extracellular alkalinization module is required for disease resistance and cell-wall damage responses initiated by ligand-RK pairs. Our findings uncover a conserved phosphorelay circuit that broadly regulates extracellular alkalinization to coordinate RK signaling, illuminating a general mechanism for RK activation and stress resilience.
Identifying tertiary structures and protein binding sites in RNA molecules remains a key challenge in RNA biology. We describe multi-site dimethyl sulfate (DMS)-mutational profiling (MaP) (msDMS-MaP), a strategy that ena...Identifying tertiary structures and protein binding sites in RNA molecules remains a key challenge in RNA biology. We describe multi-site dimethyl sulfate (DMS)-mutational profiling (MaP) (msDMS-MaP), a strategy that enables simultaneous measurement of RNA secondary, tertiary, and quaternary structures via a single DMS chemical probing experiment. Optimized reverse transcription decodes typically invisible DMS N7-methylguanine (N7-G) modifications via a tautomer-induced mutational signature concurrent with N1 and N3 modifications. We show that N7-G reactivity reports on higher-order RNA structures, revealing key functional motifs such as pseudoknots and protein binding sites. Using msDMS-MaP, we find that E. coli ribosomal RNAs encode numerous independently folding tertiary structures that coincide with binding sites for primary assembly proteins. We further apply msDMS-MaP to define the quaternary structural ensemble of the 7SK small nuclear ribonucleoprotein particle (snRNP), revealing that each of the three 7SK structural isoforms possesses distinct protein binding profiles in cells. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.
Nuclear speckles are nuclear bodies enriched with RNA-processing proteins. Highly transcribed genes and their nascent pre-mRNAs are organized around nuclear speckles, facilitating co-transcriptional RNA processing. Howev...Nuclear speckles are nuclear bodies enriched with RNA-processing proteins. Highly transcribed genes and their nascent pre-mRNAs are organized around nuclear speckles, facilitating co-transcriptional RNA processing. However, the mechanisms underlying this spatial organization remain unclear. Here, we identified a class of Alu repeat-containing RNAs that are highly enriched in nuclear speckles. These RNAs interact with actively transcribed genes through their Alu elements, partially via R-loop formation. They also engage with nuclear speckle proteins, promoting their coalescence, phase separation, and assembly into nuclear speckles, thereby contributing to the spatial organization of actively transcribed genes around these structures. Depletion of these RNAs reduces speckle size, displaces active genes, and impairs RNA processing. Moreover, this mechanism is critical for high expression of many erythroid differentiation genes during erythropoiesis. Our findings highlight an important role of Alu repeat-containing RNAs in nuclear speckles assembly and their function in mediating spatial co-transcriptional RNA processing.
Ribonuclease (RNase) III Drosha initiates microRNA (miRNA) biogenesis. Emerging evidence suggests that Drosha modulates processes beyond miRNA biogenesis, indicative of the functional complexity of Drosha. Here, we show...Ribonuclease (RNase) III Drosha initiates microRNA (miRNA) biogenesis. Emerging evidence suggests that Drosha modulates processes beyond miRNA biogenesis, indicative of the functional complexity of Drosha. Here, we show that Drosha facilitates activation of sterol regulatory element-binding protein 1 (SREBP1), the central player in triglyceride (TG) biosynthesis, in an RNase activity-independent manner. Mechanistically, cytoplasmic Drosha interacts with Sec31, a subunit of coat protein complex II (COPII), through its arginine/serine (RS)-rich domain. This interaction promotes COPII-mediated transport and activation of SREBP1. Moreover, Akt phosphorylates Drosha at Ser237 in the RS-rich domain, which is required for the Drosha-Sec31 interaction, SREBP1 processing, and TG accumulation in response to insulin. The Akt-Drosha-SREBP1 axis was hyperactivated in mice fed a high-fat, high-sugar diet, and hepatic Drosha deletion or administration of an interfering peptide that blocked Akt-mediated phosphorylation of Drosha ameliorated liver TG deposition and insulin resistance in this mouse model. Thus, our findings uncover a noncanonical function of Drosha involved in SREBP1 processing and lipogenesis.
Being able to control the complementarity and hindrance between target DNA and base editor proteins enables precise, bystander-free editing. Here, we combined combinatorial mutagenesis with machine learning to analyze an...Being able to control the complementarity and hindrance between target DNA and base editor proteins enables precise, bystander-free editing. Here, we combined combinatorial mutagenesis with machine learning to analyze and engineer these interactions at scale. By profiling DNA motif preferences across 160,000 evoAPOBEC1 and 64 million TadA variants in human cells, we used as little as 0.004% of the mutational landscape to make predictions. This identified variants with motif-specific activity and eliminated residual adenine editing in cytosine base editors. In correcting >800 disease-associated mutations, our variants outperformed previous versions in precluding unintended edits at purine motifs, achieving undetectable bystander edits in 50% of cases. Additionally, a pre-trained, structure-based deep learning model predicted functional TadA variants with 63% success across 20 variants spanning 26 amino acid sites, without experimental data and in a single prediction round. These approaches streamline the re-engineering of base editors for enhanced precision tailored to specific targets.
Mining phages for new enzymatic activities continues to be important for the development of new tools for biotechnology. In this study, we used MetaGPA-a method linking genotype to phenotype in metagenomic data-to identi...Mining phages for new enzymatic activities continues to be important for the development of new tools for biotechnology. In this study, we used MetaGPA-a method linking genotype to phenotype in metagenomic data-to identify deoxycytidine deaminases, a protein family highly associated with cytosine modifications in metaviromes. Unexpectedly, a subset of these deaminases exhibited a preference for 5-methylcytosine (5mC) over cytosine (C) in both mononucleotide and single-stranded DNA substrates. In a methylome-sequencing workflow, deamination of 5mC by these enzymes enabled direct conversion of methylated cytosine while completely eliminating any background deamination of unmodified cytosine. This direct conversion allows for precise identification of methylated sites at single-base resolution with unmatched sensitivity enabling broad applications for the simultaneous sequencing of genome and methylome.
Alternative polyadenylation (APA) generates transcript isoforms with variable 3' untranslated regions (UTR) lengths, yet its role in DNA damage response (DDR) genes is poorly understood. Here, we demonstrate that the pro...Alternative polyadenylation (APA) generates transcript isoforms with variable 3' untranslated regions (UTR) lengths, yet its role in DNA damage response (DDR) genes is poorly understood. Here, we demonstrate that the proximal polyadenylation site (pPAS) of MRE11 engages in PAS-promoter looping to facilitate RNA polymerase recycling and sustain high promoter activity-a mechanism not well characterized in mammals. Deletion of the MRE11 pPAS disrupts this looping, reduces MRE11 transcription, impairs MRE11-RAD50-NBS1 (MRN) complex levels, and phenocopies hypomorphic MRE11 mutations. MRE11pPAS cells exhibit ectopic DNA replication and reduced viability under overgrowth conditions. 5-ethynyl-2'-deoxyuridine sequencing (EdU-seq) revealed aberrant DNA synthesis occurring primarily at intronic and intergenic regions, where MRE11 chromatin immunoprecipitation sequencing (ChIP-seq) showed decreased binding correlating with elevated replication. Furthermore, multiple DDR genes with several PASs also form PAS-promoter loops, suggesting a broader regulatory mechanism. Together these findings identify the MRE11 pPAS as a critical noncoding element that maintains genome stability through transcriptional regulation via PAS-promoter looping.
The target of rapamycin complex 2 (TORC2) is a central node in signaling feedback loops, serving to maintain the biophysical homeostasis of the plasma membrane (PM). How TORC2 is regulated by mechanical perturbation of t...The target of rapamycin complex 2 (TORC2) is a central node in signaling feedback loops, serving to maintain the biophysical homeostasis of the plasma membrane (PM). How TORC2 is regulated by mechanical perturbation of the PM is not well understood. To address this, we determined the cryo-electron microscopy structure of endogenous yeast TORC2 at up to 2.2 Å resolution. Our model refines the position and interactions of TORC2-specific subunits, providing a structural basis for the differential assembly of Tor2 into TORC2. Furthermore, we observe the insertion of the pleckstrin-homology domain of the Avo1 subunit into the Tor2 active site, providing a regulatory mechanism mediated by phosphoinositides. Structure-guided functional experiments reveal a potential TORC2 membrane-binding surface and a positively charged pocket in the Avo3 subunit that is necessary for TORC2 activation. Collectively, our data suggest that signaling phosphoinositides activate TORC2 by membrane-induced structural rearrangements via the concerted action of conserved regulatory subunits.
Aberrant activation of the PI3K/AKT/mTOR signaling pathway is a common feature of cancer, but while mTOR kinase represents an attractive drug target, mTOR inhibitors have not seen broad success as single agents. To ident...Aberrant activation of the PI3K/AKT/mTOR signaling pathway is a common feature of cancer, but while mTOR kinase represents an attractive drug target, mTOR inhibitors have not seen broad success as single agents. To identify strategies to enhance the utility of third-generation bi-steric mTORC1 inhibitors, we performed genome-scale CRISPR interference chemogenomics screens, which revealed that mTORC1 inhibitor-mediated cytostasis leaves cells exquisitely dependent on the lipid peroxide scavenging enzyme GPX4. Mechanistically, using unbiased CRISPR activation chemogenomics screens, we demonstrate that mTORC1-dependent control of ferroptosis occurs, in part, through regulation of SCARB1 expression. Specifically, we find that the high-density lipoprotein (HDL) can suppress ferroptosis through interaction with its receptor SCARB1 and delivery of vitamin E to target cells. Our work highlights combining mTORC1 with GPX4 inhibition as one of the most promising combinatorial approaches for mTOR-targeted cancer therapies and defines an HDL-SCARB1 ferroptosis-suppression system that is regulated by mTORC1 activity.
Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), with core structural subunits encoded by mitochondrial DNA (mtDNA) and translated by mitochondrial ribosomes. However, how mitochondrial translation e...Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), with core structural subunits encoded by mitochondrial DNA (mtDNA) and translated by mitochondrial ribosomes. However, how mitochondrial translation elongation influences OXPHOS biogenesis remains unclear. Here, we show that in Neurospora crassa, the mitochondrial ribosomal RNA (rRNA) methyltransferase 1 (MRM1) promotes OXPHOS biogenesis by repressing translation elongation independently of its catalytic activity. The N-terminal intrinsically disordered region (IDR) of MRM1 binds simultaneously to mitochondrial ribosomes and mRNAs. Disrupting either interaction accelerates elongation and enhances synthesis of mtDNA-encoded OXPHOS subunits but impairs their co-translational folding and membrane insertion. Pharmacological slowing of mitochondrial translation partially alleviates these defects. The MRM1 IDR is conserved in Ascomycete fungi and is essential for plant infection by Magnaporthe oryzae. Together, our findings identify translation elongation control as a mechanism coordinating mitochondrial protein synthesis and folding during OXPHOS biogenesis and MRM1 as a potential target for broad-spectrum antifungal strategies.
The pro-inflammatory, senescence-associated secretory phenotype (SASP) is a hallmark of senescent cells (SnCs) that exacerbates age-related pathophysiology and chronic diseases. Although unique gene regulation is essenti...The pro-inflammatory, senescence-associated secretory phenotype (SASP) is a hallmark of senescent cells (SnCs) that exacerbates age-related pathophysiology and chronic diseases. Although unique gene regulation is essential for fulfilling the pro-inflammatory SASP, the epigenomic basis in SnCs remains largely unknown. Here, we show that FOXF1/2 define the senescence-specific enhancer landscape by shaping chromatin accessibility. FOXF1/2 interact with p300/CREB-binding protein (CBP) and stimulate H3K27 acetylation and chromatin opening at de novo enhancers of pro-inflammatory SASP genes, together with the recruitment of AP-1 c-JUN to these regions. FOXF1/2 depletion in SnCs significantly suppresses pro-inflammatory SASP, independent of persistent growth arrest, and diminishes the paracrine effect on surrounding cells. Notably, loss of FOXF1/2 leads to the redistribution of AP-1 c-JUN to regulatory elements of senescence-associated downregulated genes. Our results uncover that FOXF1/2 coordinate the pro-inflammatory SASP program, suggesting that FOXF1/2-mediated enhancer remodeling is a key target for modulating SnCs to promote healthy aging.
Phosphoinositides regulate cellular signaling. Tan et al. and Li et al. identify PtdIns(3,5)P₂ and cholesterol as key lipids that stabilize STING oligomers and enable TBK1 activation, explaining why STING must traffic fr...Phosphoinositides regulate cellular signaling. Tan et al. and Li et al. identify PtdIns(3,5)P₂ and cholesterol as key lipids that stabilize STING oligomers and enable TBK1 activation, explaining why STING must traffic from the ER to the Golgi to initiate immune signaling.
In a recent Cell paper, Yi and Zhang et al. reveal that ecDNA serves as a platform for the generation of oncogenic fusion transcripts, particularly PVT1 5' end-based fusions, which enhance oncogene expression and drive c...In a recent Cell paper, Yi and Zhang et al. reveal that ecDNA serves as a platform for the generation of oncogenic fusion transcripts, particularly PVT1 5' end-based fusions, which enhance oncogene expression and drive cancer progression beyond gene amplification alone.
In this issue of Molecular Cell, Nössing et al. demonstrate that TNF activates a species-specific cleavage of p62/SQSTM1 by caspase-8, which activates extrinsic apoptosis via a feedforward loop. Introducing this missing...In this issue of Molecular Cell, Nössing et al. demonstrate that TNF activates a species-specific cleavage of p62/SQSTM1 by caspase-8, which activates extrinsic apoptosis via a feedforward loop. Introducing this missing p62 cleavage site into mice amplifies TNF-driven inflammation, shock, and mortality.