Mitotic entry is a tightly regulated process controlled by CDK1-Cyclin B1. Its activity is negatively regulated by the WEE family kinases, WEE1, WEE2 and PKMYT1, to prevent premature mitotic entry. While WEE1 has an esta...Mitotic entry is a tightly regulated process controlled by CDK1-Cyclin B1. Its activity is negatively regulated by the WEE family kinases, WEE1, WEE2 and PKMYT1, to prevent premature mitotic entry. While WEE1 has an established role in regulating CDK2 during S-phase, the full scope of PKMYT1's roles in the cell cycle is surprisingly unexplored. Here, we show that PKMYT1 activity increases during anaphase and we reveal a novel mitotic function for PKMYT1 that is distinct from its family member WEE1. Chemical inhibition of PKMYT1 induces premature anaphase, leading to chromosome segregation errors. These errors, including chromatin bridges and micronuclei, consequently activate the cGAS-STING pathway. We further demonstrate that PKMYT1 contributes to maintaining spindle assembly checkpoint integrity, as its inhibition promotes mitotic slippage in the presence of anti-microtubule drugs. Our findings reveal that PKMYT1 provides an additional regulatory mechanism, acting alongside Cyclin B1 degradation, to control CDK1-Cyclin B1 activity and ensure the fidelity of the metaphase-to-anaphase transition.
The evolutionary timing of the origin of secretory proteins underlying post-mating reproductive processes remains uncharacterized in vertebrates. We dated the origin of 2520 human tissue-specific genes encoding secretory...The evolutionary timing of the origin of secretory proteins underlying post-mating reproductive processes remains uncharacterized in vertebrates. We dated the origin of 2520 human tissue-specific genes encoding secretory proteins across vertebrate evolution, finding that the male reproductive (MR) secretome underwent a dramatic expansion during the eutherian (placental mammal) diversification, experiencing a 6.8-fold gene gain-the largest increase compared to any other secretome or transition in vertebrate evolution. These genes are predominantly expressed in the epididymis, where they protect sperm and drive sperm maturation, influencing essential post-mating reproductive processes and male reproductive outcome. In contrast, MR secretome genes that originated along other evolutionary branches are primarily associated with sperm structure, motility, egg binding, and fusion. These findings provide molecular evidence for a major reconfiguration of male reproductive biology during placental mammal diversification through the MR secretome, affecting primarily the epididymis.
The mA methyltransferase complex ("writer") regulates mRNA stability and translation, but how its assembly is orchestrated remains incompletely understood. Wilms' tumor 1-associating protein (WTAP) is a conserved regulat...The mA methyltransferase complex ("writer") regulates mRNA stability and translation, but how its assembly is orchestrated remains incompletely understood. Wilms' tumor 1-associating protein (WTAP) is a conserved regulatory subunit essential for mA deposition and cell proliferation, yet its structural organization and mechanistic contributions remain elusive. Here, we report that WTAP dimerizes and further assembles into a stable tetramer through its middle coiled-coil domain, as revealed by high-resolution crystal structures. Disruption of this tetrameric interface abolishes WTAP's interaction with METTL3, METTL14, and ZC3H13, impairs mA deposition, and fails to rescue proliferation defects in WTAP-depleted cells. Live-cell imaging demonstrates that WTAP is required for accurate chromosome segregation, and MeRIP-seq analysis identifies WTAP-dependent mA regulation as a critical determinant sustaining the expression of mitotic regulators, including KIF20A. Together, our study defines a tetrameric scaffold function for WTAP that is essential for writer complex integrity and highlights its pivotal role in linking mA methylation to cell cycle progression.
The paracaspase MALT1 is essential for lymphocyte activation and also plays roles in non-immune cells and cancer. Its protease activity regulates immune signaling by cleaving specific substrates, making it a promising th...The paracaspase MALT1 is essential for lymphocyte activation and also plays roles in non-immune cells and cancer. Its protease activity regulates immune signaling by cleaving specific substrates, making it a promising therapeutic target. However, broad inhibition of MALT1 protease activity causes multiorgan inflammation in mice, highlighting the need to understand the effects of individual substrate cleavage. We generated CYLD(R321A) knock-in mice expressing a MALT1-resistant form of the deubiquitinase CYLD. These mice are healthy, with normal lymphocyte development and preserved immune signaling. Unlike MALT1 protease-dead mice, they do not develop spontaneous inflammation. Notably, they exhibit altered gut microbiota and reduced disease severity in a model of multiple sclerosis. Together, our work shows that blocking cleavage of a single MALT1 substrate is sufficient to modulate microbiota and neuroinflammation without causing overt defects in lymphocyte cell development or activation, providing in vivo evidence for substrate-specific targeting of MALT1 as a refined therapeutic strategy.
The p16-3MR mouse model, designed to express Renilla luciferase, mRFP, and herpes simplex virus 1 thymidine kinase (HSV-TK) under the p16 promoter, has been widely used to visualize and ablate senescent cells in vivo, bu...The p16-3MR mouse model, designed to express Renilla luciferase, mRFP, and herpes simplex virus 1 thymidine kinase (HSV-TK) under the p16 promoter, has been widely used to visualize and ablate senescent cells in vivo, but our analyses revealed critical limitations. Bioluminescence signals in p16-3MR mice were extremely weak and virtually indistinguishable from those of wild-type mice injected with coelenterazine-h, indicating that previously reported signals largely reflected substrate background rather than authentic reporter expression. Signal intensity remained unchanged with aging, doxorubicin treatment, or cutaneous wound healing, failing to replicate earlier observations. Furthermore, RFP signals were undetectable in senescent fibroblasts from p16-3MR mice, and senescent cells were not eliminated by ganciclovir treatment, suggesting poor expression and lack of functional activity of the mRFP and HSV-TK transgenes. These results demonstrate functional deficiencies in all three transgenes, highlighting the importance of using wild-type controls and calling for careful reevaluation of studies employing this system.
During embryogenesis, nascent hematopoietic stem and progenitor cells (HSPCs) arise from hemogenic endothelium via endothelial-to-hematopoietic transition (EHT). While this process is orchestrated by multiple intrinsic f...During embryogenesis, nascent hematopoietic stem and progenitor cells (HSPCs) arise from hemogenic endothelium via endothelial-to-hematopoietic transition (EHT). While this process is orchestrated by multiple intrinsic factors, the role of tRNA-mediated translational control during EHT remains poorly understood. Here, we identify tRNA mA58 as a predominant tRNA modification in specifying HSPC fate in zebrafish embryos. Depletion of trmt61a compromises HSPC production in the aorta-gonad-mesonephros (AGM) region, accompanied by attenuated tRNA mA58 levels and evident p53-dependent apoptosis. Mechanistically, Trmt61a-mediated tRNA mA58 modification enhances translation efficiency of nuclear respiratory factor 1 (Nrf1), a key regulator of mitochondrial biogenesis. Consequently, trmt61a deficiency leads to mitochondrial dysfunction and compromises cell survival, ultimately impairing HSPC production. Our findings establish that tRNA mA58 modification is essential for HSPC generation by supporting translation efficiency, providing new insights into improved strategies for in vitro HSPC induction.
Effector T lymphocytes are avid nutrient consumers, but can function in nutrient-poor tumor microenvironments. Availability of key nutrients such as glucose inside the tumor is not homogeneous, and how tumor-infiltrating...Effector T lymphocytes are avid nutrient consumers, but can function in nutrient-poor tumor microenvironments. Availability of key nutrients such as glucose inside the tumor is not homogeneous, and how tumor-infiltrating T lymphocytes (TILs) differ between regions with better and poorer blood perfusion is not well known. Here we show that in vitro-stimulated TILs can induce substantial production of hallmark glucose-dependent cytokines under glucose concentrations 20 times lower than in blood. In vivo, effector TILs in tumor regions with poor access to blood show comparable capacity for inducing IFNγ and granzyme B to TILs with fuller accessibility; exhibit an enhanced type I IFN response supported by local myeloid cells; and unexpectedly, have reduced expression of immune checkpoint and Treg-associated markers. TILs with poor blood accessibility also have lower biosynthetic activity than highly blood-accessible TILs, yet both compartments depend fundamentally on glucose for ATP production. Thus, effector T lymphocytes in poorly perfused tumor regions can maintain specific glucose-dependent responses, and might be partially protected from inhibitory and exhausting pressure from the tumor microenvironment.
DNA replication stress can generate mitotic defects because incompletely replicated chromosomes or unresolved replication intermediates tether sister chromatids and hinder their segregation. We and others recently uncove...DNA replication stress can generate mitotic defects because incompletely replicated chromosomes or unresolved replication intermediates tether sister chromatids and hinder their segregation. We and others recently uncovered a mitotic role for the oncoprotein CIP2A, which promotes chromosome stability and is essential in homologous recombination-deficient (HRD) cells. However, how CIP2A safeguards mitotic genome integrity remains unclear. Here, we investigate the role of CIP2A in mitotic responses to replication stress. We show that replication stress induces a strong increase in CIP2A foci during mitosis, highlighting its involvement in processing under-replicated DNA. In wild-type cells, CIP2A is required for efficient recruitment of the scaffold SLX4 and the nucleases MUS81 and XPF to sites of under-replicated DNA. CIP2A loss disrupts this recruitment and leads to increased anaphase lagging chromosomes and micronuclei formation. CIP2A also contributes to mitotic DNA synthesis (MiDAS), although this varies across cell lines, indicating that MiDAS and SMX complex recruitment are not strictly coupled. Together, our findings identify CIP2A as a regulator of mitotic processing of under-replicated DNA and provide a framework for understanding context-dependent vulnerabilities in cancer cells.
Histone modifiers are crucial for instructing multiple-stage cellular differentiation, yet the mechanisms underlying their temporal precision remain enigmatic. Here, we demonstrate that the H3K27 demethylase Kdm6b acts a...Histone modifiers are crucial for instructing multiple-stage cellular differentiation, yet the mechanisms underlying their temporal precision remain enigmatic. Here, we demonstrate that the H3K27 demethylase Kdm6b acts as an epigenetic regulator, coordinating stepwise motor neuron (MN) differentiation through sequential partnerships with stage-specific transcription factors (TFs). Genome-wide profiling reveals a progressive gain in Kdm6b occupancy, especially at distal regulatory elements, as differentiation proceeds. Kdm6b dynamically remodels chromatin landscapes by coordinating H3K27me3 removal with H3K27ac and H3K4me1 acquisition, thereby enabling timed gene activation from MN specification to maturation. Stage-specific inhibition of Kdm6b compromises the ordered expression of developmental genes. Mechanistically, Kdm6b interacts with temporal TFs over time to ensure precise transcriptional control and MN differentiation. Our work elucidates how a single epigenetic regulator achieves temporal fidelity of stepwise MN development, providing insight into epigenetic regulation of developmental timing.
Leucine-rich repeat kinase 2 (LRRK2) not only plays a vital role in familial forms of Parkinson's disease (PD) but also represents a risk factor for idiopathic PD. Its multi-domain architecture enables fine-tuned regulat...Leucine-rich repeat kinase 2 (LRRK2) not only plays a vital role in familial forms of Parkinson's disease (PD) but also represents a risk factor for idiopathic PD. Its multi-domain architecture enables fine-tuned regulation of its biological function by orchestrating intra- and inter-molecular interactions. Here, we present BioID proximity proteomes of LRRK2 that reveal new interactors, which we further characterize using a novel evolutionary and structural bioinformatics pipeline. Co-evolutionary analysis of the protein-protein interaction network identifies a structural and functional module enriched in cytoskeletal components associated with the centrosome and microtubules. In addition, structural modeling of binary interactions using AlphaFold-Multimer reveals distinct groups of interactors that engage LRRK2 in a manner dependent on specific conformations and epitopes. Furthermore, we identify distinct changes in the LRRK2 proximity proteome that are induced by the type I kinase inhibitor MLi-2 or by co-expression of the LRRK2 upstream effector RAB29. Depending on its activity state and conformation, these protein-protein interactions link LRRK2 to defined cellular sub-compartments, including centriolar satellites and vesicular sub-compartments.
Hasegawa K, Hama N, Amemiya M
… +13 more, Zeng C, Ito Y, Suzuki S, Nakamura K, Kondo J, Takeda C, Kurihara Y, Ikeda K, Fujita Y, Okada Y, Toyoda A, Hamada M, Kuwako KI
Brain aging is an intricate process that inevitably leads to functional deterioration. However, its molecular drivers remain unclear. Here, we show that the age-related decline in LINC complex expression on the neuronal...Brain aging is an intricate process that inevitably leads to functional deterioration. However, its molecular drivers remain unclear. Here, we show that the age-related decline in LINC complex expression on the neuronal nuclear envelope impairs axon initial segment (AIS)-mediated excitability and triggers brain aging. With aging, the expression of LINC complex components, including Sun1, decreases in various brain regions, accompanied by a reduction in AIS length. Preserving Sun1 expression rescues nuclear structural abnormalities in aged neurons, shifting chromatin dynamics and global gene expression toward those of young neurons. Particularly, it restores the expression of AIS-related molecules, including voltage-gated sodium or potassium channels essential for action potential generation. Inhibiting the LINC complex in young mice impairs AIS integrity, leading to reduced neuronal excitability and brain dysfunction. Furthermore, Sun1 administration to aged neurons prevents age-related AIS shortening, excitability impairment, and brain function changes. Thus, we uncover the mechanism of normal brain aging involving AIS dysfunction, identifying the LINC complex component Sun1 as essential for preserving brain function.
Vocal communication is fundamental for social interaction across species, yet the neural mechanisms that shape vocal circuit development remain poorly understood despite their relevance to neurodevelopmental disorders. H...Vocal communication is fundamental for social interaction across species, yet the neural mechanisms that shape vocal circuit development remain poorly understood despite their relevance to neurodevelopmental disorders. Here, we investigate vocal circuit development in mice using isolation-induced ultrasonic vocalizations (USVs) in neonates. An activity-tagging approach identifies the ventromedial prefrontal cortex (vmPFC) as a cortical region strongly activated during USV emission. We find a predictable temporal correlation between vmPFC activity and USV emission using in vivo fiber photometry. Selective activation and inhibition of vmPFC neurons establishes a causal role of vmPFC in vocalization. Interestingly, chronic activation of vmPFC neurons not only increases Foxp2, a gene implicated in childhood speech apraxia, but also Vglut1-labeled synapses in the striatum, suggesting that activity-dependent increases in Foxp2 may promote corticostriatal synaptogenesis. Consistent with this finding, neonatal vmPFC activation partially rescues USV deficits in Foxp2 heterozygous mutant mice. Collectively, our results identify the vmPFC-striatal circuit as a key regulator of neonatal vocalization and suggest that Foxp2 may mediate activity-dependent development of vocal circuits.
Seki S, Harada S, Sugimoto-Ishige A
… +18 more, Unno M, Sakuma M, Ito S, Yamamoto H, Tanabe A, Matsuoka S, Makino-Okamura C, Ki S, Fukuyama H, Harada M, Tsumagari K, Imami K, Saito T, Kubo M, Suzuki T, Koseki H, Matano T, Miyauchi K
Understanding the relationship between viral proteins and host factors is essential for developing strategies to control virus infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells u...Understanding the relationship between viral proteins and host factors is essential for developing strategies to control virus infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells using angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) as viral receptor and priming protease during the viral entry phase. Here, we report that TMPRSS2 reduces the infectivity of SARS-CoV-2 and HIV-1 during the viral production phase. Treatment of virus-producing cells with a TMPRSS2 inhibitor increases the production of infectious virions. TMPRSS2 enzymatic activity specifically disrupts the trans-Golgi by phosphorylating Golgi stacking proteins through ERK activation, which disturbs virion spike incorporation and structural maturation of the viral envelope glycoproteins, causing lower viral infectivity. We find that SARS-CoV-2 envelope protein (E protein) counteracts this TMPRSS2 activity to rescue SARS-CoV-2-S incorporation. These results demonstrate negative regulation of viral envelope glycoprotein incorporation by TMPRSS2 and reveal that SARS-CoV-2 regulates the Golgi system to create an optimal viral replication environment.
The ability to adapt to nutrient stress, such as amino acid limitation, is crucial for cell survival. The mTORC1 complex and integrated stress response (ISR) are two mechanisms that sense the availability of amino acids...The ability to adapt to nutrient stress, such as amino acid limitation, is crucial for cell survival. The mTORC1 complex and integrated stress response (ISR) are two mechanisms that sense the availability of amino acids and regulate protein synthesis. Here, we reveal a new SIRT2-mediated pathway, downstream of the ISR, that is activated under amino acids limitation to suppress global translation. Under amino acid deprivation, SIRT2 protein level is upregulated translationally by its upstream open reading frame (uORF). SIRT2 in turn suppresses translation, which helps cells to survive amino acid limitation. We identify eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), which binds to eIF4E and inhibits translation, as a substrate of SIRT2. SIRT2 deacetylates 4E-BP1 at lysine 69 and stabilizes 4E-BP1 by protecting it from proteasomal degradation, leading to suppression of global translation. Our study uncovers a role for SIRT2 in regulating translation and identifies a new regulatory mechanism of 4E-BP1 in cells.
Transcription-replication conflicts (TRCs) arise when DNA replication forks encounter actively transcribing RNA polymerases, creating a major threat to genome stability. These conflicts, which can occur in different orie...Transcription-replication conflicts (TRCs) arise when DNA replication forks encounter actively transcribing RNA polymerases, creating a major threat to genome stability. These conflicts, which can occur in different orientations, disrupt replication fork progression, impair transcriptional fidelity and reshape the chromatin landscape. In this review, we discuss emerging conceptual insights into how cells coordinate replication and transcription in space and time to minimise such encounters, and we highlight the central role of RNA polymerase II dynamics in both preventing and resolving TRCs. We further describe how TRCs engage a broad network of genome maintenance pathways that regulate R-loops, stabilize stalled forks and maintain chromatin integrity. Importantly, elevated or mismanaged TRCs create vulnerabilities that many cancers exploit, positioning conflict-resolution mechanisms as attractive therapeutic targets. Finally, we examine current challenges in detecting and analysing these transient, dynamic events and underscore the need for improved imaging and sequencing technologies to study the genome's molecular "traffic jams". A deeper mechanistic understanding of TRCs will be crucial for harnessing them in precision oncology and clarifying their broader roles in genome regulation.
Mitochondrial biogenesis and inheritance must be tightly coordinated with cell division to maintain mitochondrial function and cell survival. The dynamics of the mitochondrial network, including fusion and fission, are e...Mitochondrial biogenesis and inheritance must be tightly coordinated with cell division to maintain mitochondrial function and cell survival. The dynamics of the mitochondrial network, including fusion and fission, are essential for mitochondrial inheritance and quality control. In budding yeast, simultaneous inhibition of both processes compromises mitochondrial DNA (mtDNA) integrity, increasing the frequency of petite cells. Loss of fusion alone completely eliminates mtDNA. Although this has been known for decades, why mtDNA is lost remained unclear. Here, we examine the effects of impaired mitochondrial fusion by depleting the mitofusin Fzo1. By analyzing over thirty thousand single cells across their cell cycles, we show that Fzo1-depletion induces rapid mitochondrial fragmentation and loss of membrane potential, followed by progressive declines in mtDNA content and growth rate. During division, Fzo1-depleted daughters inherit disproportionately large mitochondrial amounts, leaving mothers with too little. This imbalance, combined with an inability to upregulate compensatory mtDNA synthesis, drives rapid mtDNA loss. Our results reveal how fusion defects cause mtDNA loss and mitochondrial dysfunction, which might have implications for diseases linked to impaired fusion.
While much is known about the effects of the chemical microenvironment on cellular metabolism, mechanical cues have emerged as critical stimuli of intracellular metabolic pathways. Mechanical signals from the extracellul...While much is known about the effects of the chemical microenvironment on cellular metabolism, mechanical cues have emerged as critical stimuli of intracellular metabolic pathways. Mechanical signals from the extracellular matrix (ECM), neighboring cells, and the microenvironment intersect with key regulators of cellular metabolism, often leading to changes in fundamental cell behaviors, including cell proliferation and migration. Here, we review recent work that has uncovered a role for mechanical cues from microenvironmental factors on cellular metabolism. We discuss how cell-ECM interactions and forces such as shear, tension, and compression affect cellular metabolic requirements and energy production. Importantly, mechanometabolism shapes both physiological homeostasis and pathological states, and further investigation has implications for understanding tissue function and disease progression and uncovering potential therapeutic strategies.
Li KL, Wang C, Li YH
… +19 more, Peng B, Zheng L, Che Q, Lu XY, Li D, Li YT, Wei HM, Chang JF, Wang WF, Liu M, Li X, Jin F, Liu K, Li Y, Liu LW, Li M, Ni JQ, Xie T, Sun FL
Maintenance of the genome and epigenome stability is vital for animal longevity. Long noncoding RNAs, roX1 and roX2, are known to be important in the male X chromosome dosage complex in Drosophila males. However, their f...Maintenance of the genome and epigenome stability is vital for animal longevity. Long noncoding RNAs, roX1 and roX2, are known to be important in the male X chromosome dosage complex in Drosophila males. However, their functions in Drosophila females have never been explored. This study demonstrates a role of roX RNAs in promoting heterochromatin formation in intestinal stem cells (ISCs) of Drosophila females under pathogen infection or aging. Increased heterochromatin formation in ISCs and progenitor enteroblasts (EBs) is associated with decreased active epigenetic modifications and global gene repression. Elevation of roX RNAs in ISCs promotes heterochromatinization and represses gene expression by recruiting heterochromatin proteins such as HP1a and Su(var)3-9. Overexpression of roX RNAs promotes ISCs hyperplasia, while their inactivation mitigates ISCs dysplasia and extends lifespan. Moreover, Xist RNA, the functional analog of roX RNAs, also promotes heterochromatin formation and ISCs hyperplasia in Drosophila, and significantly increases in aged people. Therefore, our findings reveal a role of roX RNAs in promoting heterochromatin expansion and controlling animal longevity.
STING is an important component in the host innate immune system where its activation by cyclic dinucleotides culminates in the production of interferons and pro-inflammatory cytokines that mediate host defence against i...STING is an important component in the host innate immune system where its activation by cyclic dinucleotides culminates in the production of interferons and pro-inflammatory cytokines that mediate host defence against infection. While the mechanisms that govern STING-induced interferon production have been comprehensively characterised, how pro-inflammatory cytokines are produced downstream of STING remains less understood. Here we discover that IRF3 is critical for effective STING-mediated inflammatory cytokine production from macrophages as those lacking IRF3 display significant defects. Interestingly, the loss of IRF3 does not impact the activation of the prominent pro-inflammatory transcription factor, NF-κB, but rather affects the AP-1 transcriptional complex. We further discover the role of IRF3 in STING inflammatory responses is independent of its phosphorylation and distinct from its role as a transcription factor for induction of type I interferons. This additional activity of IRF3 is dependent on its recruitment to the previously defined IRF3 binding motif within the C-terminal tail of STING. Hence, our findings reveal an unexpected noncanonical function of IRF3 that is critical for mediating STING-induced pro-inflammatory cytokines from macrophages.