CRISPR screening identifies PRMT1 as a key pro-ferroptotic gene via a two-layer regulatory mechanism.

Ferroptosis is a form of nonapoptotic cell death characterized by iron-dependent peroxidation of polyunsaturated phospholipids. However, much remains unknown about the regulators of ferroptosis. Here, using CRISPR-Cas9-mediated genetic screening, we identify protein arginine methyltransferase 1 (PRMT1) as a crucial promoter of ferroptosis. We find that PRMT1 decreases the expression of solute carrier family 7 member 11 (SLC7A11) to limit the abundance of intracellular glutathione (GSH). Moreover, we show that PRMT1 interacts with ferroptosis suppressor protein 1 (FSP1), a GSH-independent ferroptosis suppressor, to inhibit the membrane localization and enzymatic activity of FSP1 through arginine dimethylation at R316, thus reducing CoQ10H2 content and inducing ferroptosis sensitivity. Importantly, genetic depletion or pharmacological inhibition of PRMT1 in mice prevents ferroptotic events in the liver and improves the overall survival under concanavalin A (ConA) exposure. Hence, our findings suggest that PRMT1 is a key regulator of ferroptosis and a potential target for antiferroptosis therapeutics.

CCDC32 stabilizes clathrin-coated pits and drives their invagination.

Clathrin-mediated endocytosis (CME) is essential for maintaining cellular homeostasis. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. Quantitative live cell imaging reveals that CCDC32, a poorly characterized endocytic accessory protein, regulates CCP stabilization and is required for efficient CCP invagination. CCDC32 interacts with the α-appendage domain (AD) of AP2 via its coiled-coil domain to exert this function. Furthermore, we showed that the clinically observed nonsense mutations in CCDC32, which result in the development of cardio-facio-neuro-developmental syndrome (CFNDS), inhibit CME by abolishing CCDC32-AP2 interactions. Overall, our data demonstrates the function and molecular mechanism of a novel endocytic accessory protein, CCDC32, in CME regulation. Significance Statement: Clathrin-mediated endocytosis (CME) happens via the initiation, stabilization, and invagination of clathrin-coated pits (CCPs). In this study, we used a combination of quantitative live cell imaging, ultrastructure electron microscopy and biochemical experiments to show that CCDC32, a poorly studied and functional ambiguous protein, acts as an important endocytic accessory protein that regulates CCP stabilization and invagination. Specifically, CCDC32 exerts this function via its interactions with AP2, and the coiled-coil domain of CCDC32 and the α-appendage domain (AD) of AP2 are essential in mediating CCDC32-AP2 interactions. Importantly, we demonstrate that clinically observed loss-of-function mutations in CCDC32 lose AP2 interaction capacity and inhibit CME, resulting in the development of cardio-facio-neuro-developmental syndrome (CFNDS).

Hippo-signaling-controlled MHC class I antigen processing and presentation pathway potentiates antitumor immunity.

The major histocompatibility complex class I (MHC class I)-mediated tumor antigen processing and presentation (APP) pathway is essential for the recruitment and activation of cytotoxic CD8+ T lymphocytes (CD8+ CTLs). However, this pathway is frequently dysregulated in many cancers, thus leading to a failure of immunotherapy. Here, we report that activation of the tumor-intrinsic Hippo pathway positively correlates with the expression of MHC class I APP genes and the abundance of CD8+ CTLs in mouse tumors and patients. Blocking the Hippo pathway effector Yes-associated protein/transcriptional enhanced associate domain (YAP/TEAD) potently improves antitumor immunity. Mechanistically, the YAP/TEAD complex cooperates with the nucleosome remodeling and deacetylase complex to repress NLRC5 transcription. The upregulation of NLRC5 by YAP/TEAD depletion or pharmacological inhibition increases the expression of MHC class I APP genes and enhances CD8+ CTL-mediated killing of cancer cells. Collectively, our results suggest a crucial tumor-promoting function of YAP depending on NLRC5 to impair the MHC class I APP pathway and provide a rationale for inhibiting YAP activity in immunotherapy for cancer.

A Glb1-2A-mCherry reporter monitors systemic aging and predicts lifespan in middle-aged mice.

The progressive decline of physiological function and the increased risk of age-related diseases challenge healthy aging. Multiple anti-aging manipulations, such as senolytics, have proven beneficial for health; however, the biomarkers that label in vivo senescence at systemic levels are lacking, thus hindering anti-aging applications. In this study, we generate a Glb1+/m‒Glb1-2A-mCherry (GAC) reporter allele at the Glb1 gene locus, which encodes lysosomal ß-galactosidase-an enzyme elevated in tissues of old mice. A linear correlation between GAC signal and chronological age is established in a cohort of middle-aged (9 to 13 months) Glb1+/m mice. The high GAC signal is closely associated with cardiac hypertrophy and a shortened lifespan. Moreover, the GAC signal is exponentially increased in pathological senescence induced by bleomycin in the lung. Senolytic dasatinib and quercetin (D + Q) reduce GAC signal in bleomycin treated mice. Thus, the Glb1-2A-mCherry reporter mice monitors systemic aging and function decline, predicts lifespan, and may facilitate the understanding of aging mechanisms and help in the development of anti-aging interventions.

Lamin A safeguards the m6 A methylase METTL14 nuclear speckle reservoir to prevent cellular senescence.

Mutations in LMNA gene are frequently identified in patients suffering from a genetic disorder known as Hutchison-Gilford progeria syndrome (HGPS), providing an ideal model for the understanding of the mechanisms of aging. Lamin A, encoded by LMNA, is an essential component of the subnuclear domain-nuclear speckles; however, the functional significance in aging is unclear. Here, we show that Lamin A interacts with the m6 A methyltransferases, METTL3 and METTL14 in nuclear speckles. Lamin A deficiency compromises the nuclear speckle METTL3/14 reservoir and renders these methylases susceptible to proteasome-mediated degradation. Moreover, METTL3/14 levels progressively decline in cells undergoing replicative senescence. Overexpression of METTL14 attenuates both replicative senescence and premature senescence. The data reveal an essential role for Lamin A in safeguarding the nuclear speckle reservoir of the m6 A methylase METTL14 to antagonize cellular senescence.

Synergy between SIRT1 and SIRT6 helps recognize DNA breaks and potentiates the DNA damage response and repair in humans and mice.

The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR in human and mouse cells. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to γH2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair in humans and mice.

PML2-mediated thread-like nuclear bodies mark late senescence in Hutchinson-Gilford progeria syndrome.

Progerin accumulation disrupts nuclear lamina integrity and causes nuclear structure abnormalities, leading to premature aging, that is, Hutchinson-Gilford progeria syndrome (HGPS). The roles of nuclear subcompartments, such as PML nuclear bodies (PML NBs), in HGPS pathogenesis, are unclear. Here, we show that classical dot-like PML NBs are reorganized into thread-like structures in HGPS patient fibroblasts and their presence is associated with late stage of senescence. By co-immunoprecipitation analysis, we show that farnesylated Progerin interacts with human PML2, which accounts for the formation of thread-like PML NBs. Specifically, human PML2 but not PML1 overexpression in HGPS cells promotes PML thread development and accelerates senescence. Further immunofluorescence microscopy, immuno-TRAP, and deep sequencing data suggest that these irregular PML NBs might promote senescence by perturbing NB-associated DNA repair and gene expression in HGPS cells. These data identify irregular structures of PML NBs in senescent HGPS cells and support that the thread-like PML NBs might be a novel, morphological, and functional biomarker of late senescence.

Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis.

SIRT6 deacetylase activity improves stress resistance via gene silencing and genome maintenance. Here, we reveal a deacetylase-independent function of SIRT6, which promotes anti-apoptotic gene expression via the transcription factor GATA4. SIRT6 recruits TIP60 acetyltransferase to acetylate GATA4 at K328/330, thus enhancing its chromatin binding capacity. In turn, GATA4 inhibits the deacetylase activity of SIRT6, thus ensuring the local chromatin accessibility via TIP60-promoted H3K9 acetylation. Significantly, the treatment of doxorubicin (DOX), an anti-cancer chemotherapeutic, impairs the SIRT6-TIP60-GATA4 trimeric complex, blocking GATA4 acetylation and causing cardiomyocyte apoptosis. While GATA4 hyperacetylation-mimic retains the protective effect against DOX, the hypoacetylation-mimic loses such ability. Thus, the data reveal a novel SIRT6-TIP60-GATA4 axis, which promotes the anti-apoptotic pathway to prevent DOX toxicity. Targeting the trimeric complex constitutes a new strategy to improve the safety of DOX chemotherapy in clinical application.

Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria.

DNA damage accumulates with age (Lombard et al., 2005). However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that ATM-centered DNA damage response (DDR) progressively declines with senescence and age, while low dose of chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-related metabolic shift, and prolongs replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 extend lifespan in Atm-/- mice, with restored metabolic homeostasis. Moreover, the treatment with CQ remarkably extends lifespan of Caenorhabditis elegans, but not the ATM-1 mutants. In a progeria mouse model with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases.