The cardiac endothelium influences ventricular chamber development by coordinating trabeculation and compaction. However, the endothelial-specific molecular mechanisms mediating this coordination are not fully understood. Here, we identify the Sox7 transcription factor as a critical cue instructing cardiac endothelium identity during ventricular chamber development. Endothelial-specific loss of Sox7 function in mice results in cardiac ventricular defects similar to non-compaction cardiomyopathy, with a change in the proportions of trabecular and compact cardiomyocytes in the mutant hearts. This phenotype is paralleled by abnormal coronary artery formation. Loss of Sox7 function disrupts the transcriptional regulation of the Notch pathway and connexins 37 and 40, which govern coronary arterial specification. Upon Sox7 endothelial-specific deletion, single-nuclei transcriptomics analysis identifies the depletion of a subset of Sox9/Gpc3-positive endocardial progenitor cells and an increase in erythro-myeloid cell lineages. Fate mapping analysis reveals that a subset of Sox7-null endothelial cells transdifferentiate into hematopoietic but not cardiomyocyte lineages. Our findings determine that Sox7 maintains cardiac endothelial cell identity, which is crucial to the cellular cross-talk that drives ventricular compaction and coronary artery development.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Epigenetic modifications are known to be crucial for hematopoietic stem cell (HSC) differentiation, with the BET family member BRD4 playing a vital role in this as an epigenetic reader. In this issue of EMBO reports, Yang et al (2023) demonstrate that the absence of BRD4 leads to senescence in HSCs and hematopoietic progenitor cells (HPCs), affecting the expression of crucial genes involved in myeloid and erythroid development. These data suggest that BRD4 has a protective role in preserving histone tails, thereby sustaining normal HSC/HPC functions.

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.

How does one help a struggling trainee, in or out of their own lab?

Can we prevent the future misuse of gene editing of the human population?

The new biologics have great potential to help patients with hitherto uncurable diseases. But their exorbitant costs challenges the basis of health care systems based on equity.

Recalcitrant infections pose a serious challenge by prolonging antibiotic therapies and contributing to the spread of antibiotic resistance, thereby threatening the successful treatment of bacterial infections. One potential contributing factor in persistent infections is antibiotic persistence, which involves the survival of transiently tolerant subpopulations of bacteria. This review summarizes the current understanding of antibiotic persistence, including its clinical significance and the environmental and evolutionary factors at play. Additionally, we discuss the emerging concept of persister regrowth and potential strategies to combat persister cells. Recent advances highlight the multifaceted nature of persistence, which is controlled by deterministic and stochastic elements and shaped by genetic and environmental factors. To translate in vitro findings to in vivo settings, it is crucial to include the heterogeneity and complexity of bacterial populations in natural environments. As researchers continue to gain a more holistic understanding of this phenomenon and develop effective treatments for persistent bacterial infections, the study of antibiotic persistence is likely to become increasingly complex.

Long interspersed nuclear elements (LINEs) play essential roles in shaping chromatin states, while the factors that cooperate with LINEs and their roles in higher-order chromatin organization remain poorly understood. Here, we show that MATR3, a nuclear matrix protein, interplays with antisense LINE1 (AS L1) RNAs to form a meshwork via phase separation, providing a dynamic platform for chromatin spatial organization. MATR3 and AS L1 RNAs affect the nuclear localization of each other. After MATR3 depletion, the chromatin, particularly H3K27me3-modified chromatin, redistributes in the cell nuclei. Topologically associating domains (TADs) that highly transcribe MATR3-associated AS L1 RNAs show decreased intra-TAD interactions in both AML12 and ES cells. MATR3 depletion increases the accessibility of H3K27me3 domains adjacent to MATR3-associated AS L1, without affecting H3K27me3 modifications. Furthermore, amyotrophic lateral sclerosis (ALS)-associated MATR3 mutants alter biophysical features of the MATR3-AS L1 RNA meshwork and cause an abnormal H3K27me3 staining. Collectively, we reveal a role of the meshwork formed by MATR3 and AS L1 RNAs in gathering chromatin in the nucleus.

The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis strain SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic ABA-dependent manner. Application of SA190 priming on crops is demonstrated for alfalfa, showing enhanced performance under drought conditions. In summary, a single beneficial root bacterial strain can help plants to resist drought conditions.

Repetitive DNA elements are packaged in heterochromatin, but many require bursts of transcription to initiate and maintain long-term silencing. The mechanisms by which these heterochromatic genome features are transcribed remain largely unknown. Here, we show that DOT1L, a conserved histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), has a specialized role in transcription of major satellite repeats to maintain pericentromeric heterochromatin and genome stability. We find that H3K79me3 is selectively enriched relative to H3K79me2 at repetitive elements in mouse embryonic stem cells (mESCs), that DOT1L loss compromises pericentromeric satellite transcription, and that this activity involves possible coordination between DOT1L and the chromatin remodeler SMARCA5. Stimulation of transcript production from pericentromeric repeats by DOT1L participates in stabilization of heterochromatin structures in mESCs and cleavage-stage embryos and is required for preimplantation viability. Our findings uncover an important role for DOT1L as a bridge between transcriptional activation of repeat elements and heterochromatin stability, advancing our understanding of how genome integrity is maintained and how chromatin state is set up during early development.

Intratumor heterogeneity (ITH) is a barrier to effective therapy. However, it is largely unknown how ITH is established at the onset of tumor progression, such as in colorectal cancer (CRC). Here, we integrate single-cell RNA-seq and functional validation to show that asymmetric division of CRC stem-like cells (CCSC) is critical for early ITH establishment. We find that CCSC-derived xenografts contain seven cell subtypes, including CCSCs, that dynamically change during CRC xenograft progression. Furthermore, three of the subtypes are generated by asymmetric division of CCSCs. They are functionally distinct and appear at the early stage of xenografts. In particular, we identify a chemoresistant and an invasive subtype, and investigate the regulators that control their generation. Finally, we show that targeting the regulators influences cell subtype composition and CRC progression. Our findings demonstrate that asymmetric division of CCSCs contributes to the early establishment of ITH. Targeting asymmetric division may alter ITH and benefit CRC therapy.

Hexanucleotide repeat expansions within C9orf72 are a frequent cause of amyotrophic lateral sclerosis and frontotemporal dementia. Haploinsufficiency leading to reduced C9orf72 protein contributes to disease pathogenesis. C9orf72 binds SMCR8 to form a robust complex that regulates small GTPases, lysosomal integrity, and autophagy. In contrast to this functional understanding, we know far less about the assembly and turnover of the C9orf72-SMCR8 complex. Loss of either subunit causes the concurrent ablation of the respective partner. However, the molecular mechanism underlying this interdependence remains elusive. Here, we identify C9orf72 as a substrate of branched ubiquitin chain-dependent protein quality control. We find that SMCR8 prevents C9orf72 from rapid degradation by the proteasome. Mass spectrometry and biochemical analyses reveal the E3 ligase UBR5 and the BAG6 chaperone complex as C9orf72-interacting proteins, which are components of the machinery that modifies proteins with K11/K48-linked heterotypic ubiquitin chains. Depletion of UBR5 results in reduced K11/K48 ubiquitination and increased C9orf72 when SMCR8 is absent. Our data provide novel insights into C9orf72 regulation with potential implication for strategies to antagonize C9orf72 loss during disease progression.

GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2-dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors.

Skeletal muscle plays a key role in systemic energy homeostasis besides its contractile function, but what links these functions is poorly defined. Protein Arginine Methyl Transferase 5 (PRMT5) is a well-known oncoprotein but also expressed in healthy tissues with unclear physiological functions. As adult muscles express high levels of Prmt5, we generated skeletal muscle-specific Prmt5 knockout (Prmt5MKO ) mice. We observe reduced muscle mass, oxidative capacity, force production, and exercise performance in Prmt5MKO mice. The motor deficiency is associated with scarce lipid droplets in myofibers due to defects in lipid biosynthesis and accelerated degradation. Specifically, PRMT5 deletion reduces dimethylation and stability of Sterol Regulatory Element-Binding Transcription Factor 1a (SREBP1a), a master regulator of de novo lipogenesis. Moreover, Prmt5MKO impairs the repressive H4R3 symmetric dimethylation at the Pnpla2 promoter, elevating the level of its encoded protein ATGL, the rate-limiting enzyme catalyzing lipolysis. Accordingly, skeletal muscle-specific double knockout of Pnpla2 and Prmt5 normalizes muscle mass and function. Together, our findings delineate a physiological function of PRMT5 in linking lipid metabolism to contractile function of myofibers.

The counterregulatory response to hypoglycemia (CRR), which ensures a sufficient glucose supply to the brain, is an essential survival function. It is orchestrated by incompletely characterized glucose-sensing neurons, which trigger a coordinated autonomous and hormonal response that restores normoglycemia. Here, we investigate the role of hypothalamic Tmem117, identified in a genetic screen as a regulator of CRR. We show that Tmem117 is expressed in vasopressin magnocellular neurons of the hypothalamus. Tmem117 inactivation in these neurons increases hypoglycemia-induced vasopressin secretion leading to higher glucagon secretion in male mice, and this effect is estrus cycle phase dependent in female mice. Ex vivo electrophysiological analysis, in situ hybridization, and in vivo calcium imaging reveal that Tmem117 inactivation does not affect the glucose-sensing properties of vasopressin neurons but increases ER stress, ROS production, and intracellular calcium levels accompanied by increased vasopressin production and secretion. Thus, Tmem117 in vasopressin neurons is a physiological regulator of glucagon secretion, which highlights the role of these neurons in the coordinated response to hypoglycemia.

Transposable elements (TEs) are among the most dynamic parts of genomes. Since TEs are potentially deleterious, eukaryotes silence them through epigenetic mechanisms such as repressive histone modifications and DNA methylation. We previously reported that Arabidopsis TEs, called VANDALs, counteract epigenetic silencing through a group of sequence-specific anti-silencing proteins, VANCs. VANC proteins bind to noncoding regions of specific VANDAL copies and induce loss of silent chromatin marks. The VANC-target regions form tandem repeats, which diverge rapidly. Sequence-specific anti-silencing allows these TEs to proliferate with minimum host damage. Here, we show that RNA-directed DNA methylation (RdDM) efficiently targets noncoding regions of VANDAL TEs to silence them de novo. Thus, escape from RdDM could be a primary event leading to the rapid evolution and diversification of sequence-specific anti-silencing systems. We propose that this selfish behavior of TEs paradoxically could make them diverse and less harmful to the host.

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H2 O2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H2 O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention.

The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.

The mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping-pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism.

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eμ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER-endosome tethering mechanism mediated by homotypic interactions. The single-pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN-knockout (KO) cells, the ER-late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline-rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER-endosome membrane tethering in cells. A region of 28 amino acids spanning 150-177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN-KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150-177), brings two different liposomes closer in vitro. Using organelle-specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER-endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation (pUb), Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support the recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization and mt UPR-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: (i) kinetics of pUb accumulation, (ii) pUb puncta on mitochondria by super-resolution imaging, (iii) recruitment of Parkin and autophagy machinery to damaged mitochondria, (iv) mitophagic flux, and (v) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/β-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic β-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic β-catenin, and reducing its cytoplasmic levels in a GSK3β- and β-TrCP-independent manner, indicating that NEURL-β-catenin interactions can lead to a disruption of the canonical Wnt/β-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/β-catenin signalling.

Misfolded Aβ is involved in the progression of Alzheimer's disease (AD). However, the role of its polymorphic variants or conformational strains in AD pathogenesis is not fully understood. Here, we study the seeding properties of two structurally defined synthetic misfolded Aβ strains (termed 2F and 3F) using in vitro and in vivo assays. We show that 2F and 3F strains differ in their biochemical properties, including resistance to proteolysis, binding to strain-specific dyes, and in vitro seeding. Injection of these strains into a transgenic mouse model produces different pathological features, namely different rates of aggregation, formation of different plaque types, tropism to specific brain regions, differential recruitment of Aβ40 /Aβ42 peptides, and induction of microglial and astroglial responses. Importantly, the aggregates induced by 2F and 3F are structurally different as determined by ssNMR. Our study analyzes the biological properties of purified Aβ polymorphs that have been characterized at the atomic resolution level and provides relevant information on the pathological significance of misfolded Aβ strains.

Homologous recombination (HR), a form of error-free DNA double-strand break (DSB) repair, is important for the maintenance of genomic integrity. Here, we identify a moonlighting protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a regulator of HR repair, which is mediated through HDAC1-dependent regulation of RAD51 stability. Mechanistically, in response to DSBs, Src signaling is activated and mediates GAPDH nuclear translocation. Then, GAPDH directly binds with HDAC1, releasing it from its suppressor. Subsequently, activated HDAC1 deacetylates RAD51 and prevents it from undergoing proteasomal degradation. GAPDH knockdown decreases RAD51 protein levels and inhibits HR, which is re-established by overexpression of HDAC1 but not SIRT1. Notably, K40 is an important acetylation site of RAD51, which facilitates stability maintenance. Collectively, our findings provide new insights into the importance of GAPDH in HR repair, in addition to its glycolytic activity, and they show that GAPDH stabilizes RAD51 by interacting with HDAC1 and promoting HDAC1 deacetylation of RAD51.

Several homeoprotein transcription factors transfer between cells and regulate gene expression, protein translation, and chromatin organization in recipient cells. ENGRAILED-1 is one such homeoprotein expressed in spinal V1 interneurons that synapse on α-motoneurons. Neutralizing extracellular ENGRAILED-1 by expressing a secreted single-chain antibody blocks its capture by spinal motoneurons resulting in α-motoneuron loss and limb weakness. A similar but stronger phenotype is observed in the Engrailed-1 heterozygote mouse, confirming that ENGRAILED-1 exerts a paracrine neurotrophic activity on spinal cord α-motoneurons. Intrathecal injection of ENGRAILED-1 leads to its specific internalization by spinal motoneurons and has long-lasting protective effects against neurodegeneration and weakness. Midbrain dopaminergic neurons express Engrailed-1 and, similarly to spinal cord α-motoneurons, degenerate in the heterozygote. We identify genes expressed in spinal cord motoneurons whose expression changes in mouse Engrailed-1 heterozygote midbrain neurons. Among these, p62/SQSTM1 shows increased expression during aging in spinal cord motoneurons in the Engrailed-1 heterozygote and upon extracellular ENGRAILED-1 neutralization. We conclude that ENGRAILED-1 might regulate motoneuron aging and has non-cell-autonomous neurotrophic activity.

Precise regulation of mitochondrial fusion and fission is essential for cellular activity and animal development. Imbalances between these processes can lead to fragmentation and loss of normal membrane potential in individual mitochondria. In this study, we show that MIRO-1 is stochastically elevated in individual fragmented mitochondria and is required for maintaining mitochondrial membrane potential. We further observe a higher level of membrane potential in fragmented mitochondria in fzo-1 mutants and wounded animals. Moreover, MIRO-1 interacts with VDAC-1, a crucial mitochondrial ion channel located in the outer mitochondrial membrane, and this interaction depends on the residues E473 of MIRO-1 and K163 of VDAC-1. The E473G point mutation disrupts their interaction, resulting in a reduction of the mitochondrial membrane potential. Our findings suggest that MIRO-1 regulates membrane potential and maintains mitochondrial activity and animal health by interacting with VDAC-1. This study provides insight into the mechanisms underlying the stochastic maintenance of membrane potential in fragmented mitochondria.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Recent studies highlight the importance of baseline functional immunity for immune checkpoint blockade therapies. High-dimensional systemic immune profiling is performed in a cohort of non-small-cell lung cancer patients undergoing PD-L1/PD-1 blockade immunotherapy. Responders show high baseline myeloid phenotypic diversity in peripheral blood. To quantify it, we define a diversity index as a potential biomarker of response. This parameter correlates with elevated activated monocytic cells and decreased granulocytic phenotypes. High-throughput profiling of soluble factors in plasma identifies fractalkine (FKN), a chemokine involved in immune chemotaxis and adhesion, as a biomarker of response to immunotherapy that also correlates with myeloid cell diversity in human patients and murine models. Secreted FKN inhibits lung adenocarcinoma growth in vivo through a prominent contribution of systemic effector NK cells and increased tumor immune infiltration. FKN sensitizes murine lung cancer models refractory to anti-PD-1 treatment to immune checkpoint blockade immunotherapy. Importantly, recombinant FKN and tumor-expressed FKN are efficacious in delaying tumor growth in vivo locally and systemically, indicating a potential therapeutic use of FKN in combination with immunotherapy.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Hypoxia can occur in pancreatic β-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on β-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human β-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or β-cells of ob/ob mice reverses defects in insulin secretion. Mechanistically, BHLHE40 represses the expression of Mafa, encoding the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating the binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic β-cells was recovered by MAFA re-expression. Collectively, our work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in β-cells that inhibit insulin secretion by suppressing MAFA expression.

A variety of lifespans, faster ageing processes, and shared social spaces with humans mean studies of our canine friends hold great potential for shining light on ageing and cognitive decline.

Fetal development and parturition are precisely regulated processes that involve continuous crosstalk between the mother and the fetus. Our previous discovery that wild-type mice carrying steroid receptor coactivator (Src)-1 and Src-2 double-deficient fetuses exhibit impaired lung development and delayed labor, which indicates that the signals for parturition emanate from the fetus. In this study, we perform RNA sequencing and targeted metabolomics analyses of the lungs from fetal Src-1/-2 double-knockout mice and find that expression of arginase 1 (Arg1) is significantly decreased, accompanied by increased levels of the Arg1 substrate L-arginine. Knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. Moreover, treatment of human myometrial smooth muscle cells with L-arginine significantly inhibits spontaneous contractions by attenuating activation of NF-κB and downregulating expression of contraction-associated protein genes. Transcription factors GR and C/EBPβ increase transcription of Arg1 in an Src-1/Src-2-dependent manner. These findings provide new evidence that fetus-derived factors may play dual roles in coordinating fetal lung development and the initiation of labor.

53BP1 is a chromatin-binding protein that promotes DNA double-strand break repair through the recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity is largely unknown. Here, we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination and PARP inhibitor sensitivity. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.

A severe outbreak of bacterial blight in East Africa has scientists puzzled about the origin of the pathogen and how to deal with it.

Navigating the intricate interplay of competitive and co-operative interactions and the complex relationship between virulence and transmission pose challenges for our understanding of how pathogens evolve and spread.

Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. Here, we generate null alleles of TRMT9B and find it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, we find that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

Hematopoietic stem and progenitor cells (HSPCs) are cells mainly present in the bone marrow and capable of forming mature blood cells. However, the epigenetic mechanisms governing the homeostasis of HSPCs remain elusive. Here, we demonstrate an important role for histone deacetylase 6 (HDAC6) in regulating this process. Our data show that the percentage of HSPCs in Hdac6 knockout mice is lower than in wild-type mice due to decreased HSPC proliferation. HDAC6 interacts with isocitrate dehydrogenase 1 (IDH1) and deacetylates IDH1 at lysine 233. The deacetylation of IDH1 inhibits its catalytic activity and thereby decreases the 5-hydroxymethylcytosine level of ten-eleven translocation 2 (TET2) target genes, changing gene expression patterns to promote the proliferation of HSPCs. These findings uncover a role for HDAC6 and IDH1 in regulating the homeostasis of HSPCs and may have implications for the treatment of hematological diseases.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome plays an important role in the pathogenesis of a wide variety of human diseases. So far, drugs directly and specifically targeting the NLRP3 inflammasome are not available for clinical use since the safety and efficacy of new compounds are often unclear. A promising approach is thus to identify NLRP3 inhibitors from existing drugs that are already in clinical use. Here, we show that mefloquine, a well-known antimalarial drug, is a highly selective and potent NLRP3 inhibitor by screening a FDA-approved drug library. Mechanistically, mefloquine directly binds to the NLRP3 NACHT and LRR domains to prevent NLRP3 inflammasome activation. More importantly, mefloquine treatment attenuates the symptoms of lipopolysaccharide-induced systemic inflammation and Parkinson's disease-like neural damage in mice. Our findings identify mefloquine as a potential therapeutic agent for NLRP3-driven diseases and migth expand its clinical use considerably.

Owing to advances in genome sequencing, genome stability has become one of the most scrutinized cellular traits across the Tree of Life. Despite its centrality to all things biological, the mutation rate (per nucleotide site per generation) ranges over three orders of magnitude among species and several-fold within individual phylogenetic lineages. Within all major organismal groups, mutation rates scale negatively with the effective population size of a species and with the amount of functional DNA in the genome. This relationship is most parsimoniously explained by the drift-barrier hypothesis, which postulates that natural selection typically operates to reduce mutation rates until further improvement is thwarted by the power of random genetic drift. Despite this constraint, the molecular mechanisms underlying DNA replication fidelity and repair are free to wander, provided the performance of the entire system is maintained at the prevailing level. The evolutionary flexibility of the mutation rate bears on the resolution of several prior conundrums in phylogenetic and population-genetic analysis and raises challenges for future applications in these areas.

How many brilliant scientists are out there, whose ideas never gain traction because the technologies to test them aren't yet available?

Neonatal health is determined by the transfer of maternal antibodies from the mother to the fetus. Besides antibodies, maternal cells cross the placental barrier and seed into fetal organs. Contrary to maternal antibodies, maternal microchimeric cells (MMc) show a high longevity, as they can persist in the offspring until adulthood. Recent evidence highlights that MMc leukocytes promote neonatal immunity against early-life infections in mice and humans. As shown in mice, this promotion of immunity was attributable to an improved fetal immune development. Besides this indirect effect, MMc may be pathogen-specific and thus, directly clear pathogen threats in the offspring postnatally. By using ovalbumin recombinant Listeria monocytogenes (LmOVA), we here provide evidence that OVA-specific T cells are transferred from the mother to the fetus, which is associated with increased activation of T cells and a milder course of postnatal infection in the offspring. Our data highlight that maternally-derived passive immunity of the neonate is not limited to antibodies, as MMc have the potential to transfer immune memory between generations.

Plants coordinately use cell-surface and intracellular immune receptors to perceive pathogens and mount an immune response. Intracellular events of pathogen recognition are largely mediated by immune receptors of the nucleotide binding and leucine rich-repeat (NLR) classes. Upon pathogen perception, NLRs trigger a potent broad-spectrum immune reaction, usually accompanied by a form of programmed cell death termed the hypersensitive response. Some plant NLRs act as multifunctional singleton receptors which combine pathogen detection and immune signaling. However, NLRs can also function in higher order pairs and networks of functionally specialized interconnected receptors. In this article, we cover the basic aspects of plant NLR biology with an emphasis on NLR networks. We highlight some of the recent advances in NLR structure, function, and activation and discuss emerging topics such as modulator NLRs, pathogen suppression of NLRs, and NLR bioengineering. Multi-disciplinary approaches are required to disentangle how these NLR immune receptor pairs and networks function and evolve. Answering these questions holds the potential to deepen our understanding of the plant immune system and unlock a new era of disease resistance breeding.

Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. In this study, we use single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene we have named eggplant (eggpl) is highly expressed in GSCs and downregulated in early daughter cells. RNAi knockdown of eggpl causes germ cell proliferation and differentiation defects. In flies fed a rich yeast diet, the expression of eggpl is significantly lower and knockdown or knockout of eggpl phenocopies a rich diet. In addition, eggpl knockdown suppresses the reduction in germ cell proliferation caused by inhibition of the insulin effector PI3K. These findings suggest that downregulation of eggpl links nutritional status to germ cell proliferation and differentiation. Collectively, this study provides new insights into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.

IL-1 receptor (IL-1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, how these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. During IL-1R signaling, Myddosomes dynamically reorganize into multi-Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF-kB activation. Myddosomes function as scaffolds that assemble an NF-kB signalosome consisting of E3-ubiquitin ligases TRAF6 and LUBAC, K63/M1-linked polyubiquitin chains, phospho-IKK, and phospho-p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10-fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL-1R signaling.

Comment on "Asexuality in Drosophila juvenile males is organizational and independent of juvenile hormone" by Ji et al.

The formation of social memory between individuals of the opposite sex is crucial for expanding mating options or establishing monogamous pair bonding. A specialized neuronal circuit that regulates social memory could enhance an individual's mating opportunities and provide a parallel pathway for computing social behaviors. While the influence of light exposure on various forms of memory, such as fear and object memory, has been studied, its modulation of social recognition memory remains unclear. Here, we demonstrate that acute exposure to light impairs social recognition memory (SRM) in mice. Unlike sound and touch stimuli, light inhibits oxytocin neurons in the supraoptic nucleus (SON) via M1 SON-projecting intrinsically photosensitive retinal ganglion cells (ipRGCs) and GABAergic neurons in the perinuclear zone of the SON (pSON). We further show that optogenetic activation of SON oxytocin neurons using channelrhodopsin is sufficient to enhance SRM performance, even under light conditions. Our findings unveil a dedicated neuronal circuit through which luminance affects SRM, utilizing a non-image-forming visual pathway, distinct from the canonical modulatory role of the oxytocin system.

Sexuality is generally prevented in newborns and arises with organizational rewiring of neural circuitry and optimization of fitness for reproduction competition. Recent studies reported that sex circuitry in Drosophila melanogaster is developed in juvenile males but functionally inhibited by juvenile hormone (JH). Here, we find that the fly sex circuitry, mainly expressing the male-specific fruitless (fruM ) and/or doublesex (dsx), is organizationally undeveloped and functionally inoperative in juvenile males. Artificially activating all fruM neurons induces substantial courtship in solitary adult males but not in juvenile males. Synaptic transmissions between major courtship regulators and all dsx neurons are strong in adult males but either weak or undetectable in juvenile males. We further find that JH does not inhibit male courtship in juvenile males but instead promotes courtship robustness in adult males. Our results indicate that the transition to sexuality from juvenile to adult flies requires organizational rewiring of neural circuitry.

Monoamine transporters retrieve serotonin (SERT), dopamine (DAT), and norepinephrine (NET) from the synaptic cleft. Transporter internalization contributes to the regulation of their surface expression. Clathrin-mediated endocytosis of plasma membrane proteins requires adaptor protein-2 (AP2), which recruits cargo to the nascent clathrin cage. However, the intracellular portions of monoamine transporters are devoid of a conventional AP2-binding site. Here, we identify a MAD2 (mitotic arrest deficient-2) interaction motif in the C-terminus of SERT, which binds the closed conformation of MAD2 and allows for the recruitment of two additional mitotic spindle assembly checkpoint (SAC) proteins, BubR1 and p31comet , and of AP2. We visualize MAD2, BubR1, and p31comet in dorsal raphe neurons, and depletion of MAD2 in primary serotonergic rat neurons decreases SERT endocytosis in the soma. Our findings do not only provide mechanistic insights into transporter internalization but also allow for rationalizing why SAC proteins are present in post-mitotic neurons.

The complex life cycle of the human malaria parasite, Plasmodium falciparum, is driven by specific transcriptional programs, but it is unclear how most genes are activated or silenced at specific times. There is an association between transcription and spatial organization; however, the molecular mechanisms behind genome organization are unclear. While P. falciparum lacks key genome-organizing proteins found in metazoans, it has all core components of the cohesin complex. To investigate the role of cohesin in P. falciparum, we functionally characterize the cohesin subunit Structural Maintenance of Chromosomes protein 3 (SMC3). SMC3 knockdown during early stages of the intraerythrocytic developmental cycle (IDC) upregulates a subset of genes involved in erythrocyte egress and invasion, which are normally expressed at later stages. ChIP-seq analyses reveal that during the IDC, SMC3 enrichment at the promoter regions of these genes inversely correlates with gene expression and chromatin accessibility. These data suggest that SMC3 binding contributes to the repression of specific genes until their appropriate time of expression, revealing a new mode of stage-specific gene repression in P. falciparum.

Mitochondrial replacement technology (MRT) aims to reduce the risk of serious disease in children born to women who carry pathogenic mitochondrial DNA (mtDNA) variants. By transplanting nuclear genomes from eggs of an affected woman to enucleated eggs from an unaffected donor, MRT creates new combinations of nuclear and mtDNA. Based on sets of shared sequence variants, mtDNA is classified into ~30 haplogroups. Haplogroup matching between egg donors and women undergoing MRT has been proposed as a means of reducing mtDNA sequence divergence between them. Here we investigate the potential effect of mtDNA haplogroup matching on clinical delivery of MRT and on mtDNA sequence divergence between donor/recipient pairs. Our findings indicate that haplogroup matching would limit the availability of egg donors such that women belonging to rare haplogroups may have to wait > 4 years for treatment. Moreover, we find that intra-haplogroup sequence variation is frequently within the range observed between randomly matched mtDNA pairs. We conclude that haplogroup matching would restrict the availability of MRT, without necessarily reducing mtDNA sequence divergence between donor/recipient pairs.

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.

Accumulation of excess nutrients hampers proper liver function and is linked to nonalcoholic fatty liver disease (NAFLD) in obesity. However, the signals responsible for an impaired adaptation of hepatocytes to obesogenic dietary cues remain still largely unknown. Post-translational modification by the small ubiquitin-like modifier (SUMO) allows for a dynamic regulation of numerous processes including transcriptional reprogramming. We demonstrate that specific SUMOylation of transcription factor Prox1 represents a nutrient-sensitive determinant of hepatic fasting metabolism. Prox1 is highly SUMOylated on lysine 556 in the liver of ad libitum and refed mice, while this modification is abolished upon fasting. In the context of diet-induced obesity, Prox1 SUMOylation becomes less sensitive to fasting cues. The hepatocyte-selective knock-in of a SUMOylation-deficient Prox1 mutant into mice fed a high-fat/high-fructose diet leads to a reduction of systemic cholesterol levels, associated with the induction of liver bile acid detoxifying pathways during fasting. The generation of tools to maintain the nutrient-sensitive SUMO-switch on Prox1 may thus contribute to the development of "fasting-based" approaches for the preservation of metabolic health.