Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption.

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. VIDEO ABSTRACT.

Tolerance thresholds underlie responses to DNA damage during germline development.

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.

CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis.

Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4Cdt2 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Here, using quantitative microscopy and in-vitro-reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.

m6A-driven SF3B1 translation control steers splicing to direct genome integrity and leukemogenesis.

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

Hallmarks of DNA replication stress.

Faithful DNA replication is critical for the maintenance of genomic integrity. Although DNA replication machinery is highly accurate, the process of DNA replication is constantly challenged by DNA damage and other intrinsic and extrinsic stresses throughout the genome. A variety of cellular stresses interfering with DNA replication, which are collectively termed replication stress, pose a threat to genomic stability in both normal and cancer cells. To cope with replication stress and maintain genomic stability, cells have evolved a complex network of cellular responses to alleviate and tolerate replication problems. This review will focus on the major sources of replication stress, the impacts of replication stress in cells, and the assays to detect replication stress, offering an overview of the hallmarks of DNA replication stress.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

Metabolite-Sensing Receptor Ffar2 Regulates Colonic Group 3 Innate Lymphoid Cells and Gut Immunity.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals that are critical for gut homeostasis and host defense. However, the metabolite-sensing G-protein-coupled receptors that regulate colonic ILC3s remain poorly understood. We found that colonic ILC3s expressed Ffar2, a microbial metabolite-sensing receptor, and that Ffar2 agonism promoted ILC3 expansion and function. Deficiency of Ffar2 in ILC3s decreased their in situ proliferation and ILC3-derived interleukin-22 (IL-22) production. This led to impaired gut epithelial function characterized by altered mucus-associated proteins and antimicrobial peptides and increased susceptibility to colonic injury and bacterial infection. Ffar2 increased IL-22+ CCR6+ ILC3s and influenced ILC3 abundance in colonic lymphoid tissues. Ffar2 agonism differentially activated AKT or ERK signaling and increased ILC3-derived IL-22 via an AKT and STAT3 axis. Our findings suggest that Ffar2 regulates colonic ILC3 proliferation and function, and they identify an ILC3-receptor signaling pathway modulating gut homeostasis and pathogen defense.

Nuclear PARPs and genome integrity.

Effective maintenance and stability of our genomes is essential for normal cell division, tissue homeostasis, and cellular and organismal fitness. The processes of chromosome replication and segregation require continual surveillance to insure fidelity. Accurate and efficient repair of DNA damage preserves genome integrity, which if lost can lead to multiple diseases, including cancer. Poly(ADP-ribose) a dynamic and reversible posttranslational modification and the enzymes that catalyze it (PARP1, PARP2, tankyrase 1, and tankyrase 2) function to maintain genome stability through diverse mechanisms. Here we review the role of these enzymes and the modification in genome repair, replication, and resolution in human cells.

Making sense of proprioception.

Proprioception - the sense of body position in space - is intimately linked to motor control. Here, we briefly review the current knowledge of the proprioceptive system and how advances in the genetic characterisation of proprioceptive sensory neurons in mice promise to dissect its role in health and disease.

Human Enteric α-Defensin 5 Promotes Shigella Infection by Enhancing Bacterial Adhesion and Invasion.

Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection in vitro in a structure-dependent manner. Shigella, commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium through interactions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella-induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen.

Mouse ANKRD31 Regulates Spatiotemporal Patterning of Meiotic Recombination Initiation and Ensures Recombination between X and Y Sex Chromosomes.

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.

Screening p-hackers: Dissemination noise as bait.

We show that adding noise before publishing data effectively screens [Formula: see text]-hacked findings: spurious explanations produced by fitting many statistical models (data mining). Noise creates "baits" that affect two types of researchers differently. Uninformed [Formula: see text]-hackers, who are fully ignorant of the true mechanism and engage in data mining, often fall for baits. Informed researchers, who start with an ex ante hypothesis, are minimally affected. We show that as the number of observations grows large, dissemination noise asymptotically achieves optimal screening. In a tractable special case where the informed researchers' theory can identify the true causal mechanism with very few data, we characterize the optimal level of dissemination noise and highlight the relevant trade-offs. Dissemination noise is a tool that statistical agencies currently use to protect privacy. We argue this existing practice can be repurposed to screen [Formula: see text]-hackers and thus improve research credibility.

Blood endothelium transition and phenotypic plasticity: A key regulator of integrity/permeability in response to ischemia.

In the human body, the 1013 blood endothelial cells (ECs) which cover a surface of 500-700 m2 (Mai et al., 2013) are key players of tissue homeostasis, remodeling and regeneration. Blood vessel ECs play a major role in the regulation of metabolic and gaz exchanges, cell trafficking, blood coagulation, vascular tone, blood flow and fluid extravasation (also referred to as blood vascular permeability). ECs are heterogeneous in various capillary beds and have the exquisite capacity to cope with environmental changes by regulating their gene expression. Ischemia has major detrimental effects on the endothelium and ischemia-induced regulation of vascular integrity is of paramount importance for human health, as small amounts of fluid accumulation in the interstitium may be responsible for major effects on organ functions and patients outcome. In this review, we will here focus on the stimuli and the molecular mechanisms that control blood endothelium maintenance and phenotypic plasticity/transition involved in controlling blood capillary leakage that might open new avenues for therapeutic applications.

Cell line authentication: a necessity for reproducible biomedical research.

Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific reproducibility. Based on the experiences of mandatory cell line authentication at the International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi-layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.

Critical importance of DNA binding for CSL protein functions in fission yeast.

CSL proteins [named after the homologs CBF1 (RBP-Jκ in mice), Suppressor of Hairless and LAG-1] are conserved transcription factors found in animals and fungi. In the fission yeast Schizosaccharomyces pombe, they regulate various cellular processes, including cell cycle progression, lipid metabolism and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central ß-trefoil domain. Here, we investigated the importance of DNA binding for CSL protein functions in fission yeast. We created CSL protein mutants with disrupted DNA binding and found that the vast majority of CSL protein functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse phenotypes associated with CSL protein functions. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast.

Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase and the PCM.

Centrioles are part of centrosomes and cilia, which are microtubule organising centres (MTOC) with diverse functions. Despite their stability, centrioles can disappear during differentiation, such as in oocytes, but little is known about the regulation of their structural integrity. Our previous research revealed that the pericentriolar material (PCM) that surrounds centrioles and its recruiter, Polo kinase, are downregulated in oogenesis and sufficient for maintaining both centrosome structural integrity and MTOC activity. We now show that the expression of specific components of the centriole cartwheel and wall, including ANA1/CEP295, is essential for maintaining centrosome integrity. We find that Polo kinase requires ANA1 to promote centriole stability in cultured cells and eggs. In addition, ANA1 expression prevents the loss of centrioles observed upon PCM-downregulation. However, the centrioles maintained by overexpressing and tethering ANA1 are inactive, unlike the MTOCs observed upon tethering Polo kinase. These findings demonstrate that several centriole components are needed to maintain centrosome structure. Our study also highlights that centrioles are more dynamic than previously believed, with their structural stability relying on the continuous expression of multiple components.

PHF2-mediated H3K9me balance orchestrates heterochromatin stability and neural progenitor proliferation.

Heterochromatin stability is crucial for progenitor proliferation during early neurogenesis. It relays on the maintenance of local hubs of H3K9me. However, understanding the formation of efficient localized levels of H3K9me remains limited. To address this question, we used neural stem cells to analyze the function of the H3K9me2 demethylase PHF2, which is crucial for progenitor proliferation. Through mass-spectroscopy and genome-wide assays, we show that PHF2 interacts with heterochromatin components and is enriched at pericentromeric heterochromatin (PcH) boundaries where it maintains transcriptional activity. This binding is essential for silencing the satellite repeats, preventing DNA damage and genome instability. PHF2's depletion increases the transcription of heterochromatic repeats, accompanied by a decrease in H3K9me3 levels and alterations in PcH organization. We further show that PHF2's PHD and catalytic domains are crucial for maintaining PcH stability, thereby safeguarding genome integrity. These results highlight the multifaceted nature of PHF2's functions in maintaining heterochromatin stability and regulating gene expression during neural development. Our study unravels the intricate relationship between heterochromatin stability and progenitor proliferation during mammalian neurogenesis.

A Scientist's Oath.

Data on the perceptions of scientists suggest a moderate public distrust of scientist's motivations. Bettridge et al. suggest scientist's reluctance to engage the public on controversial ethical issues may be a contributing factor. The authors propose a Scientist's Oath to send a clear message to the public about our ideals.

Functions of FACT in Breaking the Nucleosome and Maintaining Its Integrity at the Single-Nucleosome Level.

The human FACT (facilitates chromatin transcription) complex, composed of two subunits SPT16 (Suppressor of Ty 16) and SSRP1 (Structure-specific recognition protein-1), plays essential roles in nucleosome remodeling. However, the molecular mechanism of FACT reorganizing the nucleosome still remains elusive. In this study, we demonstrate that FACT displays dual functions in destabilizing the nucleosome and maintaining the original histones and nucleosome integrity at the single-nucleosome level. We found that the subunit SSRP1 is responsible for maintenance of nucleosome integrity by holding the H3/H4 tetramer on DNA and promoting the deposition of the H2A/H2B dimer onto the nucleosome. In contrast, the large subunit SPT16 destabilizes the nucleosome structure by displacing the H2A/H2B dimers. Our findings provide mechanistic insights by which the two subunits of FACT coordinate with each other to fulfill its functions and suggest that FACT may play essential roles in preserving the original histones with epigenetic identity during transcription or DNA replication.