bims-ginsta Biomed News
on Genome instability
Issue of 2024‒01‒07
28 papers selected by
Jinrong Hu, National University of Singapore

  1. Genome Biol. 2024 Jan 02. 25(1): 2
      BACKGROUND: Formation of tissue-specific transcriptional programs underlies multicellular development, including dorsoventral (DV) patterning of the Drosophila embryo. This involves interactions between transcriptional enhancers and promoters in a chromatin context, but how the chromatin landscape influences transcription is not fully understood.RESULTS: Here we comprehensively resolve differential transcriptional and chromatin states during Drosophila DV patterning. We find that RNA Polymerase II pausing is established at DV promoters prior to zygotic genome activation (ZGA), that pausing persists irrespective of cell fate, but that release into productive elongation is tightly regulated and accompanied by tissue-specific P-TEFb recruitment. DV enhancers acquire distinct tissue-specific chromatin states through CBP-mediated histone acetylation that predict the transcriptional output of target genes, whereas promoter states are more tissue-invariant. Transcriptome-wide inference of burst kinetics in different cell types revealed that while DV genes are generally characterized by a high burst size, either burst size or frequency can differ between tissues.
    CONCLUSIONS: The data suggest that pausing is established by pioneer transcription factors prior to ZGA and that release from pausing is imparted by enhancer chromatin state to regulate bursting in a tissue-specific manner in the early embryo. Our results uncover how developmental patterning is orchestrated by tissue-specific bursts of transcription from Pol II primed promoters in response to enhancer regulatory cues.
  2. Dev Cell. 2023 Dec 23. pii: S1534-5807(23)00651-2. [Epub ahead of print]
      Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.
    Keywords:  C. elegans; MAP9; axoneme; cilia; dynein; kinesin; microtubule; microtubule doublet; microtubule-associated protein; polyglutamylation
  3. PLoS Biol. 2024 Jan;22(1): e3002453
      To achieve a stable size distribution over multiple generations, proliferating cells require a means of counteracting stochastic noise in the rate of growth, the time spent in various phases of the cell cycle, and the imprecision in the placement of the plane of cell division. In the most widely accepted model, cell size is thought to be regulated at the G1/S transition, such that cells smaller than a critical size pause at the end of G1 phase until they have accumulated mass to a predetermined size threshold, at which point the cells proceed through the rest of the cell cycle. However, a model, based solely on a specific size checkpoint at G1/S, cannot readily explain why cells with deficient G1/S control mechanisms are still able to maintain a very stable cell size distribution. Furthermore, such a model would not easily account for stochastic variation in cell size during the subsequent phases of the cell cycle, which cannot be anticipated at G1/S. To address such questions, we applied computationally enhanced quantitative phase microscopy (ceQPM) to populations of cultured human cell lines, which enables highly accurate measurement of cell dry mass of individual cells throughout the cell cycle. From these measurements, we have evaluated the factors that contribute to maintaining cell mass homeostasis at any point in the cell cycle. Our findings reveal that cell mass homeostasis is accurately maintained, despite disruptions to the normal G1/S machinery or perturbations in the rate of cell growth. Control of cell mass is generally not confined to regulation of the G1 length. Instead mass homeostasis is imposed throughout the cell cycle. In the cell lines examined, we find that the coefficient of variation (CV) in dry mass of cells in the population begins to decline well before the G1/S transition and continues to decline throughout S and G2 phases. Among the different cell types tested, the detailed response of cell growth rate to cell mass differs. However, in general, when it falls below that for exponential growth, the natural increase in the CV of cell mass is effectively constrained. We find that both mass-dependent cell cycle regulation and mass-dependent growth rate modulation contribute to reducing cell mass variation within the population. Through the interplay and coordination of these 2 processes, accurate cell mass homeostasis emerges. Such findings reveal previously unappreciated and very general principles of cell size control in proliferating cells. These same regulatory processes might also be operative in terminally differentiated cells. Further quantitative dynamical studies should lead to a better understanding of the underlying molecular mechanisms of cell size control.
  4. J Cell Biol. 2024 Mar 04. pii: e202211122. [Epub ahead of print]223(3):
      Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation by a poorly understood pathway. Here, we identify a linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ contribute to the linkage. Furthermore, a minimal segment of Spc105 with a propensity to multimerize and which contains protein binding motifs is sufficient to link Spc105 to RZZ/dynein. Deletion of the minimal region from Spc105 compromises the recruitment of its binding partners to kinetochores and elevates chromosome missegregation due to merotelic attachments. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 contributes to localizing a core pool of RZZ that promotes accurate chromosome segregation.
  5. J Cell Biol. 2024 Mar 04. pii: e202211048. [Epub ahead of print]223(3):
      Lateral diffusion barriers compartmentalize membranes to generate polarity or asymmetrically partition membrane-associated macromolecules. Budding yeasts assemble such barriers in the endoplasmic reticulum (ER) and the outer nuclear envelope at the bud neck to retain aging factors in the mother cell and generate naïve and rejuvenated daughter cells. However, little is known about whether other organelles are similarly compartmentalized. Here, we show that the membranes of mitochondria are laterally compartmentalized at the bud neck and near the cell poles. The barriers in the inner mitochondrial membrane are constitutive, whereas those in the outer membrane form in response to stresses. The strength of mitochondrial diffusion barriers is regulated positively by spatial cues from the septin axis and negatively by retrograde (RTG) signaling. These data indicate that mitochondria are compartmentalized in a fission-independent manner. We propose that these diffusion barriers promote mitochondrial polarity and contribute to mitochondrial quality control.
  6. Nat Commun. 2024 Jan 05. 15(1): 315
      The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.
  7. Nat Struct Mol Biol. 2024 Jan 04.
      As embryonic stem cells (ESCs) transition from naive to primed pluripotency during early mammalian development, they acquire high DNA methylation levels. During this transition, the germline is specified and undergoes genome-wide DNA demethylation, while emergence of the three somatic germ layers is preceded by acquisition of somatic DNA methylation levels in the primed epiblast. DNA methylation is essential for embryogenesis, but the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Here, using culture modeling of cellular transitions, we found that DNA methylation-free mouse ESCs with triple DNA methyltransferase knockout (TKO) progressed through the continuum of pluripotency states but demonstrated skewed differentiation abilities toward neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells, even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that neural and germline lineages are linked by a similar enhancer dynamic upon exit from the naive state, defined by common sets of transcription factors, including methyl-sensitive ones, that fail to be decommissioned in the absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of the preferred differentiation route during early development.
  8. Nat Cell Biol. 2024 Jan 02.
      To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.
  9. Nat Aging. 2024 Jan 04.
      The decline in stem cell function during aging may affect the regenerative capacity of mammalian organisms; however, the gene regulatory mechanism underlying this decline remains unclear. Here we show that the aging of neural stem and progenitor cells (NSPCs) in the male mouse brain is characterized by a decrease in the generation efficacy of proliferative NSPCs rather than the changes in lineage specificity of NSPCs. We reveal that the downregulation of age-dependent genes in NSPCs drives cell aging by decreasing the population of actively proliferating NSPCs while increasing the expression of quiescence markers. We found that epigenetic deregulation of the MLL complex at promoters leads to transcriptional inactivation of age-dependent genes, highlighting the importance of the dynamic interaction between histone modifiers and gene regulatory elements in regulating transcriptional program of aging cells. Our study sheds light on the key intrinsic mechanisms driving stem cell aging through epigenetic regulators and identifies potential rejuvenation targets that could restore the function of aging stem cells.
  10. Nat Commun. 2024 Jan 02. 15(1): 73
      Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely unclear. We previously identified a chloroplast-localized RNase H1 protein AtRNH1C that can remove R-loops and relax HO-TRCs for genome integrity. Through the mutagenesis screen, we identify a mutation in chloroplast-localized primase ATH that weakens the binding affinity of DNA template and reduces the activities of RNA primer synthesis and delivery. This slows down DNA replication, and reduces competition of transcription-replication, thus rescuing the developmental defects of atrnh1c. Strand-specific DNA damage sequencing reveals that HO-TRCs cause DNA damage at the end of the transcription unit in the lagging strand and overexpression of ATH can boost HO-TRCs and exacerbates DNA damage. Furthermore, mutation of plastid DNA polymerase Pol1A can similarly rescue the defects in atrnh1c mutants. Taken together these results illustrate a potentially conserved mechanism among organisms, of which the primase activity can promote the occurrence of transcription-replication conflicts leading to HO-TRCs and genome instability.
  11. Nat Commun. 2024 Jan 02. 15(1): 90
      Embryonic cells exhibit diverse metabolic states. Recent studies have demonstrated that metabolic reprogramming drives changes in cell identity by affecting gene expression. However, the connection between cellular metabolism and gene expression remains poorly understood. Here we report that glycolysis-regulated histone lactylation couples the metabolic state of embryonic cells with chromatin organization and gene regulatory network (GRN) activation. We found that lactylation marks genomic regions of glycolytic embryonic tissues, like the neural crest (NC) and pre-somitic mesoderm. Histone lactylation occurs in the loci of NC genes as these cells upregulate glycolysis. This process promotes the accessibility of active enhancers and the deployment of the NC GRN. Reducing the deposition of the mark by targeting LDHA/B leads to the downregulation of NC genes and the impairment of cell migration. The deposition of lactyl-CoA on histones at NC enhancers is supported by a mechanism that involves transcription factors SOX9 and YAP/TEAD. These findings define an epigenetic mechanism that integrates cellular metabolism with the GRNs that orchestrate embryonic development.
  12. Immunity. 2023 Dec 22. pii: S1074-7613(23)00532-0. [Epub ahead of print]
      The dominant risk factors for late-onset Alzheimer's disease (AD) are advanced age and the APOE4 genetic variant. To examine how these factors alter neuroimmune function, we generated an integrative, longitudinal single-cell atlas of brain immune cells in AD model mice bearing the three common human APOE alleles. Transcriptomic and chromatin accessibility analyses identified a reactive microglial population defined by the concomitant expression of inflammatory signals and cell-intrinsic stress markers whose frequency increased with age and APOE4 burden. An analogous population was detectable in the brains of human AD patients, including in the cortical tissue, using multiplexed spatial transcriptomics. This population, which we designate as terminally inflammatory microglia (TIM), exhibited defects in amyloid-β clearance and altered cell-cell communication during aducanumab treatment. TIM may represent an exhausted-like state for inflammatory microglia in the AD milieu that contributes to AD risk and pathology in APOE4 carriers and the elderly, thus presenting a potential therapeutic target for AD.
    Keywords:  APOE; Alzheimer's disease; TIM; microglia; single cell
  13. Nat Metab. 2024 Jan 03.
      Mammalian preimplantation development is associated with marked metabolic robustness, and embryos can develop under a wide variety of nutrient conditions, including even the complete absence of soluble amino acids. Here we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of preimplantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis, alongside a robust lysosomal digestive system. Following transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant on exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the preimplantation epiblast. More broadly, we find that all lineages of preimplantation blastocysts exhibit constitutive macropinocytic protein uptake and digestion. Taken together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of preimplantation development and provide insight into the catabolic strategies that enable embryos to sustain viability before implantation.
  14. Nat Commun. 2024 Jan 02. 15(1): 167
      Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.
  15. Endocrinology. 2024 Jan 05. pii: bqad200. [Epub ahead of print]
      Signaling in the granulosa cells of mammalian ovarian follicles is necessary for maintaining prophase arrest in the oocyte and for mediating the resumption of meiosis in response to luteinizing hormone (LH). However, the follicle also includes an outer layer of theca cells, some of which express receptors for LH. To investigate whether the theca cells are required for maintaining meiotic arrest and reinitiating meiosis in response to LH, we mechanically separated the granulosa cells and oocyte from the theca and basal lamina. This was accomplished by cutting a slit in the outer surface of isolated follicles such that the mural granulosa cells and cumulus-oocyte complex were extruded from the theca shell, forming a lawn of cells on an organotypic membrane. The remnant of theca cells and basal lamina was then removed. The separation of the granulosa cells from the theca cells and basal lamina was demonstrated by immunofluorescence localization of endomucin (blood vessels of the theca) and laminin gamma (basal lamina). Cells comprising these granulosa cell-oocyte complexes expressed LH receptors and were connected by gap junctions. Oocytes within these granulosa cell complexes maintained meiotic arrest, and resumed meiosis in response to LH, showing that the granulosa cells alone, without theca cells, transduce these signals. This semi-intact and mostly 2-dimensional preparation could facilitate imaging studies of follicle physiology.
    Keywords:  Ovarian follicle; granulosa cells; luteinizing hormone; meiotic resumption; theca
  16. Cell. 2024 Jan 04. pii: S0092-8674(23)01319-3. [Epub ahead of print]187(1): 110-129.e31
      X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.
    Keywords:  X chromosome dampening; X chromosome inactivation; XIST; dispersed localization; dosage compensation; human pluripotent stem cells; lncRNA; polycomb-repressive complexes; sexual dimorphism; trans-acting
  17. Nat Commun. 2024 Jan 02. 15(1): 93
      Lysosomes have emerged as critical regulators of cellular homeostasis. Here we show that the lysosomal protein TMEM55B contributes to restore cellular homeostasis in response to oxidative stress by three different mechanisms: (1) TMEM55B mediates NEDD4-dependent PLEKHM1 ubiquitination, causing PLEKHM1 proteasomal degradation and halting autophagosome/lysosome fusion; (2) TMEM55B promotes recruitment of components of the ESCRT machinery to lysosomal membranes to stimulate lysosomal repair; and (3) TMEM55B sequesters the FLCN/FNIP complex to facilitate translocation of the transcription factor TFE3 to the nucleus, allowing expression of transcriptional programs that enable cellular adaptation to stress. Knockout of tmem55 genes in zebrafish embryos increases their susceptibility to oxidative stress, causing early death of tmem55-KO animals in response to arsenite toxicity. Altogether, our work identifies a role for TMEM55B as a molecular sensor that coordinates autophagosome degradation, lysosomal repair, and activation of stress responses.
  18. Cell Metab. 2024 Jan 02. pii: S1550-4131(23)00446-1. [Epub ahead of print]36(1): 21-35
      Mitochondria are central hubs of cellular metabolism and are tightly connected to signaling pathways. The dynamic plasticity of mitochondria to fuse, divide, and contact other organelles to flux metabolites is central to their function. To ensure bona fide functionality and signaling interconnectivity, diverse molecular mechanisms evolved. An ancient and long-overlooked mechanism is the generation of mitochondrial-derived vesicles (MDVs) that shuttle selected mitochondrial cargoes to target organelles. Just recently, we gained significant insight into the mechanisms and functions of MDV transport, ranging from their role in mitochondrial quality control to immune signaling, thus demonstrating unexpected and diverse physiological aspects of MDV transport. This review highlights the origin of MDVs, their biogenesis, and their cargo selection, with a specific focus on the contribution of MDV transport to signaling across cell and organ barriers. Additionally, the implications of MDVs in peroxisome biogenesis, neurodegeneration, metabolism, aging, and cancer are discussed.
  19. Nat Cancer. 2024 Jan 02.
      Availability of the essential amino acid methionine affects cellular metabolism and growth, and dietary methionine restriction has been implicated as a cancer therapeutic strategy. Nevertheless, how liver cancer cells respond to methionine deprivation and underlying mechanisms remain unclear. Here we find that human liver cancer cells undergo irreversible cell cycle arrest upon methionine deprivation in vitro. Blocking methionine adenosyl transferase 2A (MAT2A)-dependent methionine catabolism induces cell cycle arrest and DNA damage in liver cancer cells, resulting in cellular senescence. A pharmacological screen further identified GSK3 inhibitors as senolytics that selectively kill MAT2A-inhibited senescent liver cancer cells. Importantly, combined treatment with MAT2A and GSK3 inhibitors therapeutically blunts liver tumor growth in vitro and in vivo across multiple models. Together, methionine catabolism is essential for liver tumor growth, and its inhibition can be exploited as an improved pro-senescence strategy for combination with senolytic agents to treat liver cancer.
  20. Cell Rep. 2023 Dec 28. pii: S2211-1247(23)01634-0. [Epub ahead of print]43(1): 113622
      While ATM loss of function has long been identified as the genetic cause of ataxia-telangiectasia (A-T), how it leads to selective and progressive degeneration of cerebellar Purkinje and granule neurons remains unclear. ATM expression is enriched in microglia throughout cerebellar development and adulthood. Here, we find evidence of microglial inflammation in the cerebellum of patients with A-T using single-nucleus RNA sequencing. Pseudotime analysis revealed that activation of A-T microglia preceded upregulation of apoptosis-related genes in granule and Purkinje neurons and that microglia exhibited increased neurotoxic cytokine signaling to granule and Purkinje neurons in A-T. To confirm these findings experimentally, we performed transcriptomic profiling of A-T induced pluripotent stem cell (iPSC)-derived microglia, which revealed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, A-T microglia co-culture with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may promote neurodegeneration in A-T.
    Keywords:  CP: Neuroscience; ataxia-telangiectasia; cerebellar degeneration; microglial activation; single-nucleus RNA-sequencing
  21. Cell Rep Methods. 2023 Dec 27. pii: S2667-2375(23)00353-3. [Epub ahead of print] 100674
      Transcription by RNA polymerase II (RNA Pol II) is crucial for cellular function, but DNA damage severely impedes this process. Thus far, transcription-blocking DNA lesions (TBLs) and their repair have been difficult to quantify in living cells. To overcome this, we generated, using CRISPR-Cas9-mediated gene editing, mScarletI-tagged Cockayne syndrome group B protein (CSB) and UV-stimulated scaffold protein A (UVSSA) knockin cells. These cells allowed us to study the binding dynamics of CSB and UVSSA to lesion-stalled RNA Pol II using fluorescence recovery after photobleaching (FRAP). We show that especially CSB mobility is a sensitive transcription stress marker at physiologically relevant DNA damage levels. Transcription-coupled nucleotide excision repair (TC-NER)-mediated repair can be assessed by studying CSB immobilization over time. Additionally, flow cytometry reveals the regulation of CSB protein levels by CRL4CSA-mediated ubiquitylation and deubiquitylation by USP7. This approach allows the sensitive detection of TBLs and their repair and the study of TC-NER complex assembly and stability in living cells.
    Keywords:  CP: Imaging; CP: Molecular biology; CSB; DNA damage; FRAP; TC-NER; UVSSA; nucleotide excision repair; transcription-blocking DNA damage; transcription-coupled repair
  22. J Clin Invest. 2024 Jan 04. pii: e174483. [Epub ahead of print]
      Aneuploidy, a deviation from the normal chromosome copy number, is common in human embryos and is considered a primary cause of implantation failure and early pregnancy loss. Meiotic errors lead to uniformly abnormal karyotypes, while mitotic errors lead to chromosomal mosaicism: the presence of cells with at least two different karyotypes within an embryo. Knowledge about mosaicism in blastocysts mainly derives from bulk DNA sequencing of multicellular trophectoderm (TE) and/or inner cell mass (ICM) samples. However, this can only detect an average net gain or loss of DNA above a detection threshold of 20-30%. To accurately assess mosaicism, we separated the TE and ICM of 55 good quality surplus blastocysts and successfully applied single-cell whole genome sequencing (scKaryo-seq) on 1057 cells. Mosaicism involving numerical and structural chromosome abnormalities was detected in 82% of the embryos, where most abnormalities affected less than 20% of the cells. Structural abnormalities, potentially caused by replication stress and DNA damage, were observed in 69% of the embryos. In conclusion, our findings indicated that mosaicism is prevalent in good-quality blastocysts, while these blastocysts would likely be identified as normal with current bulk DNA sequencing techniques used for preimplantation genetic testing for aneuploidy (PGT-A).
    Keywords:  Embryonic development; Fertility; Genetic instability; Genetics; Reproductive Biology
  23. Cell Stem Cell. 2024 Jan 04. pii: S1934-5909(23)00432-0. [Epub ahead of print]31(1): 106-126.e13
      Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression, but tissue-specific factors linking these associations remain ill-defined. We observed that stiff breast tissue from women with high mammographic density, who exhibit increased lifetime risk for breast cancer, associates with abundant stem-progenitor epithelial cells. Using genetically engineered mouse models of elevated integrin mechanosignaling and collagen density, syngeneic manipulations, and spheroid models, we determined that a stiff matrix and high mechanosignaling increase mammary epithelial stem-progenitor cell frequency and enhance tumor initiation in vivo. Augmented tissue mechanics expand stemness by potentiating extracellular signal-related kinase (ERK) activity to foster progesterone receptor-dependent RANK signaling. Consistently, we detected elevated phosphorylated ERK and progesterone receptors and increased levels of RANK signaling in stiff breast tissue from women with high mammographic density. The findings link fibrosis and mechanosignaling to stem-progenitor cell frequency and breast cancer risk and causally implicate epidermal growth factor receptor-ERK-dependent hormone signaling in this phenotype.
    Keywords:  RANK; RANKL; breast cancer risk; extracellular matrix stiffness; integrin signaling; mammary progenitor cells; mammographic density; mechanobiology
  24. Nat Struct Mol Biol. 2024 Jan 04.
      Pioneer transcription factors direct cell differentiation by deploying new enhancer repertoires through their unique ability to target and initiate remodelling of closed chromatin. The initial steps of their action remain undefined, although pioneers have been shown to interact with nucleosomal target DNA and with some chromatin-remodeling complexes. We now define the sequence of events that enables the pioneer Pax7 with its unique abilities. Chromatin condensation exerted by linker histone H1 is the first constraint on Pax7 recruitment, and this establishes the initial speed of chromatin remodeling. The first step of pioneer action involves recruitment of the KDM1A (LSD1) H3K9me2 demethylase for removal of this repressive mark, as well as recruitment of the MLL complex for deposition of the activating H3K4me1 mark. Further progression of pioneer action requires passage through cell division, and this involves dissociation of pioneer targets from perinuclear lamin B. Only then are the SWI-SNF remodeling complex and the coactivator p300 recruited, leading to nucleosome displacement and enhancer activation. Thus, the unique features of pioneer actions are those occurring in the lamin-associated compartment of the nucleus. This model is consistent with previous work that showed a dependence on cell division for establishment of new cell fates.
  25. Dev Cell. 2023 Dec 23. pii: S1534-5807(23)00652-4. [Epub ahead of print]
      The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.
    Keywords:  NOTCH signaling; PDE2A; cGMP; cell contact inhibition; claudin 5; lymphangiogenesis; lymphatic endothelial cells; p38; phosphodiesterase 2A; proliferation; vessel maturation
  26. Cell Rep. 2023 Dec 30. pii: S2211-1247(23)01640-6. [Epub ahead of print]43(1): 113629
      The interplay between metabolism and chromatin signaling is implicated in cancer progression. However, whether and how metabolic reprogramming in tumors generates chromatin vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway, which drives aggressive and chemo-resistant disease. Using a chromatin-focused CRISPR screen, we report that NRF2 activation sensitizes LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDACs). This association is observed across cultured cells, mouse models, and patient-derived xenografts. Integrative epigenomic, transcriptomic, and metabolomic analysis demonstrates that HDAC inhibition causes widespread redistribution of H4ac and its reader protein, which transcriptionally downregulates metabolic enzymes. This results in reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest NRF2 activation as a potential biomarker for effective repurposing of HDAC inhibitors to treat solid tumors.
    Keywords:  CP: Cancer; HDAC inhibitors; NRF2 pathway; cancer epigenetics; cancer metabolism; cancer targeted therapy; epigenetic reprogramming; lung cancer
  27. bioRxiv. 2023 Dec 05. pii: 2023.12.04.569944. [Epub ahead of print]
      Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that: a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane; and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.
  28. Sci Immunol. 2024 Jan 05. 9(91): eadh0152
      Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (Tregs) only in skin. Selective depletion of skin Tregs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin Tregs, functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin Tregs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of Tregs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.