bims-ginsta 2024-04-07 papers

bims-ginsta

Biomed News

on Genome instability

Issue of 2024–04–07
thirty papers selected by
Jinrong Hu, National University of Singapore

CDK activity at the centrosome regulates the cell cycle.
Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates.
Basal delamination during mouse gastrulation primes pluripotent cells for differentiation.
Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration.
The variation and evolution of complete human centromeres.
p53 rapidly restructures 3D chromatin organization to trigger a transcriptional response.
Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation.
Bioelectric stimulation controls tissue shape and size.
Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation.
Functional genetics reveals modulators of anti-microtubule drug sensitivity.
Immune microniches shape intestinal Treg function.
Aging-related defects in macrophage function are driven by MYC and USF1 transcriptional programs.
The mechanical influence of densification on epithelial architecture.
BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks.
Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.
Cell-cycle and Age-Related Modulations in Mouse Chromosome Stiffness.
Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.
Nuclear actin dynamics and functions at a glance.
Endogenous Nodal switches Wnt interpretation from posteriorization to germ layer differentiation in geometrically constrained human pluripotent cells.
Intrinsically disordered regions (IDRs): A vague and confusing concept for protein function.
Proliferation-driven mechanical compression induces signalling centre formation during mammalian organ development.
Oct4 controls basement membrane development during human embryogenesis.
NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.
The genetics of cardiomyocyte polyploidy.
The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.
Cardiomyocyte DNA Damage Predicts Functional Recovery in Heart Failure Patients.
Changing structures, changing paradigms: NELF helps regulate paused or elongating RNA polymerase II.
A spatiotemporal proteomic map of human adipogenesis.
14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.
The Notch-PDGFRβ axis suppresses brown adipocyte progenitor differentiation in early post-natal mice.

Cell Rep. 2024 Apr 04. pii: S2211-1247(24)00394-2. [Epub ahead of print]43(4): 114066

CDK activity at the centrosome regulates the cell cycle.

Emma L Roberts, Jessica Greenwood, Nitin Kapadia, Tania Auchynnikava, Souradeep Basu, Paul Nurse.

  In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis.

Keywords:  CDK; CP: Cell biology; cell cycle; centrosome; cyclin B; hydrophobic patch; mitosis

DOI:  https://doi.org/10.1016/j.celrep.2024.114066
Cell Rep. 2024 Apr 04. pii: S2211-1247(24)00392-9. [Epub ahead of print]43(4): 114064

Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates.

Tom Egger, Laura Morano, Marie-Pierre Blanchard, Jihane Basbous, Angelos Constantinou.

  Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

Keywords:  ATR/Chk1 signaling pathway; CP: Molecular biology; DNA damage response; DNA replication stress; biomolecular condensates; optogenetics

DOI:  https://doi.org/10.1016/j.celrep.2024.114064
Dev Cell. 2024 Apr 02. pii: S1534-5807(24)00176-X. [Epub ahead of print]

Basal delamination during mouse gastrulation primes pluripotent cells for differentiation.

Nanami Sato, Viviane S Rosa, Aly Makhlouf, Helene Kretzmer, Abhishek Sampath Kumar, Stefanie Grosswendt, Alexandra L Mattei, Olivia Courbot, Steffen Wolf, Jerome Boulanger, Frederic Langevin, Michal Wiacek, Daniel Karpinski, Alberto Elosegui-Artola, Alexander Meissner, Magdalena Zernicka-Goetz, Marta N Shahbazi.

  The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.

Keywords:  3D culture; Wnt signaling; delamination; differentiation; embryonic stem cells; gastrulation; morphogenesis; mouse embryo; pluripotency; proliferation

DOI:  https://doi.org/10.1016/j.devcel.2024.03.008
bioRxiv. 2024 Mar 13. pii: 2024.03.12.584570. [Epub ahead of print]

Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration.

Florian Constanty, Bailin Wu, Ke-Hsuan Wei, I-Ting Lin, Julia Dallmann, Stefan Guenther, Till Lautenschlaeger, Rashmi Priya, Shih-Lei Lai, Didier Y R Stainier, Arica Beisaw.

Despite numerous advances in our understanding of zebrafish cardiac regeneration, an aspect that remains less studied is how regenerating cardiomyocytes invade, and eventually replace, the collagen-containing fibrotic tissue following injury. Here, we provide an in-depth analysis of the process of cardiomyocyte invasion using live-imaging and histological approaches. We observed close interactions between protruding cardiomyocytes and macrophages at the wound border zone, and macrophage-deficient irf8 mutant zebrafish exhibited defects in extracellular matrix (ECM) remodeling and cardiomyocyte protrusion into the injured area. Using a resident macrophage ablation model, we show that defects in ECM remodeling at the border zone and subsequent cardiomyocyte protrusion can be partly attributed to a population of resident macrophages. Single-cell RNA-sequencing analysis of cells at the wound border revealed a population of cardiomyocytes and macrophages with fibroblast-like gene expression signatures, including the expression of genes encoding ECM structural proteins and ECM-remodeling proteins. The expression of mmp14b , which encodes a membrane-anchored matrix metalloproteinase, was restricted to cells in the border zone, including cardiomyocytes, macrophages, fibroblasts, and endocardial/endothelial cells. Genetic deletion of mmp14b led to a decrease in 1) macrophage recruitment to the border zone, 2) collagen degradation at the border zone, and 3) subsequent cardiomyocyte invasion. Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion into the injured tissue and along the apical surface of the wound. Altogether, our data shed important insights into the process of cardiomyocyte invasion of the collagen-containing injured tissue during cardiac regeneration. They further suggest that cardiomyocytes and resident macrophages contribute to ECM remodeling at the border zone to promote cardiomyocyte replenishment of the fibrotic injured tissue.

DOI: https://doi.org/10.1101/2024.03.12.584570
Nature. 2024 Apr 03.

The variation and evolution of complete human centromeres.

Glennis A Logsdon, Allison N Rozanski, Fedor Ryabov, Tamara Potapova, Valery A Shepelev, Claudia R Catacchio, David Porubsky, Yafei Mao, DongAhn Yoo, Mikko Rautiainen, Sergey Koren, Sergey Nurk, Julian K Lucas, Kendra Hoekzema, Katherine M Munson, Jennifer L Gerton, Adam M Phillippy, Mario Ventura, Ivan A Alexandrov, Evan E Eichler.

Human centromeres have been traditionally very difficult to sequence and assemble owing to their repetitive nature and large size1. As a result, patterns of human centromeric variation and models for their evolution and function remain incomplete, despite centromeres being among the most rapidly mutating regions2,3. Here, using long-read sequencing, we completely sequenced and assembled all centromeres from a second human genome and compared it to the finished reference genome4,5. We find that the two sets of centromeres show at least a 4.1-fold increase in single-nucleotide variation when compared with their unique flanks and vary up to 3-fold in size. Moreover, we find that 45.8% of centromeric sequence cannot be reliably aligned using standard methods owing to the emergence of new α-satellite higher-order repeats (HORs). DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by >500 kb. To understand evolutionary change, we selected six chromosomes and sequenced and assembled 31 orthologous centromeres from the common chimpanzee, orangutan and macaque genomes. Comparative analyses reveal a nearly complete turnover of α-satellite HORs, with characteristic idiosyncratic changes in α-satellite HORs for each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the short (p) and long (q) arms across centromeres and reveals that novel α-satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

DOI: https://doi.org/10.1038/s41586-024-07278-3
Nat Commun. 2024 Apr 01. 15(1): 2821

p53 rapidly restructures 3D chromatin organization to trigger a transcriptional response.

François Serra, Andrea Nieto-Aliseda, Lucía Fanlo-Escudero, Llorenç Rovirosa, Mónica Cabrera-Pasadas, Aleksey Lazarenkov, Blanca Urmeneta, Alvaro Alcalde-Merino, Emanuele M Nola, Andrei L Okorokov, Peter Fraser, Mariona Graupera, Sandra D Castillo, Jose L Sardina, Alfonso Valencia, Biola M Javierre.

Activation of the p53 tumor suppressor triggers a transcriptional program to control cellular response to stress. However, the molecular mechanisms by which p53 controls gene transcription are not completely understood. Here, we uncover the critical role of spatio-temporal genome architecture in this process. We demonstrate that p53 drives direct and indirect changes in genome compartments, topologically associating domains, and DNA loops prior to one hour of its activation, which escort the p53 transcriptional program. Focusing on p53-bound enhancers, we report 340 genes directly regulated by p53 over a median distance of 116 kb, with 74% of these genes not previously identified. Finally, we showcase that p53 controls transcription of distal genes through newly formed and pre-existing enhancer-promoter loops in a cohesin dependent manner. Collectively, our findings demonstrate a previously unappreciated architectural role of p53 as regulator at distinct topological layers and provide a reliable set of new p53 direct target genes that may help designs of cancer therapies.

DOI: https://doi.org/10.1038/s41467-024-46666-1
Dev Cell. 2024 Mar 29. pii: S1534-5807(24)00190-4. [Epub ahead of print]

Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation.

Yusheng Shen, Kassandra M Ori-McKenney.

  Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live-cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs). We find that human epithelial cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification and find that MAP7 promotes acetylation and inhibits detyrosination. Our data identify MAP7 in modulating the tubulin code, resulting in microtubule cytoskeleton remodeling and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Keywords:  cellular adaptation; cytoplasmic density; macromolecular crowding; microtubule; microtubule motor transport; microtubule-associated proteins; osmolarity challenges; tubulin posttranslational modifications

DOI:  https://doi.org/10.1016/j.devcel.2024.03.022
Nat Commun. 2024 Apr 05. 15(1): 2938

Bioelectric stimulation controls tissue shape and size.

Gawoon Shim, Isaac B Breinyn, Alejandro Martínez-Calvo, Sameeksha Rao, Daniel J Cohen.

Epithelial tissues sheath organs and electro-mechanically regulate ion and water transport to regulate development, homeostasis, and hydrostatic organ pressure. Here, we demonstrate how external electrical stimulation allows us to control these processes in living tissues. Specifically, we electrically stimulate hollow, 3D kidneyoids and gut organoids and find that physiological-strength electrical stimulation of ∼ 5 - 10 V/cm powerfully inflates hollow tissues; a process we call electro-inflation. Electro-inflation is mediated by increased ion flux through ion channels/transporters and triggers subsequent osmotic water flow into the lumen, generating hydrostatic pressure that competes against cytoskeletal tension. Our computational studies suggest that electro-inflation is strongly driven by field-induced ion crowding on the outer surface of the tissue. Electrically stimulated tissues also break symmetry in 3D resulting from electrotaxis and affecting tissue shape. The ability of electrical cues to regulate tissue size and shape emphasizes the role and importance of the electrical micro-environment for living tissues.

DOI: https://doi.org/10.1038/s41467-024-47079-w
bioRxiv. 2024 Mar 12. pii: 2024.03.08.584115. [Epub ahead of print]

Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation.

Elizabeth M Black, Carlos Andrés Ramírez Parrado, Isabelle Trier, Wenxue Li, Yoon Ki Joo, Jennifer Pichurin, Yansheng Liu, Lilian Kabeche.

Polo-like kinase 1 (PLK1) protects against genome instability by ensuring timely and accurate mitotic cell division. PLK1 activity is tightly regulated throughout the cell cycle. Although the pathways that initially activate PLK1 in G2 are well-characterized, the factors that directly regulate PLK1 in mitosis remain poorly understood. Here, we identify that human PLK1 activity is sustained by the DNA damage response kinase Checkpoint kinase 2 (Chk2) in mitosis. Chk2 directly phosphorylates PLK1 T210, a residue on its T-loop whose phosphorylation is essential for full PLK1 kinase activity. Loss of Chk2-dependent PLK1 activity causes increased mitotic errors, including chromosome misalignment, chromosome missegregation, and cytokinetic defects. Moreover, Chk2 deficiency increases sensitivity to PLK1 inhibitors, suggesting that Chk2 status may be an informative biomarker for PLK1 inhibitor efficacy. This work demonstrates that Chk2 sustains mitotic PLK1 activity and protects genome stability through discrete functions in interphase DNA damage repair and mitotic chromosome segregation.

DOI: https://doi.org/10.1101/2024.03.08.584115
bioRxiv. 2024 Mar 13. pii: 2024.03.12.584469. [Epub ahead of print]

Functional genetics reveals modulators of anti-microtubule drug sensitivity.

Kuan-Chung Su, Elena Radul, Nolan K Maier, Mary-Jane Tsang, Claire Goul, Brittania Moodie, Heather R Keys, Iain M Cheeseman.

  Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for ~1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.

Keywords:  Microtubule; dynamic instability; mitosis; nocodazole; taxol

DOI:  https://doi.org/10.1101/2024.03.12.584469
Nature. 2024 Apr 03.

Immune microniches shape intestinal Treg function.

Yisu Gu, Raquel Bartolomé-Casado, Chuan Xu, Alice Bertocchi, Alina Janney, Cornelia Heuberger, Claire F Pearson, Sarah A Teichmann, Emily E Thornton, Fiona Powrie.

The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identified3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103+SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies.

DOI: https://doi.org/10.1038/s41586-024-07251-0
Cell Rep. 2024 Apr 04. pii: S2211-1247(24)00401-7. [Epub ahead of print]43(4): 114073

Aging-related defects in macrophage function are driven by MYC and USF1 transcriptional programs.

Charlotte E Moss, Simon A Johnston, Joshua V Kimble, Martha Clements, Veryan Codd, Stephen Hamby, Alison H Goodall, Sumeet Deshmukh, Ian Sudbery, Daniel Coca, Heather L Wilson, Endre Kiss-Toth.

  Macrophages are central innate immune cells whose function declines with age. The molecular mechanisms underlying age-related changes remain poorly understood, particularly in human macrophages. We report a substantial reduction in phagocytosis, migration, and chemotaxis in human monocyte-derived macrophages (MDMs) from older (>50 years old) compared with younger (18-30 years old) donors, alongside downregulation of transcription factors MYC and USF1. In MDMs from young donors, knockdown of MYC or USF1 decreases phagocytosis and chemotaxis and alters the expression of associated genes, alongside adhesion and extracellular matrix remodeling. A concordant dysregulation of MYC and USF1 target genes is also seen in MDMs from older donors. Furthermore, older age and loss of either MYC or USF1 in MDMs leads to an increased cell size, altered morphology, and reduced actin content. Together, these results define MYC and USF1 as key drivers of MDM age-related functional decline and identify downstream targets to improve macrophage function in aging.

Keywords:  CP: Immunology; MYC; USF1; aging; innate immune function; longevity; macrophage; monocyte; upstream stimulatory factor

DOI:  https://doi.org/10.1016/j.celrep.2024.114073
PLoS Comput Biol. 2024 Apr 01. 20(4): e1012001

The mechanical influence of densification on epithelial architecture.

Christian Cammarota, Nicole S Dawney, Philip M Bellomio, Maren Jüng, Alexander G Fletcher, Tara M Finegan, Dan T Bergstralh.

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayered epithelial tissue-whether protective, secretory, absorptive, or filtrative-relies on the side-by-side arrangement of its component cells. The mechanical parameters that determine the shape of epithelial cells in the apical-basal plane are not well-understood. Epithelial tissue architecture in culture is intimately connected to cell density, and cultured layers transition between architectures as they proliferate. This prompted us to ask to what extent epithelial architecture emerges from two mechanical considerations: A) the constraints of densification and B) cell-cell adhesion, a hallmark feature of epithelial cells. To address these questions, we developed a novel polyline cell-based computational model and used it to make theoretical predictions about epithelial architecture upon changes to density and cell-cell adhesion. We tested these predictions using cultured cell experiments. Our results show that the appearance of extended lateral cell-cell borders in culture arises as a consequence of crowding-independent of cell-cell adhesion. However, cadherin-mediated cell-cell adhesion is associated with a novel architectural transition. Our results suggest that this transition represents the initial appearance of a distinctive epithelial architecture. Together our work reveals the distinct mechanical roles of densification and adhesion to epithelial layer formation and provides a novel theoretical framework to understand the less well-studied apical-basal plane of epithelial tissues.

DOI: https://doi.org/10.1371/journal.pcbi.1012001
Cell Rep. 2024 Mar 29. pii: S2211-1247(24)00334-6. [Epub ahead of print]43(4): 114006

BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks.

Daniela Georgieva, Ning Wang, Angelo Taglialatela, Stepan Jerabek, Colleen R Reczek, Pei Xin Lim, Julie Sung, Qian Du, Michiko Horiguchi, Maria Jasin, Alberto Ciccia, Richard Baer, Dieter Egli.

  Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Keywords:  BRCA1; BRCA2; CP: Molecular biology; double-strand break; pluripotency; replication gap suppression; replication stress; somatic cell reprogramming; stalled replication fork

DOI:  https://doi.org/10.1016/j.celrep.2024.114006
Dev Cell. 2024 Apr 01. pii: S1534-5807(24)00143-6. [Epub ahead of print]

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

Jonas L Fowler, Sherry Li Zheng, Alana Nguyen, Angela Chen, Xiaochen Xiong, Timothy Chai, Julie Y Chen, Daiki Karigane, Allison M Banuelos, Kouta Niizuma, Kensuke Kayamori, Toshinobu Nishimura, M Kyle Cromer, David Gonzalez-Perez, Charlotte Mason, Daniel Dan Liu, Leyla Yilmaz, Lucile Miquerol, Matthew H Porteus, Vincent C Luca, Ravindra Majeti, Hiromitsu Nakauchi, Kristy Red-Horse, Irving L Weissman, Lay Teng Ang, Kyle M Loh.

  The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Keywords:  artery; developmental biology; hematopoietic stem cell; human pluripotent stem cell differentiation

DOI:  https://doi.org/10.1016/j.devcel.2024.03.003
bioRxiv. 2024 Mar 11. pii: 2024.03.06.583771. [Epub ahead of print]

Cell-cycle and Age-Related Modulations in Mouse Chromosome Stiffness.

Ning Liu, Wenan Qiang, Philip Jordan, John Marko, Huanyu Qiao.

  The intricate structure of chromosomes is complex, and many aspects of chromosome configuration/organization remain to be fully understood. Measuring chromosome stiffness can provide valuable insights into their structure. However, the nature of chromosome stiffness, whether static or dynamic, remains elusive. In this study, we analyzed chromosome stiffness in MI and MII oocytes. We revealed that MI oocytes had a ten-fold increase in stiffness compared to mitotic chromosomes, whereas chromosome stiffness in MII oocytes was relatively low chromosome. We then investigated the contribution of meiosis-specific cohesin complexes to chromosome stiffness in MI and MII oocytes. Surprisingly, the Young's modulus of chromosomes from the three meiosis-specific cohesin mutants did not exhibit significant differences compared to the wild type, indicating that these proteins may not play a substantial role in determining chromosome stiffness. Additionally, our findings revealed an age-related increase in chromosome stiffness in MI oocytes. Age correlates with elevated DNA damage levels, so we investigated the impact of etoposide-induced DNA damage on chromosome stiffness, discovering a reduction in stiffness in response to such damage in MI oocytes. Overall, our study underscores the dynamic nature of chromosome stiffness, subject to changes influenced by the cell cycle and age.

Keywords:  age; cell cycle; chromosome stiffness; cohesin protein; meiosis; oocyte; spermatocyte

DOI:  https://doi.org/10.1101/2024.03.06.583771
Dev Cell. 2024 Apr 03. pii: S1534-5807(24)00192-8. [Epub ahead of print]

Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.

Adriano Bolondi, Benjamin K Law, Helene Kretzmer, Seher Ipek Gassaloglu, René Buschow, Christina Riemenschneider, Dian Yang, Maria Walther, Jesse V Veenvliet, Alexander Meissner, Zachary D Smith, Michelle M Chan.

  Embryogenesis requires substantial coordination to translate genetic programs to the collective behavior of differentiating cells, but understanding how cellular decisions control tissue morphology remains conceptually and technically challenging. Here, we combine continuous Cas9-based molecular recording with a mouse embryonic stem cell-based model of the embryonic trunk to build single-cell phylogenies that describe the behavior of transient, multipotent neuro-mesodermal progenitors (NMPs) as they commit into neural and somitic cell types. We find that NMPs show subtle transcriptional signatures related to their recent differentiation and contribute to downstream lineages through a surprisingly broad distribution of individual fate outcomes. Although decision-making can be heavily influenced by environmental cues to induce morphological phenotypes, axial progenitors intrinsically mature over developmental time to favor the neural lineage. Using these data, we present an experimental and analytical framework for exploring the non-homeostatic dynamics of transient progenitor populations as they shape complex tissues during critical developmental windows.

Keywords:  cell plasticity; embryonic development; lineage tracing; molecular recording; morphogenesis; neuro-mesodermal progenitor dynamics; single-cell phylogenies; stem cell embryoids

DOI:  https://doi.org/10.1016/j.devcel.2024.03.024
J Cell Sci. 2024 Mar 15. pii: jcs261630. [Epub ahead of print]137(6):

Nuclear actin dynamics and functions at a glance.

Svenja Ulferts, Massimo Lopes, Kei Miyamoto, Robert Grosse.

  Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

Keywords:  Chromatin dynamics; DNA damage; DNA repair; Nuclear actin; Nuclear architecture; Oocyte development; Transcription

DOI:  https://doi.org/10.1242/jcs.261630
bioRxiv. 2024 Mar 14. pii: 2024.03.13.584912. [Epub ahead of print]

Endogenous Nodal switches Wnt interpretation from posteriorization to germ layer differentiation in geometrically constrained human pluripotent cells.

Miguel Angel Ortiz-Salazar, Elena Camacho-Aguilar, Aryeh Warmflash.

The Wnt pathway is essential for inducing the primitive streak, the precursor of the mesendoderm, as well as setting anterior-posterior coordinates. How Wnt coordinates these diverse activities remains incompletely understood. Here, we show that in Wnt-treated human pluripotent cells, endogenous Nodal signaling is a crucial switch between posteriorizing and primitive streak-including activities. While treatment with Wnt posteriorizes cells in standard culture, in micropatterned colonies, higher levels of endogenously induced Nodal signaling combine with exogenous Wnt to drive endoderm differentiation. Inhibition of Nodal signaling restores dose-dependent posteriorization by Wnt. In the absence of Nodal inhibition, micropatterned colonies undergo spontaneous, elaborate morphogenesis concomitant with endoderm differentiation even in the absence of added extracellular matrix proteins like Matrigel. Our study shows how Wnt and Nodal combinatorially coordinate germ layer differentiation with AP patterning and establishes a system to study a natural self-organizing morphogenetic event in in vitro culture.

DOI: https://doi.org/10.1101/2024.03.13.584912
Mol Cell. 2024 Apr 04. pii: S1097-2765(24)00142-4. [Epub ahead of print]84(7): 1186-1187

Intrinsically disordered regions (IDRs): A vague and confusing concept for protein function.

Kevin Struhl.

The term "intrinsically disordered region" (IDR) in proteins has been used in numerous publications. However, most proteins contain IDRs, the term refers to very different types of structures and functions, and many IDRs become structured upon interaction with other biomolecules. Thus, IDR is an unnecessary, vague, and ultimately confusing concept.

DOI: https://doi.org/10.1016/j.molcel.2024.02.023
Nat Cell Biol. 2024 Apr 03.

Proliferation-driven mechanical compression induces signalling centre formation during mammalian organ development.

Neha Pincha Shroff, Pengfei Xu, Sangwoo Kim, Elijah R Shelton, Ben J Gross, Yucen Liu, Carlos O Gomez, Qianlin Ye, Tingsheng Yu Drennon, Jimmy K Hu, Jeremy B A Green, Otger Campàs, Ophir D Klein.

Localized sources of morphogens, called signalling centres, play a fundamental role in coordinating tissue growth and cell fate specification during organogenesis. However, how these signalling centres are established in tissues during embryonic development is still unclear. Here we show that the main signalling centre orchestrating development of rodent incisors, the enamel knot (EK), is specified by a cell proliferation-driven buildup in compressive stresses (mechanical pressure) in the tissue. Direct mechanical measurements indicate that the stresses generated by cell proliferation are resisted by the surrounding tissue, creating a circular pattern of mechanical anisotropy with a region of high compressive stress at its centre that becomes the EK. Pharmacological inhibition of proliferation reduces stresses and suppresses EK formation, and application of external pressure in proliferation-inhibited conditions rescues the formation of the EK. Mechanical information is relayed intracellularly through YAP protein localization, which is cytoplasmic in the region of compressive stress that establishes the EK and nuclear in the stretched anisotropic cells that resist the pressure buildup around the EK. Together, our data identify a new role for proliferation-driven mechanical compression in the specification of a model signalling centre during mammalian organ development.

DOI: https://doi.org/10.1038/s41556-024-01380-4
Dev Cell. 2024 Apr 01. pii: S1534-5807(24)00175-8. [Epub ahead of print]

Oct4 controls basement membrane development during human embryogenesis.

Margit Rosner, Markus Hengstschläger.

  Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling. They are key regulators of cell behavior and tissue functions, and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material, and the inaccessibility of the in vivo condition, BM regulation during human embryo development has remained elusive. Using the post-implantation amniotic sac embryoid (PASE), we delineate BM assembly upon post-implantation development and BM disassembly during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development by regulating Akt signaling and the small GTPase Rac1. These results represent a relevant step toward a more comprehensive understanding of early human development.

Keywords:  Akt; Oct4; Rac1; basement membrane; human embryogenesis; human embryoid

DOI:  https://doi.org/10.1016/j.devcel.2024.03.007
Cell Stem Cell. 2024 Apr 04. pii: S1934-5909(24)00084-5. [Epub ahead of print]31(4): 554-569.e17

NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

Kenji Namoto, Clara Baader, Vanessa Orsini, Alexandro Landshammer, Eva Breuer, Kieu Trinh Dinh, Rosemarie Ungricht, Monika Pikiolek, Stephane Laurent, Bo Lu, Alexandra Aebi, Katharina Schönberger, Eric Vangrevelinghe, Olivera Evrova, Tianliang Sun, Stefano Annunziato, Julie Lachal, Emily Redmond, Louis Wang, Kristie Wetzel, Paola Capodieci, Jonathan Turner, Gabi Schutzius, Vincent Unterreiner, Markus Trunzer, Nicole Buschmann, Dirk Behnke, Rainer Machauer, Clemens Scheufler, Christian N Parker, Magali Ferro, Armelle Grevot, Armin Beyerbach, Wei-Yu Lu, Stuart J Forbes, Jürgen Wagner, Tewis Bouwmeester, Jun Liu, Bindi Sohal, Sukhdeep Sahambi, Linda E Greenbaum, Felix Lohmann, Philipp Hoppe, Feng Cong, Andreas W Sailer, Heinz Ruffner, Ralf Glatthar, Bostjan Humar, Pierre-Alain Clavien, Michael T Dill, Elizabeth George, Jürgen Maibaum, Prisca Liberali, Jan S Tchorz.

  The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Keywords:  LATS kinase inhibitor; YAP agonist; YAP/Hippo pathway; drug development; liver regeneration; organoid expansion; regenerative medicine; tissue regeneration; tissue stem cells; wound healing

DOI:  https://doi.org/10.1016/j.stem.2024.03.003
Curr Top Dev Biol. 2024 ;pii: S0070-2153(24)00008-5. [Epub ahead of print]156 245-295

The genetics of cardiomyocyte polyploidy.

Tyler Buddell, Alexandra L Purdy, Michaela Patterson.

  The regulation of ploidy in cardiomyocytes is a complex and tightly regulated aspect of cardiac development and function. Cardiomyocyte ploidy can range from diploid (2N) to 8N or even 16N, and these states change during key stages of development and disease progression. Polyploidization has been associated with cellular hypertrophy to support normal growth of the heart, increased contractile capacity, and improved stress tolerance in the heart. Conversely, alterations to ploidy also occur during cardiac pathogenesis of diseases, such as ischemic and non-ischemic heart failure and arrhythmia. Therefore, understanding which genes control and modulate cardiomyocyte ploidy may provide mechanistic insight underlying cardiac growth, regeneration, and disease. This chapter summarizes the current knowledge regarding the genes involved in the regulation of cardiomyocyte ploidy. We discuss genes that have been directly tested for their role in cardiomyocyte polyploidization, as well as methodologies used to identify ploidy alterations. These genes encode cell cycle regulators, transcription factors, metabolic proteins, nuclear scaffolding, and components of the sarcomere, among others. The general physiological and pathological phenotypes in the heart associated with the genetic manipulations described, and how they coincide with the respective cardiomyocyte ploidy alterations, are further discussed in this chapter. In addition to being candidates for genetic-based therapies for various cardiac maladies, these genes and their functions provide insightful evidence regarding the purpose of widespread polyploidization in cardiomyocytes.

Keywords:  Cardiomyocyte; Polyploidy, Cell cycle, Endoreduplication, Endomitosis, Regeneration

DOI:  https://doi.org/10.1016/bs.ctdb.2024.01.008
Cell. 2024 Mar 22. pii: S0092-8674(24)00250-2. [Epub ahead of print]

The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.

Vidhya Krishnamoorthy, Martina Foglizzo, Robert L Dilley, Angela Wu, Arindam Datta, Parul Dutta, Lisa J Campbell, Oksana Degtjarik, Laura J Musgrove, Antonio N Calabrese, Elton Zeqiraj, Roger A Greenberg.

  Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

Keywords:  55LCC; AAA+ ATPase; C1orf109; CINP; SPATA5; SPATA5L1; genome instability; replication stress response; replisome regulation/proteostasis; unfoldase

DOI:  https://doi.org/10.1016/j.cell.2024.03.002
JACC Heart Fail. 2024 Apr;pii: S2213-1779(24)00142-2. [Epub ahead of print]12(4): 662-664

Cardiomyocyte DNA Damage Predicts Functional Recovery in Heart Failure Patients.

Shah R Ali, Hesham A Sadek.



Keywords:  DNA damage; biopsy; heart failure; precision medicine; risk prediction

DOI:  https://doi.org/10.1016/j.jchf.2024.01.012
Mol Cell. 2024 Apr 04. pii: S1097-2765(24)00190-4. [Epub ahead of print]84(7): 1180-1182

Changing structures, changing paradigms: NELF helps regulate paused or elongating RNA polymerase II.

William F Richter, Dylan J Taatjes.

Using cryo-EM and biochemical methods, Su and Vos1 discover an alternative NELF structural state that enables transcription and switches NELF-RNA polymerase II (RNAPII) compatibility with other RNAPII-associated factors that regulate pausing, elongation, termination, and transcription-coupled DNA repair.

DOI: https://doi.org/10.1016/j.molcel.2024.03.008
Nat Metab. 2024 Apr 02.

A spatiotemporal proteomic map of human adipogenesis.

Felix Klingelhuber, Scott Frendo-Cumbo, Muhmmad Omar-Hmeadi, Lucas Massier, Pamela Kakimoto, Austin J Taylor, Morgane Couchet, Sara Ribicic, Martin Wabitsch, Ana C Messias, Arcangela Iuso, Timo D Müller, Mikael Rydén, Niklas Mejhert, Natalie Krahmer.

White adipocytes function as major energy reservoirs in humans by storing substantial amounts of triglycerides, and their dysfunction is associated with metabolic disorders; however, the mechanisms underlying cellular specialization during adipogenesis remain unknown. Here, we generate a spatiotemporal proteomic atlas of human adipogenesis, which elucidates cellular remodelling as well as the spatial reorganization of metabolic pathways to optimize cells for lipid accumulation and highlights the coordinated regulation of protein localization and abundance during adipocyte formation. We identify compartment-specific regulation of protein levels and localization changes of metabolic enzymes to reprogramme branched-chain amino acids and one-carbon metabolism to provide building blocks and reduction equivalents. Additionally, we identify C19orf12 as a differentiation-induced adipocyte lipid droplet protein that interacts with the translocase of the outer membrane complex of lipid droplet-associated mitochondria and regulates adipocyte lipid storage by determining the capacity of mitochondria to metabolize fatty acids. Overall, our study provides a comprehensive resource for understanding human adipogenesis and for future discoveries in the field.

DOI: https://doi.org/10.1038/s42255-024-01025-8
Cell Rep. 2024 Apr 04. pii: S2211-1247(24)00382-6. [Epub ahead of print]43(4): 114054

14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.

Liu Liu, Ienglam Lei, Shuo Tian, Wenbin Gao, Yijing Guo, Zhaokai Li, Ziad Sabry, Paul Tang, Y Eugene Chen, Zhong Wang.

  Cell fate conversion is associated with extensive post-translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and chromatin modifiers. Akt1 kinase and protein phosphatase 2A are the key writer and key eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3-embedded factors, such as histone deacetylase 4 (Hdac4), Mef2c, and Foxo1, form Hdac4-organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming.

Keywords:  14-3-3; CP: Molecular biology; biomolecular condensate; cardiac reprogramming; epigenetic code; post-translational modification

DOI:  https://doi.org/10.1016/j.celrep.2024.114054
Dev Cell. 2024 Mar 26. pii: S1534-5807(24)00181-3. [Epub ahead of print]

The Notch-PDGFRβ axis suppresses brown adipocyte progenitor differentiation in early post-natal mice.

Zuoxiao Shi, Shaolei Xiong, Ruoci Hu, Zilai Wang, Jooman Park, Yanyu Qian, Jaden Wang, Pratibha Bhalla, Nipun Velupally, Qing Song, Zhenyuan Song, Minsun Stacey Jeon, Ke Kurt Zhang, Linlin Xie, Brian T Layden, Sang-Ging Ong, Yuwei Jiang.

  De novo brown adipogenesis holds potential in combating the epidemics of obesity and diabetes. However, the identity of brown adipocyte progenitor cells (APCs) and their regulation have not been extensively explored. Here, through in vivo lineage tracing and mouse modeling, we observed that platelet-derived growth factor receptor beta (PDGFRβ)+ pericytes give rise to developmental brown adipocytes but not to those in adult homeostasis. By contrast, T-box 18 (TBX18)+ pericytes contribute to brown adipogenesis throughout both developmental and adult stages, though in a depot-specific manner. Mechanistically, Notch inhibition in PDGFRβ+ pericytes promotes brown adipogenesis by downregulating PDGFRβ. Furthermore, inhibition of Notch signaling in PDGFRβ+ pericytes mitigates high-fat, high-sucrose (HFHS)-induced glucose and metabolic impairment in mice during their development and juvenile phases. Collectively, these findings show that the Notch/PDGFRβ axis negatively regulates developmental brown adipogenesis, and its repression promotes brown adipose tissue expansion and improves metabolic health.

Keywords:  PDGFRβ; Tbx18; brown adipocyte progenitor cells; development; obesity; pericytes

DOI:  https://doi.org/10.1016/j.devcel.2024.03.012