EMBO Journal最新文献

In mouse early pachytene spermatocytes, the X and Y chromosomes undergo rapid non-homologous (NH) synapsis and desynapsis, but the functional significance remains unknown. Here, we report that pachynema-specific knockout of Speedy A (SpdyA) from telomeres caused persistent Y-X NH synapsis, with the entire Y axis synapsed onto the X axis. This persistent Y-X NH synapsis did not interrupt meiotic sex chromosome inactivation, recombination, or sex body formation, but it disrupted X-Y loop-axis organization and homologous X-Y desynapsis, leading to spermatocyte death. Similarly, persistent Y-X NH synapsis was also observed in pachytene spermatocytes lacking TRF1, where SpdyA was frequently lost from the X-Y non-pseudoautosomal region (non-PAR) telomeres. Mechanistic studies revealed that Serine 48 of SUN1 is a key SpdyA/CDK2 phosphorylation site required for Y-X NH desynapsis. We propose that SpdyA governs Y-X NH desynapsis by stabilizing the linkage between the X-Y non-PAR telomeres and their LINC complexes, and that this process is regulated independently from other aspects of pachynema progression. Our findings suggest a key role for Y-X NH desynapsis in establishing proper X-Y loop-axis organization.

Tendon injury promotes aberrant osteochondral differentiation of tendon stem cells (TSCs) and results in disability. However, the cellular subsets within the osteochondral lineage involved in this process and associated mechanisms remain unclear. Here, we found that, following Achilles tenotomy, murine Gli1⁺ tendon sheath cells expanded rapidly, transitioning into tenogenic and osteochondrogenic cells. Lineage tracing, together with single-cell RNA sequencing, revealed that osteochondrogenic Gli1⁺ tendon sheath cells originate from Scx⁺ tendon stem/progenitor cells, preferentially differentiate into osteochondral lineage tendon progenitors at 7 dpi, subsequently undergoing aberrant chondrogenesis and osteogenesis at 21dpi and 63dpi, respectively. In addition, Acvr1^R206H/+ robustly accelerates osteochondral differentiation in Gli1⁺ tendon sheath progenitors. Furthermore, GNAS/PKA signaling was significantly activated in osteochondral differentiation of Gli1⁺ tendon sheath progenitors. Alternatively, treatment with the G_sα antagonist, NF449, or genetic inhibition of the PKA subunit, Prkaca, in Gli1⁺ sheath progenitors significantly alleviated aberrant osteochondral differentiation. NF449 also prevented osteochondral differentiation of human tendon stem cells. These findings identify Gli1⁺ tendon sheath progenitors with osteochondral differentiation capacity during heterotopic ossification via activation of GNAS/PKA signaling, suggesting PKA as a potentially effective therapeutic target to treat tendon ossification.

Formation of skin epithelial appendages like hair follicles requires hedgehog (Hh) signal reception, its conduction through the primary cilium and activation of Gli transcription factors. How Hh signalling induces cell-type-specific responses through Gli transcription factors in hair follicle stem cells and their cilia-dependence remains unclear. Here, we use conditional mouse mutants to genetically dissect the roles of Gli2 and Gli3 transcription factors and cilia in the skin epithelium. Upon keratinocyte-specific depletion of Gli2, hair follicle morphogenesis is delayed whereas sebaceous gland formation is enhanced, suggesting a dual role for Gli2 during appendage development. Gli2 promotes proliferation of sebaceous gland stem cells, impacting the number and size of individual sebaceous gland lobes. While ablation of Gli3 shows no detectable phenotypes, hair follicle cell fate is blocked in Gli2/Gli3 double knockout (dKO) mice, suggesting functional compensation. Finally, loss of cilia phenocopies the depletion of Gli2 but not the Gli2/3 dKO mutants. Our study reveals compartment-specific regulation of murine skin morpohogenesis by Gli2 and cilia-independent activator functions of Gli3 in the absence of Gli2.

The primary cilium plays a crucial role in regulating whole-body energy metabolism, as reflected in Bardet-Biedl syndrome (BBS), where ciliary dysfunction leads to obesity due to hyperphagia and white adipose tissue (WAT) remodeling. Regulation of the fate and differentiation of adipocyte precursor cells (APCs) is essential for maintaining WAT homeostasis during obesity. Using Bbs8^-/- mice that recapitulate the BBS patient phenotype, we demonstrate that primary cilia dysfunction reduces the stem-cell-like P1 APC subpopulation by inducing a phenotypic switch to a fibrogenic progenitor state. This switch is characterized by extracellular matrix (ECM) remodeling and upregulation of the fibrosis marker CD9, even before the onset of obesity. Single-cell RNA sequencing reveals a direct transition of P1 APCs into fibrogenic progenitors, bypassing the committed P2 progenitor state. Ectopic ciliary Hedgehog signaling upon loss of BBS8 appears as a central driver of the molecular changes in Bbs8^-/- APCs, altering their differentiation into adipocytes and promoting their lipid uptake. These findings unravel a novel role for primary cilia in governing APC fate by determining the balance between adipogenesis and fibrogenesis, and suggest potential therapeutic targets for obesity.

The DNA Damage Response (DDR) is a highly regulated process that safeguards genomic integrity against DNA lesions. Increasing evidence supports a reciprocal relationship between damaged chromatin architecture and the signalling pathways that coordinate the DDR. However, the mechanisms underlying this interplay in response to transcription-blocking DNA lesions remain largely unexplored. Here, we show that stalling of RNA polymerase II (RNAPII) at such lesions induces local chromatin acetylation, mediated primarily by the histone acetyltransferase p300. The resulting chromatin relaxation stimulates the dissociation of mature co-transcriptional spliceosomes from nascent RNA and promotes RNA:DNA hybrid (R-loop) formation, leading to ATM activation. In turn, activated ATM modulates chromatin conformation by phosphorylating histone H2A.X and triggering p38MAPK/MSK1-dependent histone H3S10 phosphorylation. Our findings highlight the cross-regulation between chromatin state and ATM signalling as a key component of the cellular response to transcription stress.

Loss of pluripotency is an essential step in post-implantation development that facilitates the emergence of somatic cell identities essential for gastrulation. Before implantation, pluripotent cell identity is governed by a gene regulatory network that includes the key transcription factors SOX2 and NANOG. However, it is unclear how the pluripotency gene regulatory network is dissolved to enable lineage restriction. Here, we show that SOX2 is required for post-implantation pluripotent identity in the mouse, and cells that lose SOX2 expression in the posterior epiblast are no longer pluripotent. Using in vitro and in vivo analyses, we demonstrate anticorrelated expression of NANOG and SOX2 preceding gastrulation, culminating in an early disappearance of pluripotent identity from posterior NANOG^high/SOX2^low epiblast. Surprisingly, Sox2 expression is repressed by NANOG and embryos with post-implantation deletion of Nanog maintain posterior SOX2 expression. Our results demonstrate that the distinctive features of post-implantation pluripotency are underpinned by altered functionality of pluripotency transcription factors, ensuring correct spatio-temporal loss of embryonic pluripotency.

A biochemical deficiency of mitochondrial complex I (CI) underlies approximately 30% of cases of primary mitochondrial disease, yet the inventory of molecular machinery required for CI assembly remains incomplete. We previously characterised patients with isolated CI deficiency caused by segregating variants in RTN4IP1, a gene that encodes a mitochondrial NAD(P)H oxidoreductase. Here, we demonstrate that RTN4IP1 deficiency causes a CI assembly defect in both patient fibroblasts and knockout cells, and report that RTN4IP1 is a bona fide CI assembly factor. Complexome profiling revealed accumulation of unincorporated ND5-module and impaired N-module production. RTN4IP1 patient fibroblasts also exhibited defective coenzyme Q biosynthesis, substantiating a second function of RTN4IP1. Thus, our data reveal RTN4IP1 plays necessary and independent roles in both the terminal stages of CI assembly and in coenzyme Q metabolism, and that pathogenic RTN4IP1 variants impair both functions in patients with mitochondrial disease.

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is unclear. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 is required for lysophagic flux after lysosomal damage but is not involved in the initial phagophore formation on terminally damaged lysosomes. Instead, ATXN3 is recruited to a distinct subset of lysosomes that are decorated with phosphatidylinositol-(4,5)-bisphosphate and that are not yet fully reacidified. There, ATXN3, along with its partner VCP/p97, targets and turns over K48-K63-branched ubiquitin conjugates. ATXN3 thus facilitates degradation of a fraction of LAMP2 via microautophagy to regenerate the lysosomal membrane and to thereby reestablish degradative capacity needed also for completion of lysophagy. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.

The cell envelope of gram-negative bacteria is composed of an inner and an outer membrane. In Escherichia coli, several pathways mediate phospholipid transport between the two membranes, including the Mla (i.e., maintenance of lipid asymmetry) and Pqi (i.e., paraquat inducible) systems. Here, we identify and characterise in the intracellular pathogen Brucella abortus a complex named Mpc, which exhibits homology to both Mla and Pqi components. Mpc is required for bacterial growth under envelope stress conditions, and for survival within macrophages during the early stages of infection. Analyses of protein-protein interactions and structural predictions suggest that the Mpc complex bridges the two membranes of the bacterial cell envelope. Absence of this system results in altered lipid composition of the outer membrane vesicles, indicating that Mpc plays a role in lipid transport between the membranes. Our sequence comparisons reveal that Mpc is conserved across numerous species of Hyphomicrobiales. The discovery of this novel lipid-trafficking system expands our understanding of the diversity and evolution of lipid-transport mechanisms in diderm bacteria.