Biology subjects

Settembre, C.

Publications and source records attributed to Settembre, C..

5 recordsLinked to original sources

bioRxiv2026

ZNF687 couples bone marrow myeloid progenitor dynamics toosteoclastogenesis in severe Paget's disease of bone

Pagets disease of bone (PDB) is a late-onset skeletal disorder characterized by excessive osteoclast-mediated bone remodelling and disorganized bone deposition. The P937R mutation in the ZNF687 gene causes a severe form of PDB complicated by giant cell tumour transformation. Although ZNF687 has been implicated in osteoclastogenesis, whether it regulates upstream haematopoietic progenitor dynamics and bone marrow myeloid output remains unclear. Using a constitutive Zfp687 knock-out mouse model, we showed that Zfp687 loss causes postnatal growth restriction, reduced bone marrow cellularity, impaired osteoclast differentiation in vitro and in vivo, and increased trabecular bone mass during adulthood. Flow cytometry revealed a marked reduction in osteoclast progenitors and macrophages in Zfp687-deficient bone marrow, whereas the pagetic P937R mutation promoted the expansion of the same myeloid populations in the Zfp687P937R knock-in mouse model. Single-cell RNA sequencing of bone marrow-derived c-Kit+ haematopoietic progenitors further demonstrated that Zfp687 loss selectively disrupted the myeloid progenitor compartment. This analysis identified 22 transcriptionally distinct populations and revealed a significant depletion of the early cycling granulocyte-monocyte progenitor cluster, without evidence of a global block in myeloid differentiation. Mechanistically, Zfp687 deficiency impaired the Brd4-c-Myc-NFATc1 axis in osteoclastogenic precursors and reduced Csf1 expression in bone marrow stromal and osteoblastic cells, linking intrinsic transcriptional competence to niche-derived M-CSF support. In pagetic patient iPSCs-derived haematopoietic progenitors, the P937R mutation enhanced clonogenic haematopoietic output, accelerated colony formation, and promoted the expansion of primitive/multipotent colony-forming progenitors, leading to hypercellular myeloid colonies. Together, our findings establish ZNF687 as a regulator of haematopoietic progenitor dynamics that couples bone marrow myeloid output to osteoclastogenesis, providing a progenitor-level mechanism for severe ZNF687-related PDB.

cancer biology↗

bioRxiv2026

LC3C-ATG4D regulatory axis supports coronavirus replication organelle formation independently of canonical autophagy

Coronaviruses hijack host membranes to assemble ER-derived double-membrane vesicles (DMVs) that shield viral RNA replication from the cell intrinsic surveillance. Although DMVs morphologically resemble autophagosomes, whether and how autophagy factors actively support their biogenesis has remained elusive. Here, we identify a non-canonical requirement for the autophagy protein LC3C in {beta}-coronavirus replication. Loss of LC3s impaired viral RNA replication, whereas genetic ablation of ATG7 did not, indicating that canonical ATG7-dependent lipidation is dispensable in this context. Reconstitution experiments showed that only LC3C substantially restored replication in LC3-deficient cells and that LC3C phospho-mutants, differing in accessibility to ATG4-mediated processing, displayed distinct proviral activities. Additionally, ATG4D, the main protease responsible for maintaining the LC3 non-lipidated pool, is selectively required for viral replication. Both ATG4D and LC3s depletion triggers formation of aberrant DMV-like structures and potently suppresses SARS-CoV-2 replication. Ultrastructural analysis of nsp3-nsp4-induced membranes showed that depletion of LC3s or ATG4 proteases altered DMV abundance and morphology, supporting a role for the LC3C-ATG4D axis in replication organelle biogenesis. These data establish that {beta}-coronaviruses repurpose ATG4D-driven LC3C de-lipidation for non-canonical LC3 recruitment to replication organelles, identifying the lipidation state of LC3 as a molecular determinant of replication organelle biogenesis and efficient viral replication. HighlightsThe manuscript shows that {beta}-coronavirus replication depends on LC3 proteins and particularly on LC3C in reconstitution experiments, that this dependency is independent of ATG7-mediated lipidation, and that ATG4D promotes efficient replication and replication organelle morphology. Together, the data support a model in which a non-canonical LC3C-ATG4D pathway contributes to DMV biogenesis and viral RNA replication.

cell biology↗

bioRxiv2025

Arylsulfatase I is a novel lysosomal chondroitin sulfatase regulating endochondral ossification

During endochondral ossification, chondrocytes undergo maturation and biochemically modify the collagenous extracellular matrix of cartilage. Similar modifications to cartilage proteoglycans (PGs), which are predominantly chondroitin sulfate, have not been characterized. Using synchrotron X-ray fluorescence imaging, we demonstrated that PG sulfation significantly decreased during cartilage maturation of chick embryos. Laser-capture microdissection and RNAseq revealed upregulation of Arylsulfatase I (Arsi) in mature cartilage of mouse. ARSI protein also increased in mature cartilage of mouse and chick in vivo and during maturation of ATDC5 chondrocytes in vitro, whereas expression of the two known chondroitin sulfate PG sulfatases (ARSB and GALNS) was not specific to mature cartilage. Colocalization studies suggested that ARSI is lysosomal, and functional assays revealed that ARSI impacts lysosome homeostasis in chondrocytes. Biochemical analyses of ARSI gain and loss of function cell lines and isolated cell-free systems revealed that ARSI is a novel chondroitin sulfatase, specifically desulfating GalNAc4S at the nonreducing terminal of CS/DS. Finally, Arsi knockout in RCS chondrocytes caused increased expression of maturation genes, such as Col10a1 and Mmp13. In total, these data identify ARSI as a novel PG sulfatase regulating endochondral ossification.

cell biology↗

bioRxiv2022

The ZNF687 mutation of Paget's disease associated with giant cell tumour causes severe bone remodelling alteration as a result of a deregulated osteoclast transcriptional program

Pagets disease (PDB) is a late-onset bone remodelling disorder with a broad spectrum of symptoms and complications. One of the most aggressive forms is caused by the P937R mutation in the ZNF687 gene. Although the genetic involvement of ZNF687 in PDB has been extensively studied, the molecular mechanisms underlying this association remains unclear. Here, we describe the first Zfp687 knock-in mouse model and demonstrate that the mutation recapitulates the PDB phenotype, showing a severe bone remodelling alteration. Through micro-computed tomography analysis, we observed that 8-month-old mice showed a mainly osteolytic phase, with a significant decrease in the trabecular bone volume affecting the femurs and the vertebrae of both heterozygous and homozygous mutant mice. In contrast, osteoblast activity was deregulated, beginning to produce disorganised bone. Noteworthy, this phenotype became pervasive in 16-month-old mice, where osteoblast function overtook bone resorption as the predominant event, as highlighted by the presence of woven bone in histological analyses, consistent with the PDB phenotype. Furthermore, we detected osteophytes and intervertebral disc degeneration, outlining for the first time the link between osteoarthritis and PDB in a PDB mouse model. Finally, we generated CRISPR-Cas9-based Zfp687 knock-out RAW 264.7 cells, and noted a remarkable impairment of osteoclast differentiation capacity, reinforcing the relevance of Zfp687 during this process. RNA-sequencing on wild type and KO clones identified a set of genes involved in osteoclastogenesis under the control of Zfp687, i.e., Tspan7, Cpe, Vegfc, and Ggt1. Thus, this study established an essential role of Zfp687 in the regulation of bone remodelling, and may offer the potential to therapeutically treat PDB.

cell biology↗

bioRxiv2020

Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders.

Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unraveled Beclin-1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell penetrating autophagy inducing Tat-Beclin-1 (TB-1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB-1 reduced urinary orotic acid and hyperammonemia, and improved survival under protein-rich diet in spf-ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB-1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin-1-dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.

genetics↗