Valentina Proserpio

Adult-onset Autosomal Dominant Leukodystrophy (ADLD) is a rare fatal leukodystrophy caused by increased LMNB1 gene dosage, most commonly resulting from duplication of the LMNB1 locus. Because ADLD is a gene dosage disorder, selective reduction of pathological LMNB1 expression represents a rational therapeutic strategy. Although allele-specific RNA interference has previously been shown to lower LMNB1 levels in patient-derived fibroblasts and directly reprogrammed neurons, its therapeutic effects have not been evaluated in disease-relevant human glial cells or using functional efficacy endpoints. Here, we established human induced pluripotent stem cell-derived astrocytes from ADLD patients as a human glial model in which to validate allele-specific LMNB1 silencing across molecular, cellular, and functional readouts. ADLD astrocytes recapitulated increased LMNB1 expression and characteristic nuclear abnormalities and displayed transcriptional alterations affecting extracellular matrix organization, calcium homeostasis, metabolism and RNA processing. Functionally, these cells also exhibited functional phenotypes suitable for therapeutic evaluation: astrocyte-conditioned medium impaired the viability of both murine and human oligodendroglial cultures, while conditioned-medium and direct astrocyte-seeding paradigms revealed impaired post-lesion myelin recovery in lysolecithin-treated cerebellar organotypic slices. Allele-specific LMNB1 silencing restored physiological LMNB1 levels, corrected nuclear abnormalities, attenuated astrocyte-mediated oligodendroglial toxicity, improved post-lesion myelin recovery, and was associated with selective transcriptional programs associated with extracellular support and cholesterol metabolism. Together, these findings provide molecular, cellular, and functional validation of allele-specific LMNB1 dosage correction in patient-derived human astrocytes and offer key support for LMNB1-lowering strategies in disease-relevant human glial cells.

In response to stress, cells exploit plasticity to self-activate and form protrusions that explore the environment and guide migration. While protrusion initiation is well characterized, their maturation and functional composition remain poorly understood. Here, using a new pooled genetic approach (ReGenT-seq) to interrogate chromatin factors controlling epidermal progenitor activation, we unexpectedly identified Cbx3 as a critical determinant of protrusion formation. Beyond its nuclear activities, we revealed a cytoplasmic moonlighting function of CBX3 that governs the subcellular localization of specific mRNAs within polarizing protrusions of migrating epidermal progenitor cells, as well as in invadopodia of squamous carcinoma cells. CBX3-dependent mRNA transport promotes the localized establishment of multiple organelles, including the endoplasmic reticulum and lysosomes, within maturing protrusions, and regulates focal adhesion dynamics. Together, our findings uncover a multilayered gene regulatory mechanism controlling cell motility through protrusionogenesis and identify a chromatin-to-cell-protrusion mRNA transport pathway that represents a potential therapeutic target for enhancing wound healing and limiting cancer invasion.

Skin homeostasis depends on interactions between epithelial cells and the microbiome mediated by molecular and biochemical factors. Perturbations of this interplay are linked to inflammatory disorders, including wound healing and cancer. While research has mainly illuminated shifts in microbial community composition, novel computational approaches are starting to reveal the host-microbe functional interactome in the cutaneous ecosystem. In this review, we specifically focus on known molecular and metabolic mechanisms linking skin epithelial cells and microorganisms in health and disease. Additionally, we summarise computational tools available to investigate these interactions integrating omics data. Furthermore, we present potential applications of this functional crosstalk to advance therapies targeting skin pathologies. Finally, we propose a comparative interactomics approach to envision the existence of ecological memories in the skin ecosystem, in parallel with the one described in the gut, hypothesising a link between epithelial and microbial memories in barrier tissues.

Pluripotency, the ability to generate all body cell types, emerges in a disorganized embryonic cell mass. After implantation, these cells form a columnar epithelium and initiate lumenogenesis. During gastrulation, some undergo epithelial-to-mesenchymal transition to form the primitive streak (PS). The signals controlling these events in humans are largely unknown. Here, to study them, we developed a chemically defined 3D model where conventional pluripotent stem cells self-organize into a columnar epithelium with a lumen, from which PS-like cells emerge. We show that early TGFβ family inhibition prevents epithelial identity, also in murine 3D embryo models and in embryos. ZNF398 acts downstream of TGFβ1, activating the epithelial master regulator ESRP1 while repressing mesenchymal factors CDH2 and ZEB2. After epithelium formation, TGFβ1 stimulation is dispensable for its maintenance. However, treatment via ACTIVIN—a distinct TGFβ family ligand—induces PS efficiently. Thus, signalling of the TGFβ family dynamically governs pluripotent epiblast epithelial identity.

Skeletal muscle wasting is a defining feature of cancer cachexia, a multifactorial syndrome that drastically compromises patient quality of life and treatment outcomes. Mitochondrial dysfunction is a major contributor to skeletal muscle wasting in cancer cachexia, yet the upstream molecular drivers remain elusive. Here we show that cancer impairs the activity of cAMP-dependent protein kinase A (PKA) and of its transcriptional effector CREB1 in skeletal muscle, ultimately contributing to the downregulation of a core transcriptional network that supports mitochondrial integrity and function. The restoration of cAMP–PKA–CREB1 signalling through pharmacological inhibition of the cAMP-hydrolysing phosphodiesterase 4 (PDE4) rescues the expression of mitochondrial-related genes, improves mitochondrial function and mitigates skeletal muscle wasting in male mice. Altogether, our data identify tumour-induced suppression of the cAMP–PKA–CREB1 axis as a central mechanism contributing to mitochondrial dysfunction in skeletal muscle during cancer cachexia. Furthermore, these findings highlight PDE4, particularly the PDE4D isoform, as a potential therapeutic target to preserve muscle mitochondrial function and counteract muscle wasting in cancer cachexia.

The epigenome coordinates spatial-temporal specific gene expression during development and in adulthood, for the maintenance of homeostasis and upon tissue repair. The upheaval of the epigenetic landscape is a key event in the onset of many pathologies including tumours, where epigenetic changes cooperate with genetic aberrations to establish the neoplastic phenotype and to drive cell plasticity during its evolution. DNA methylation, histone modifiers and readers or other chromatin components are indeed often altered in cancers, such as carcinomas that develop in epithelia. Lining the surfaces and the cavities of our body and acting as a barrier from the environment, epithelia are frequently subjected to acute or chronic tissue damages, such as mechanical injuries or inflammatory episodes. These events can activate plasticity mechanisms, with a deep impact on cells’ epigenome. Despite being very effective, tissue repair mechanisms are closely associated with tumour onset. Here we review the similarities between tissue repair and carcinogenesis, with a special focus on the epigenetic mechanisms activated by cells during repair and opted by carcinoma cells in multiple epithelia. Moreover, we discuss the recent findings on inflammatory and wound memory in epithelia and describe the epigenetic modifications that characterise them. Finally, as wound memory in epithelial cells promotes carcinogenesis, we highlight how it represents an early step for the establishment of field cancerization.

Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset.

It is believed epithelial cells that have participated in a wound repair elicit a more efficient but locally restricted response to future injuries. However here we show that the cell adaptation resulting from a localised tissue damage has a wide spatial impact at a scale not previously noticed. We demonstrate that away from injured site, after a first injury a specific epithelial stem cell population gives rise to long term wound-memory progenitors residing in their own niche of origin. Notably these progenitors have not taken part in the first wound healing but become pre-activated through priming . This adaptation differs from classical features of trained immunity previously shown to be adopted by other epithelial stem cells. Our newly identified wound-distal memory cells display a cell-autonomous transcriptional pre-activated state leading to an enhanced wound repair ability that can be partially recapitulated through epigenetic perturbation even in absence of an injury. Importantly, the harmful consequences of wound repair, such as exacerbated tumorigenesis, occur within these primed cells and follow their spatial distribution. Overall, we show that sub-organ scale adaptation of an injury relies on spatially organised and memory-dedicated progenitors, characterised by an epigenetic actionable cell state, that predisposes to tumour onset.

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination.