Biology subjects

Neri, F.

Publications and source records attributed to Neri, F..

10 recordsLinked to original sources

bioRxiv2025

Opposing CTCF and GATA4 activities set the pace of chromatin topology remodeling during cardiomyogenesis

Reorganization of the three-dimensional chromatin structure is a critical feature of human embryonic development. Yet, the mechanisms regulating integrative remodelling of local structures (e.g., loops) and global architecture (e.g., A/B compartmentalization) remain un-clear. Here, we investigate this aspect in the context of cardiomyogenesis, characterized by pronounced B-to-A remodelling of several cardiac-specific genes such as TTN. We focus on the roles of the pioneer transcription factor GATA4 and the architectural protein CTCF. Using an inducible knockdown system during human induced pluripotent stem cell differ-entiation, we show that GATA4 is essential for timely topological activation of key cardiac genes, while partial depletion of CTCF, anticipating physiological downregulation during de-velopment, enhances this process. Deletion of a single CTCF binding site on TTN leads to modest gene decompaction and transcriptional activation. Bulk and single-cell RNA se-quencing of chamber-specific cardiac organoids reveals that loss of GATA4 delays differ-entiation and sustains proliferation of early cardiomyocytes, whereas premature CTCF de-pletion accelerates yet alters cardiomyocyte maturation. These findings suggest that CTCF and GATA4 have antagonistic roles on chromatin dynamics during cardiomyogenesis, form-ing a rheostat that maintains accurate developmental tempo. Disruption of this mecha-nism may contribute to congenital heart defects caused by mutations in these factors.

molecular biology↗

bioRxiv2025

Oxidative stress causes a reversible decrease of deubiquitylases activity in old vertebrate brains

The ubiquitin-proteasome system is essential for neuronal proteostasis, and its activity declines with age. How deubiquitylating enzymes (DUBs) are affected by aging in the vertebrate brain remains unclear. Here, we profiled cysteine protease DUBs using activity-based proteomics in aging mouse and killifish brains. Despite stable protein levels, we identified a subset of DUBs that progressively lose catalytic activity with age. We demonstrated that oxidative stress impairs DUB function through thiol oxidation and that antioxidant treatment restores their activity in vitro and in vivo. Further, inhibition of DUBs in human iPSC-derived neurons significantly recapitulated ubiquitylation changes observed in aged brains, and temporal analysis in mice revealed that DUB inhibition precedes proteasome decline in the brain during aging. Together, these findings indicate a redox-sensitive subset of DUBs that undergo an age-associated decline in activity and suggest that impaired deubiquitylation is an early, yet potentially reversible, driver of proteostasis decline in the aging brain.

biochemistry↗

bioRxiv2024

Epigenetic clock and lifespan prediction in the short-lived killifish Nothobranchius furzeri

Aging, characterized by a gradual decline in organismal fitness, is the primary risk factor for numerous diseases including cancer, cardiovascular, and neurodegenerative disorders. The inter-individual variability in aging and disease susceptibility has led to the concept of "biological age," an indirect measure of an individuals relative fitness. Epigenetic changes, particularly DNA methylation, have emerged as reliable biomarkers for estimating biological age, leading to the development of predictive models known as epigenetic clocks. Initially created for humans, these clocks have been extended to various mammalian species. Here we set to expand these tools for the short-lived killifish, Nothobranchius furzeri. This species, with its remarkably short lifespan and expression of canonical aging hallmarks, offers a unique model for experimental aging studies. We developed an epigenetic clock for N. furzeri using reduced-representation bisulfite sequencing (RRBS) to analyze DNA methylation in brain and caudal fin tissues across different ages. Our study involved generating comprehensive DNA methylation datasets and employing machine learning to create predictive models based on individual CpG sites and co-methylation modules. These models demonstrated high accuracy in estimating chronological age, with a median absolute error of 3 weeks (7.5% of median lifespan) for a clock based on methylation of individual CpG and 1.5 weeks (3.7% of median lifespan) for an eigenvector-based clock. Our investigation extended to assessing epigenetic age acceleration in different strains and the potential resetting effect of regeneration on fin tissue. Notably, our models indicated that a shorter-lived strain has accelerated epigenetic aging and that regeneration does not reset, but may decelerate epigenetic aging. Additionally, we used longitudinal data to develop an "epigenetic timer" for direct prediction of individual lifespan based on fin biopsies and eigenvector-based method, achieving a median absolute error of 4.5 weeks in the prediction of actual age of death. This surprising result underscores the existence of intrinsic determinants of lifespan established early in life. This study presents the first epigenetic clocks and lifespan predictors for N. furzeri, highlighting their potential as aging biomarkers and sets the stage for future research on life-extending interventions in this model organism.

genomics↗

bioRxiv2024

Polyamines sustain epithelial regeneration in aged intestines by modulating protein homeostasis

Aging hampers the regenerative potential of intestinal epithelium across species including humans, yet the underlying causes remain elusive. Here, using proteomic and metabolomic profiling of intestinal tissues together with functional assays, we characterized the temporal dynamics of regeneration following injury induced by 5-fluorouracil, a commonly used chemotherapeutic agent. Comparison of regeneration dynamics in mice of different ages revealed the emergence of a proteostasis stress signature and increased levels of polyamines following injury exclusively in old epithelia. Mechanistically, we show that delayed regeneration is an intrinsic feature of aged epithelial cells that display reduced protein synthesis and accumulation of ubiquitylated proteins. Notably, dietary restriction followed by re-feeding prior to injury increases polyamine pathway activation, enhances protein synthesis, and restores the regenerative capacity of aged intestines. Our findings highlight promising epithelial targets for interventions aimed at tackling the decline in tissue repair mechanisms associated with aging.

molecular biology↗

bioRxiv2024

Senescent cell heterogeneity and responses to senolytic treatment are related to cell cycle status during cell growth arrest.

Cellular senescence has been strongly linked to aging and age-related diseases. It is well established that the phenotype of senescent cells is highly heterogeneous and influenced by their cell type and senescence-inducing stimulus. Recent single-cell RNA-sequencing studies identified heterogeneity within senescent cell populations. However, proof of functional differences between such subpopulations is lacking. To identify functionally distinct senescent cell subpopulations, we employed high-content image analysis to measure senescence marker expression in primary human endothelial cells and fibroblasts. We found that G2-arrested senescent cells feature higher senescence marker expression than G1-arrested senescent cells. To investigate functional differences, we compared IL-6 secretion and response to ABT263 senolytic treatment in G1 and G2 senescent cells. We determined that G2-arrested senescent cells secrete more IL-6 and are more sensitive to ABT263 than G1-arrested cells. We hypothesize that cell cycle dependent DNA content is a key contributor to the heterogeneity within senescent cell populations. This study demonstrates the existence of functionally distinct senescent subpopulations even in culture. This data provides the first evidence of selective cell response to senolytic treatment among senescent cell subpopulations. Overall, this study emphasizes the importance of considering the senescent cell heterogeneity in the development of future senolytic therapies.

cell biology↗

bioRxiv2023

A Fully-Automated Senescence Test (FAST) for the high-throughput quantification of senescence-associated markers

Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated {beta}-galactosidase activity (SA-{beta}-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by i) quantifying colorimetric SA-{beta}-Gal via optical density; ii) implementing staining background controls; iii) automating image acquisition, image processing, and data analysis. In addition to the automated threshold-based scoring, a multivariate machine learning approach is provided. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions. Using FAST, we compared X-Gal with fluorescent C12FDG live-cell SA-{beta}-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that modify senescence burden. This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.

cell biology↗

bioRxiv2022

Nr2f1-dependent shaping of mitochondrial architecture in newborn mouse hippocampal neurons unravels novel insights into the neurodevelopmental disorder BBSOAS

The nuclear receptor Nr2f1 plays a key role during cortical development by acting as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, its mutations cause the Bosch-Boonstra-Schaaf optic atrophy-intellectual syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including optic nerve atrophy, intellectual disability, and autistic traits. In this study by genome-wide and in silico analyses we found that key factors for mitochondrial function and dynamics represent potential genomic targets under direct Nr2f1 transcriptional control in neurons. Although mitochondrial dysfunction is increasingly implicated in neurodevelopmental disorders, whether and how Nr2f1 can regulate mitochondria function in neural cells is still completely unknown. To address these questions, we have combined mouse genetics, neuroanatomical and imaging approaches. To genetically manipulate Nr2f1 function in newborn neurons we focused on the adult mouse hippocampal dentate gyrus, a site of persistent neurogenesis where Nr2f1 is highly expressed. We showed that conditional Nr2f1 loss-of-function within the hippocampal neurogenic niche lead to a reduced mitochondrial mass associated to mitochondrial fragmentation in newborn neurons, likely reflecting alteration in mitochondrial dynamics. Accordingly, we showed that the fusion factor Mfn2, which is as a putative Nr2f1 target, is downregulated following both Nr2f1 adult depletion and embryonic haploinsufficiency in mice. Overall, our study provides the first evidence of a crucial role of Nr2f1 in shaping mitochondria in neurons and opens a promising avenue for the identification of new mechanisms implicated in BBSOAS pathogenesis involving mitochondrial dysfunction.

neuroscience↗

bioRxiv2021

Comprehensive Profiling of Plasma Exosomes Using Data Independent Acquisitions, New Tools for Aging Cohort Studies

Aging is a complex biological process associated with progressive loss of physiological function and susceptibility to several diseases, such as cancer and neurodegeneration. Exosomes are involved in many cellular signaling pathways, and their cargo may serve as promising disease or aging biomarkers. These membrane-bound extracellular vesicles facilitate the transport of intracellular contents to proximal and distal cells in the body. Here, we investigated two omics approaches for exosome analysis. To overcome the challenges of plasma exosome contamination with abundant soluble plasma proteins, we developed a high-throughput method to isolate highly purified exosomes from human plasma by sequential size-exclusion chromatography and ultrafiltration. First, we used data-dependent acquisitions from offline high-pH reversed-phase fractions of exosome lysate to generate a deep spectral library comprising [~]2,300 exosome proteins. Second, in a pilot aging study, we used comprehensive data-independent acquisitions to compare plasma exosomes from young (20-26 yrs) and old (60-66 yrs) individuals. We quantified 1,318 exosome proteins, and levels of 144 proteins were significantly different in young and old plasma groups (Q<0.05 and >1.5-fold change). We also analyzed exosome miRNA cargo and detected 331 miRNAs. Levels of several were significantly different in young and old individuals. In addition, 88 and 17 miRNAs were unique to old and young individuals, respectively. Plasma exosome biomarkers have great potential for translational studies investigating biomarkers of aging and age-related diseases and to monitor therapeutic aging interventions.

cell biology↗

bioRxiv2020

Atlas and developmental dynamics of mouse DNase I hypersensitive sites

Early mammalian development is orchestrated by genome-encoded regulatory elements populated by a changing complement of regulatory factors, creating a dynamic chromatin landscape. To define the spatiotemporal organization of regulatory DNA landscapes during mouse development and maturation, we generated nucleotide-resolution DNA accessibility maps from 15 tissues sampled at 9 intervals spanning post-conception day 9.5 through early adult, and integrated these with 41 adult-stage DNase-seq profiles to create a global atlas of mouse regulatory DNA. Collectively, we delineated >1.8 million DNase I hypersensitive sites (DHSs), with the vast majority displaying temporal and tissue-selective patterning. Here we show that tissue regulatory DNA compartments show sharp embryonic-to-fetal transitions characterized by wholesale turnover of DHSs and progressive domination by a diminishing number of transcription factors. We show further that aligning mouse and human fetal development on a regulatory axis exposes disease-associated variation enriched in early intervals lacking human samples. Our results provide an expansive new resource for decoding mammalian developmental regulatory programs.

genomics↗

bioRxiv2020

Global reference mapping and dynamics of human transcription factor footprints

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, yet it remains challenging to distinguish variants that impact regulatory function2. Genomic DNase I footprinting enables quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3-5. However, to date only a small fraction of such sites have been precisely resolved on the human genome sequence5. To enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate at nucleotide resolution ~4.5 million compact genomic elements encoding transcription factor occupancy. We map the fine-scale structure of ~1.6 million DHS and show that the overwhelming majority is populated by well-spaced sites of single transcription factor:DNA interaction. Cell context-dependent cis-regulation is chiefly executed by wholesale actuation of accessibility at regulatory DNA versus by differential transcription factor occupancy within accessible elements. We show further that the well-described enrichment of disease- and phenotypic trait-associated genetic variants in regulatory regions1,6 is almost entirely attributable to variants localizing within footprints, and that functional variants impacting transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find that the global density of human genetic variation is markedly increased within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a new framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.

genomics↗