Biology subjects

Papantonis, A.

Publications and source records attributed to Papantonis, A..

34 records · Page 2Linked to original sources

bioRxiv2022

circRAB3IP modulates cell proliferation by reorganizing gene expression and mRNA processing in a paracrine manner

Circular RNAs are endogenous long-lived and abundant non-coding species. Despite their prevalence, only few circRNAs have been dissected mechanistically to date. Here, we catalogued nascent RNA-enriched circRNAs from primary human cells and functionally assign a role to circRAB3IP in sustaining cellular homeostasis. We combined "omics" and functional experiments to show how circRAB3IP depletion deregulates hundreds of genes, suppresses cell cycle progression, and induces senescence-associated gene expression changes. Conversely, excess circRAB3IP delivered to endothelial cells via extracellular vesicles suffices for accelerating their division. We attribute these effects to the interplay between circRAB3IP and the general splicing factor SF3B1, which impacts transcript usage of cell cycle-related genes. Together, our findings link the maintenance of cell homeostasis to the tightly regulated titers of a single circRNA.

genomics↗

bioRxiv2021

Translational specialization in pluripotency by RBPMS poises future lineage-decisions

The blueprints for developing organs are preset at the early stages of embryogenesis. Transcriptional and epigenetic mechanisms are proposed to preset developmental trajectories. However, we reveal that the competence for future cardiac fate of human embryonic stem cells (hESCs) is preset in pluripotency by a specialized mRNA translation circuit controlled by RBPMS. RBPMS is recruited to active ribosomes in hESCs to control the translation of essential factors needed for cardiac commitment program, including WNT signaling. Consequently, RBPMS loss specifically and severely impedes cardiac mesoderm specification leading to patterning and morphogenesis defects in human cardiac organoids. Mechanistically, RBPMS specializes mRNA translation, selectively via 3UTR binding and globally by promoting translation initiation. Accordingly, RBPMS loss causes translation initiation defects highlighted by aberrant retention of the EIF3 complex and depletion of EIF5A from mRNAs, thereby abrogating ribosome recruitment. We reveal how future fate trajectories are preprogrammed during embryogenesis by specialized mRNA translation. Teaser: Cardiac fate competence is preprogrammed in pluripotency by specialized mRNA translation of factors initiating cardiogenesis

developmental biology↗

bioRxiv2021

Human brain organoids assemble functionally integrated bilateral optic vesicles

During embryogenesis, optic vesicles develop from the diencephalon via a complex process of organogenesis. Using iPSC-derived human brain organoids, we attempted to simplify the complexities and demonstrate the formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day thirty, brain organoids could assemble optic vesicles, which progressively develop as visible structures within sixty days. These optic vesicle-containing brain organoids (OVB-Organoids) constitute a developing optic vesicles cellular components, including the primitive cornea and lens-like cells, developing photoreceptors, retinal pigment epithelia, axon-like projections, and electrically active neuronal networks. Besides, OVB-Organoids also display synapsin-1, CTIP-positive, myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photoreceptor activity of OVB-Organoids, and light sensitivities could be reset after a transient photo bleach blinding. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow conducting interorgan interaction studies within a single organoid.

developmental biology↗

bioRxiv2020

Cilium induction triggers differentiation of glioma stem cells

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. GBM has a poor prognosis, and currently, there are no curative options exist. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of the fundamental biology of GSCs is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit an elevated level of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components at the ciliary base is sufficient to induce ciliogenesis in a subset of GSCs. Importantly, restoring ciliogenesis caused GSCs to behave like healthy NPCs switching from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a crucial role for cilium as a molecular switch in determining GSCs fate and suggest that cilium induction is an attractive strategy to intervene in GSCs proliferation.

cell biology↗

bioRxiv2020

Genomic instability is an early event driving chromatin reorganization and escape from oncogene-induced senescence

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not timely removed via immune surveillance, senescent cells will also present a detrimental side. Although this has mostly been attributed to the senescence-associated-secretory-phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state represents another unfavorable outcome. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion, which harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and for driving cell cycle re-entry and malignant transformation. In summary, we now provide strong evidence in support of genomic instability underlying "escape" from oncogene-induced senescence. HIGHLIGHTSO_LIOncogene driven error-prone repair produces early genetic lesions allowing escape from senescence C_LIO_LICells escaping oncogene-induced senescence display mutational signatures observed in cancer patients C_LIO_LIA single recurrent inversion harboring a circadian TF gene suffices for bypassing oncogene-induced senescence C_LIO_LIChromatin loop and compartment remodeling support the "escape" transcriptional program C_LI

cell biology↗

bioRxiv2020

Retrieving high-resolution chromatin interactionsand decoding enhancer regulatory potential in silico

The advent of the chromosome conformation capture (3C) and related technologies has profoundly renewed our understaning of three-dimensional chromatin organization in mammalian nuclei. Alongside these experimental approaches, numerous computational tools for handling, normalizing, visualizing, and ultimately detecting interactions in 3C-type datasets are being developed. Here, we present Bloom, a comprehensive method for the analysis of 3C-type data matrices on the basis of Dirichlet process mixture models that addresses two important open issues. First, it retrieves occult interaction patterns from sparse data, like those derived from single-cell Hi-C experiments; thus, bloomed sparse data can now be used to study interaction landscapes at sub-kbp resolution. Second, it detects enhancer-promoter interactions with high sensitivity and inherently assigns an interaction frequency score (IFS) to each contact. Using enhancer perturbation data of different throughput, we show that IFS accurately quantifies the regulatory influence of each enhancer on its target promoter. As a result, Bloom allows decoding of complex regulatory landscapes by generating functionally-relevant enhancer atlases solely on the basis of 3C-type of data.

bioinformatics↗

bioRxiv2020

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to reestablish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation, super-resolution imaging and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon reentry into G1. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

genomics↗

bioRxiv2020

Complex small-world regulatory networks emerge from the 3D organisation of the human genome

The discovery that overexpressing one or a few critical transcription factors can switch cell state suggests that gene regulatory networks are relatively simple. In contrast, genome-wide association studies (GWAS) point to complex phenotypes being determined by hundreds of loci that rarely encode transcription factors and which individually have small effects. Here, we use computer simulations and a simple fitting-free polymer model of chromosomes to show that spatial correlations arising from 3D genome organisation naturally lead to stochastic and bursty transcription plus complex small-world regulatory networks (where the transcriptional activity of each genomic region subtly affects almost all others). These effects require factors to be present at sub-saturating levels; increasing levels dramatically simplifies networks as more transcription units are pressed into use. Consequently, results from GWAS can be reconciled with those involving overexpression. We apply this pan-genomic model to predict patterns of transcriptional activity in whole human chromosomes, and, as an example, the effects of the deletion causing the diGeorge syndrome.

biophysics↗

bioRxiv2020

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers critically implicated in human pathologies, with CHD6 being one of its least studied members. Here, we discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. We show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS-mutation affects CHD6 protein folding and impairs its ability to recruit co-factors in response to DNA damage or autophagy stimulation. This leads to an accumulation of DNA damage burden and to senescence-like phenotypes. By combining genomics and functional assays, we describe for the first time a molecular mechanism for the chromatin control of autophagic flux and genotoxic stress surveillance that applies broadly to human cell types and explains HSS onset.

cell biology↗

bioRxiv2019

Aging-associated changes in transcriptional elongation influence metazoan longevity.

Physiological homeostasis becomes compromised during aging, as a result of impairment of cellular processes, including transcription and RNA splicing. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here, we profiled and analyzed genome-wide, aging-related changes in transcriptional processes across different organisms: nematode worms, fruit flies, mice, rats and humans. The average transcriptional elongation speed (Pol-II speed) increased with age in all five species. Along with these changes in elongation speed we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin/Igf signaling, both reversed most of these aging-related changes. Genetic variants in Pol-II that reduced its speed in worms and flies increased their lifespan. Similarly, reducing Pol-II speed by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal aging and lifespan-extending interventions, and point to possible preventative measures.

genomics↗

bioRxiv2019

Redundant and specific roles of cohesin STAG subunits in chromatin looping and transcription control

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A and RAD21, cohesin contains in somatic cells one of two orthologous STAG subunits, SA1 or SA2. How these variable subunits affect the function of the cohesin complex is still unclear. SA1- and SA2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of SA1 and SA2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either SA1 or SA2. Following rapid depletion of either subunit, we perform high resolution Hi-C, RNA-sequencing and sequential ChIP studies to show that SA1 and SA2 do not co-occupy individual binding sites and have distinct ways how they affect looping and gene expression. These findings are supported at the single cell level by single-molecule localizations via dSTORM super-resolution imaging. Since somatic and congenital mutations of the SA subunits are associated with cancer (SA2) and intellectual disability syndromes with congenital abnormalities (SA1 and SA2), we verified SA1-/SA2-dependencies using human neural stem cells, hence highlighting their importance for understanding particular disease contexts.

cell biology↗

bioRxiv2019

HMGB1 as a rheostat of chromatin topology and RNA homeostasis on the path to senescence

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is however disturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from senescent cell nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin or about its roles in the nucleus. To address this, we mapped HMGB1 binding genome-wide in different primary cells using a tailored protocol. We integrated ChIP-seq and Hi-C data with a graph theory approach to uncover HMGB1 demarcation of a subset of topologically-associating domains (TADs) that harbor genes required for paracrine senescence. Moreover, using sCLIP, knock-down and overexpression experiments, we now show that HMGB1 is a bona fide RNA-binding protein (RBP) bound to senescence-relevant mRNAs and affecting splicing. HMGB1 also has an interactome rich in RBPs, many of which are implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and concertedly regulate the availability of SASP-relevant transcripts. Our findings highlight a broader than hitherto assumed role for HMGB1. It coordinates chromatin folding and RNA homeostasis as part of a feedforward loop controlling both cell-autonomous and paracrine senescence inside and outside of cells.

genomics↗

bioRxiv2018

Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations

How spatial chromosome organization influences genome integrity is still poorly understood. Here we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities, are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CTCF/cohesin bound sites at the bases of chromatin loops and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias, are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hot spots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability, and are key contributors to the occurrence of genome rearrangements that drive cancer.

cell biology↗

bioRxiv2018

DNA methylation patterns of replicative senescence are strand-specific and reflect changes in chromatin conformation

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes at specific sites in the genome. These changes might be due to an directly regulated epigenetic process, or to gradual deregulation of the epigenetic state, which is often referred to as "epigenetic drift". We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated in the course of culture expansion of mesenchymal stem cells (MSCs) and other cell types. During reprogramming into induced pluripotent stem cells (iPSCs) particularly the culture-associated hypomethylation is reversed simultaneously with age-associated and pluripotency-associated DNAm changes. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that upon passaging the DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpolulations of MSCs at later passages. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no preferential interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results indicate that DNAm changes during culture-expansion resembles epigenetic drift, which seems to occur in relation to chromatin conformation.

genomics↗

bioRxiv2017

Shaping Epigenetic Memory via Genomic Bookmarking

Reconciling the stability of epigenetic patterns with the rapid turnover of histone modifications and their adaptability to external stimuli is an outstanding challenge. Here, we propose a new biophysical mechanism that can establish and maintain robust yet plastic epigenetic domains via genomic bookmarking (GBM). We model chromatin as a recolourable polymer whose segments bear non-permanent histone marks (or colours) which can be modified by \"writer\" proteins. The three-dimensional chromatin organisation is mediated by protein bridges, or \"readers\", such as Polycomb Repressive Complexes and Transcription Factors. The coupling between readers and writers drives spreading of biochemical marks and sustains the memory of local chromatin states across replication and mitosis. In contrast, GBM-targeted perturbations destabilise the epigenetic patterns. Strikingly, we demonstrate that GBM alone can explain the full distribution of Polycomb marks in a whole Drosophila chromosome. We finally suggest that our model provides a starting point for an understanding of the biophysics of cellular differentiation and reprogramming.

biophysics↗

bioRxiv2017

Topological Demarcation By HMGB2 Is Disrupted Early Upon Senescence Entry Across Cell Types And Induces CTCF Clustering

Ageing-relevant processes, like cellular senescence, are characterized by complex, often stochastic, events giving rise to heterogeneous cell populations. We hypothesized that entry into senescence of different primary human cells can be triggered by one early molecular event affecting the spatial organization of chromosomes. To test this, we combined whole-genome chromosome conformation capture, population and single-cell transcriptomics, super-resolution imaging, and functional analyses applied on proliferating and replicatively-senescent populations from three distinct human cell types. We found a number of genes involved in DNA conformation maintenance being suppressed upon senescence across cell types. Of these, the abundant high mobility group (HMG) B1 and B2 nuclear factors are quantitatively removed from cell nuclei before typical senescence markers appear, and mark a subset of topologically-associating domain (TAD) boundaries. Their loss coincides with obvious reorganization of chromatin interactions via the dramatic spatial clustering of CTCF foci. HMGB2 knock-down recapitulates this senescence-induced CTCF clustering, while also affecting insulation at TAD boundaries. We accordingly propose that HMGB-mediated deregulation of chromosome conformation constitutes a primer for the ensuing senescent program across cell types.

genomics↗