Biology subjects

Zirkel, A.

Publications and source records attributed to Zirkel, A..

7 recordsLinked to original sources

bioRxiv2023

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is now acknowledged as a key contributor to organismal ageing and late-life disease. Although popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and its paracrine effects. To address these issues, we repurposed the small molecule inhibitor inflachromene (ICM) to induce senescence to human primary cells. Within six days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered significant similarity of the two states. Notably though, ICM suppresses the proinflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM induces a senescence-like phenotype rapidly and synchronously thereby allowing the study of its core regulatory program without any confounding heterogeneity.

genomics↗

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↗

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↗

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↗