Biology subjects

Forest, A.

Publications and source records attributed to Forest, A..

6 recordsLinked to original sources

bioRxiv2025

Non-centromeric CENP-A epigenetically regulates epithelial-mesenchymal plasticity and heterogeneity in human cells

The centromeric histone variant CENP-A, highly expressed in aggressive cancers, can promote an epithelial-mesenchymal transition (EMT) whose underlying mechanisms remain to decipher. Here, we tracked both the temporal dynamics of EMT states and CENP-A localization over time using a reversible high CENP-A expression system in human cells. Cell populations presenting hybrid EMT states at start, when exposed to high CENP-A levels, progressively accumulated mesenchymal states and displayed increased centromeric and ectopic CENP-A incorporation. Mechanistically, we reveal ectopic CENP-A gains at EMT genes by chromatin immunoprecipitation and identify two distinct EMT programs activated at different stages of the cell cycle by single-nucleus multi-omics. Importantly, while a pre-existent inflammatory program got amplified, high CENP-A induced a new developmental program. Remarkably, interrupting high CENP-A provision erased induced programs along with ectopic CENP-A incorporation, in line with non-genetic alterations. Our findings uncover an unconventional, non-centromeric function for CENP-A in epigenetically modulating epithelial-mesenchymal plasticity.

molecular biology↗

bioRxiv2025

Mitotic slippage causes nuclear instability in polyploid cells

Whole-genome duplication (WGD), leading to polyploidy can arise in physiological and pathological contexts1-5. WGD can occur via non-canonical cell cycles such as mitotic slippage, cytokinesis failure or endoreplication1,3. Whether the routes to WGD influence the behaviour of the resulting polyploid cells remains unclear. Here, we compared these routes under both physiological and non-physiological conditions. Remarkably, only mitotic slippage led to widespread nuclear abnormalities defined by highly variable nuclear deformations that we termed nuclear instability. Mechanistically, we found that these nuclei were softer - due to high levels of histone 3 phosphorylation in G1 altering chromatin compaction - and thus more vulnerable to microtubule-driven deformations. The resulting nuclear instability leads to local nuclear reorganisation and changes in 3D genome organisation impacting ultimately gene expression. Importantly, we observed similar nuclear instability in megakaryocytes, which are physiological polyploid cells that we show here to be generated by mitotic slippage, providing a molecular mechanism for their atypical nuclear architecture6,7. In striking contrast, nuclear shape was stable in different physiological polyploid cells generated by cytokinesis failure and endoreplication. Overall, our findings highlight that the route towards WGD matters and that mitotic slippage uniquely destabilizes nuclear architecture, with implications for both physiology and disease.

cell biology↗

bioRxiv2024

HIRA defines early replication initiation zones independently of their genome compartment

Chromatin states and 3D architecture have been used as proxy to identify replication initiation zones (IZs) in mammalian cells. While they do often correlate, their functional interconnections remain a puzzle. Here, we dissect these relationships by focusing on the histone H3.3 chaperone HIRA, which plays a role in both early initiation zone (IZ) definition and higher-order organisation of active chromatin. We monitored in parallel early replication initiation, chromatin accessibility, histone post-translational modifications (PTMs) and 3D organisation in wild-type cells, HIRA knock-out cells and HIRA knock-out cells complemented with HIRA. In the absence of HIRA, impaired early firing at HIRA-dependent IZs does not correspond to changes in chromatin accessibility or patterns of histone H3 PTMs. With respect to 3D organisation, a small subset of early IZs initially in compartment A switched to B and lost early initiation in the absence of HIRA. Critically, HIRA complementation restores these early IZ (and H3.3 variant enrichment) without substantial compartment reversal. Thus, our work reveals that regulation of early replication initiation by HIRA can be uncoupled from accessibility, histone mark patterns and compartment organisation.

cell biology↗

bioRxiv2024

HIRA-dependent provision of histone H3.3 in active chromatin ensures genome compartmentalisation

The mammalian genome, organised into chromatin, adopts a three-dimensional (3D) folding within the cell nucleus with spatially segregated active and repressed compartments, termed A and B. However, how nucleosome deposition impacts these levels of organisation is unknown. Here, we monitored changes in 3D genome folding by Hi-C after impairing the chaperone HIRA, involved in histone H3.3 deposition. In the absence of HIRA, H3.3 enrichment decreases in compartment A that also shows weaker interactions. At this scale, histone post-translational modifications (PTMs) do not follow H3.3 changes. In line with impaired H3.3 nucleosome maintenance, compartment A accessibility measured by ATAC-seq increases. Specifically, at active genes, accessibility increases in gene bodies but decreases at promoters where compensation by H3.1 reduces nucleosome turnover. Notably, regions flanking active genes show reduced insulation. We conclude that the HIRA-dependent pathway involved in H3.3 deposition is key to maintain higher order organisation in active regions and impact compartmentalisation independently of histone PTMs.

cell biology↗

bioRxiv2024

H3.3 deposition counteracts the replication-dependent enrichment of H3.1 at chromocenters in embryonic stem cells

Chromocenters in mouse cells are membrane-less nuclear compartments that represent typical heterochromatin stably maintained during the cell cycle. Here, we explore how histone H3 variants, replicative H3.1/H3.2 or replacement H3.3, mark these domains during the cell cycle. In mouse embryonic stem cells (ESCs), neuronal precursor cells (NPCs) as well as immortalized 3T3 cells, we find a strong and distinct H3.1 enrichment at chromocenters, with some variation in ESCs. Mechanistically, this H3.1 selective enrichment depends on the DNA Synthesis Coupled (DSC) deposition pathway operating in S phase. Yet, this selective enrichment is challenged when we target H3.3 deposition through the DNA Synthesis Independent (DSI) deposition pathway mediated by HIRA. Altering the H3.1/H3.3 equilibrium at chromocenters in ESCs affects its heterochromatin properties leading to mitotic defects. We thus reveal opposing mechanisms for H3.1 and H3.3 deposition with different enforcement according to cell cycle and potency which determine their ratio at chromocenters and are critical for genome stability and cell survival.

cell biology↗

bioRxiv2023

Microglial adipose triglyceride lipase regulates neuroinflammatory and behavioural responses to LPS

Adipose triglyceride lipase (ATGL), the enzyme that catalyses the rate-limiting step of triglyceride lipolysis, regulates inflammation in peripheral tissues. ATGL has been associated with both pro- and anti-inflammatory responses in different tissues suggesting its actions are dependent on cell type. Recent studies in microglia and macrophages suggest that lipid droplets (LD), a triglyceride storing organelle, and LD lipolysis via ATGL are important components of inflammatory responses. Here, we determined the impact of ATGL inhibition and microglia-specific ATGL loss-of-function on inflammatory and behavioural responses to acute pro-inflammatory insult. First, we evaluated the impact of lipolysis inhibition on lipopolysaccharide (LPS)-induced expression and secretion of cytokines in mouse primary microglia cultures. LPS led to LD accumulation in microglia and altered the expression of lipolysis regulators. The pan-lipase inhibitor ORlistat alleviated LPS-induced expression of IL-1{beta} and IL-6. Specific inhibition of ATGL by ATGListatin had similar anti-inflammatory action on cytokines expression and secretion in both neonatal and adult microglia cultures. Second, targeted and untargeted lipidomic studies revealed that ATGL inhibition reduced LPS-induced generation of pro-inflammatory prostanoids and affected ceramide profile. Finally, the role of ATGL in neuroinflammation was assessed in a novel mouse model with inducible ATGL deletion specifically in microglia. Loss of microglial ATGL in adult male mice dampened LPS-induced expression of IL-6 and reduced LPS-induced sickness behaviour. Together, our results demonstrate that pharmacological inhibition or loss of ATGL-mediated triglyceride lipolysis reduces LPS-induced inflammation to suggest that inhibition of lipolysis plays a beneficial role in neuroinflammation.

neuroscience↗