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Forne, I.

Publications and source records attributed to Forne, I..

At least 19 recordsLinked to original sources

Hybrid Drosophila cell lines show selective genome instabilities

Background: Post-zygotic reproductive isolation is thought to be caused by genomic conflict. However, the exact nature of these detrimental incompatibilities is largely unknown. To identify genomic regions that became incompatible during the formation of species and are therefore prone to get lost in hybrids, we established hybrid cell lines derived from an authentic interspecies cross between Drosophila melanogaster and Drosophila simulans. Results: All four independently derived lines selectively lost the D. melanogaster allele of the hybrid incompatibility gene Hmr, confirming its incompatibility with cellular viability. Moreover, each line underwent distinct but directionally biased erosion of additional D. melanogaster chromosomal regions, preferentially affecting the X chromosome, chromosome 4, and chromosome 3R. Loss of the D. melanogaster X was dosage compensated by upregulation of genes on the hemizygous D. simulans X, while D. melanogaster rDNA was paradoxically amplified across all lines. Nucleolar dominance was driven by relative rDNA copy number rather than species identity, overturning the prevailing view of fixed species-based dominance. Finally, the measurements of the species-specific proteome revealed species specific differences in the RNA to protein coefficient K_p as novel and yet undescribed sources of molecular conflict in hybrids. Conclusions: These findings reveal a hierarchical, constraint-driven mechanism of hybrid genome resolution.

evolutionary biology↗

Multi-omic dissection of reversible and persistent molecular alterations in diet-induced obesity

Diet-induced obesity drives broad molecular remodeling across host and microbial systems, but why lifestyle intervention reverses some of these alterations while others persist remains unclear. To address this, we performed a multi-omic characterization of diet-induced obese mice after a combined nutritional and exercise intervention, integrating liver transcriptomics, epigenomics, metabolomics and metallomics with gut metagenomics and metallomics. Multi-omics factor analysis resolved two dominant axes of variation. The first captured a broadly reversible response (~72% of altered variables), restored by dietary restriction and exercise, involving coordinated remodeling of hepatic metal homeostasis, epigenetic regulation, and immune and cell-turnover pathways. The second comprised persistent alterations resistant to intervention and driven primarily by microbial functional profiles: notably, functional diversity remained reduced despite substantial taxonomic recovery. Using germ-free mice to define the microbiota-responsive hepatic space, we found that a significant fraction of the persistent liver features fell within it, enriched in lipid metabolism (PPAR signaling, steroid and cholesterol biosynthesis, peroxisomal activity) and retinol metabolism. Sequential correlation analysis traced these changes to the loss of specific low-abundance taxa and their biosynthetic capacities, particularly vitamin (folate, biotin, cobalamin, pantothenate, thiamine) and cofactor metabolism, implicating microbiota-derived vitamin metabolism in sustained hepatic dysfunction. Cobalt was the sole essential element that remained persistently dysregulated and tracked dietary cobalt content, suggesting a diet--microbiota route to the persistent phenotype. These findings establish a dual regulatory framework in which metabolic plasticity is governed by reversible host-intrinsic and persistent microbiota-dependent processes, providing a systems-level explanation for obesogenic memory.

systems biology↗

RNA-dependent association of the pyruvate dehydrogenase complex with mtDNA-containing assemblies supports mitochondrial translation

Mitochondrial DNA (mtDNA) encodes core subunits of the oxidative phosphorylation machinery, and its expression is spatially organized, with the genome packaged into nucleoids around which transcription and mitoribosome assembly are concentrated. How this architecture is built, and how it is linked to the metabolic state of the organelle, remains poorly understood. Here we show that the pyruvate dehydrogenase complex (PDHc), which supplies acetyl-CoA to the tricarboxylic acid cycle, is a component of this machinery in Saccharomyces cerevisiae. PDHc co-purifies with mtDNA and resides in high-molecular-weight assemblies whose integrity requires RNA rather than DNA, and proximity labeling places it selectively adjacent to the mitoribosome. The E1 subunit Pda1 concentrates into discrete foci that depend on mtDNA and disperse reversibly upon inhibition of mitochondrial translation. Loss of Pda1, Pdb1 or Lat1, but not of the E3-binding protein Pdx1, compromises the maintenance of mtDNA; deletion of PDA1, PDB1 or LAT1 also reduces output from a mitochondrially encoded reporter, and PDHc-deficient cells are hypersensitive to translational inhibition. Catalytically inactive Pda1 and Lat1 variants rescue both defects as effectively as the wild-type proteins, demonstrating a function genetically separable from acetyl-CoA synthesis. PDHc therefore acts as a non-catalytic component of the RNA-dependent architecture that supports mitochondrial gene expression.

cell biology↗

Preimplantation factor (PIF) is an endogenous inhibitor of potassium channel KV1.3 regulating neutrophil function during pregnancy

Pregnancy is a unique period regarding immune cell regulation. Within the placenta, maternal immune cells play a central role in immune surveillance and tissue remodeling. However, regulatory mechanisms of systemic immunity during pregnancy are less clear. Here, we show that neutrophil function is altered in pregnant mice (E13.5), indicated by increased slow rolling velocity and reduced adhesion. Mechanistically, PreImplantation factor (PIF), a 15 amino acid peptide which is produced by human and murine trophoblast cells of the placenta, is continuously secreted into the maternal circulation and plays a key role in modulating neutrophil function via blocking the voltage-gated potassium channel KV1.3. This resulted in impaired intracellular Ca2+ signaling and subsequently disturbance of neutrophil post-arrest modifications and a higher susceptibility to physiological shear forces in vivo and in vitro. Furthermore, PIF-mediated KV1.3 blockade impaired E-selectin-mediated release of S100A8/A9 and phagocytosis. Taken together, we have identified PIF as an important modulator of neutrophil function during pregnancy suggesting a critical role in regulating innate immune responses throughout gestation.

immunology↗

Dual apical methyltransferases orchestrate motility initiation in apicomplexan parasites

Apicomplexan parasites such as Toxoplasma gondii initiate motility through rapid, spatially confined cytoskeletal activation at their apical end. While calcium-, lipid-, and kinase-based signalling pathways have been partially elucidated, how these cues are translated into mechanical force remains unclear. Here, we uncover a dual methyltransferase mechanism that orchestrates this process. We identify PCKMT, a preconoidal ring-associated lysine methyltransferase, as an essential upstream regulator of motility. PCKMT anchors the actin nucleator Formin-1 (FRM1) at the conoid, enabling conoid protrusion and F-actin assembly. Loss of PCKMT abolishes FRM1 recruitment, blocks conoid extrusion, and arrests invasion and egress despite preserved conoid structure. In contrast, the apical methyltransferase AKMT, previously linked to motility through recruitment of the glideosome-associated connector (GAC), acts downstream, disengaging from the conoid upon activation and likely promoting GAC-dependent force transmission. PCKMT depletion prevents AKMT translocation, revealing that actin assembly and lysine methylation are mechanistically coupled. Together, these findings define a two-step methylation checkpoint that coordinates actin nucleation with force propagation, uncovering methylation as a central regulatory axis for motility initiation in apicomplexan parasites.

microbiology↗

A time-resolved atlas of histone modifications during mitotic entry

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms--particularly those involving phosphorylation and deacetylation of specific histone residues--have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

cell biology↗

SCAMP3 is essential for proper formation and function of neutrophil granules

Host defense functions of neutrophils during infection critically depend on microbicidal and proteolytic proteins stored in primary, secondary and tertiary granules that are released into the phagosome or into the extracellular space upon degranulation. Granules are generated during granulopoiesis and impaired granule production or granule protein sorting has been linked to inefficient pathogen clearance resulting in recurrent bacterial and fungal infections. Here, we studied the role of the membrane protein secretory carrier associated membrane protein 3 (SCAMP3) for neutrophil defense functions. We generated Scamp3 knockout (KO) Hoxb8 cells and found that killing of Escherichia coli by Scamp3 KO Hoxb8 cell-derived neutrophils (dHoxb8 cells) was compromised as compared to control dHoxb8 cells in vitro. Mass spectrometric and Western blot analyses revealed a significant reduction of primary, secondary, and tertiary granule proteins in the genetic absence of Scamp3, resulting in a reduced overall granularity of these cells. Accordingly, degranulation was reduced in Scamp3 KO dHoxb8 cells compared to control dHoxb8 cells. Similarly, SCAMP3 deficiency in zebrafish resulted in reduced neutrophil granularity in comparison to wild-type animals. However, neutrophil migration towards sites of E. coli infection was unaffected in scamp3 KO zebrafish larvae. In summary, SCAMP3 represents an important novel player in granule equipment and degranulation, with key functions in neutrophil defense mechanisms during host-pathogen interactions in vitro. Brief summary sentence: The membrane protein secretory carrier associated membrane protein 3 (SCAMP3) is essential for proper protein equipment of neutrophil granules and Scamp3-deficient neutrophils have an impaired bacterial killing capacity in vitro.

physiology↗

Spatially resolved mapping of histones reveal selective neuronal response in Rett syndrome

Rett Syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in the X-linked MECP2 gene, results in profound life-long neurological dysfunction. RTT patients live an apparently normal initial life until 12-18 months of age following which, a progressive accumulation of a wide range of phenotypic manifestations sets in. While MeCP2 is known to regulate chromatin, its impact on global histone composition and dynamics remains poorly understood. Here, we combine mass spectrometry imaging (MSI) and laser capture microdissection (LCM) coupled to LC-MS/MS to systematically profile histone proteoforms in three key brain regions: the dentate gyrus (DG) and cornu ammonis (CA) of the hippocampus, and the cerebellum (Cb). Our analysis reveals striking neuron-specific differences in histone composition between Mecp2-deficient and wildtype (WT) mice. Interestingly, the expression of a pathogenic Mecp2 missense mutant (Y120D) results in subtler changes in histone composition that are distinct from the null mutations. This study provides the first spatially resolved epigenetic atlas of histone proteoforms in RTT and suggests that Mecp2 loss perturbs chromatin homeostasis in a neuron- and mutation-dependent manner. Our findings underscore the critical need for cell-type-resolved analyses to unravel the mechanistic underpinnings of RTT and emphasize the importance of personalised therapeutic strategies that consider both the affected cell-type and particular Mecp2 mutation.

neuroscience↗

Hangover regulates gene expression by limiting NSL-mediated H4K16 acetylation

The RNA-binding protein hangover is essential for several stress responses in Drosophila melanogaster. Here, we discover a novel function of hangover in the regulation of gene expression. Hangover binds to more than 2.000 genes in the Drosophila genome and modulates transcription. We identify a diverse set of chromatin regulators as hangover interactors, including NSL, dMec, Sin3A, dREAM and Ino80. Among these, the non-specific lethal complex (NSL) is the most prominent one. We show that hangover attenuates NSL-mediated H4K16 acetylation at transcriptional start sites to downregulate gene expression. Our work uncovers novel roles for hangover in epigenetic gene regulation and suggests that it coordinates the function of multiple chromatin regulators. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/649938v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@193e682org.highwire.dtl.DTLVardef@1028cb2org.highwire.dtl.DTLVardef@632bdforg.highwire.dtl.DTLVardef@d57617_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

A molecular switch at the yeast mitoribosomal tunnel exit controls cytochrome b synthesis

Mitochondrial gene expression needs to be balanced with cytosolic translation to produce oxidative phosphorylation complexes. In yeast, translational feedback loops involving lowly expressed proteins called translational activators help to achieve this balance. Synthesis of cytochrome b (Cytb or COB), a core subunit of complex III in the respiratory chain, is controlled by three translational activators and the assembly factor Cbp3-Cbp6. However, the molecular interface between the COB translational feedback loop and complex III assembly is yet unknown. Here, using protein-proximity mapping combined with selective mitoribosome profiling, we reveal the components and dynamics of the molecular switch controlling COB translation. Specifically, we demonstrate that Mrx4, a previously uncharacterized ligand of the mitoribosomal polypeptide tunnel exit, interacts with either the assembly factor Cbp3-Cbp6 or with the translational activator Cbs2. These reciprocal interactions determine whether the translational activator complex with bound COB mRNA can interact with the mRNA channel exit on the small ribosomal subunit for translation initiation. Organization of the feedback loop at the tunnel exit therefore orchestrates mitochondrial translation with respiratory chain biogenesis.

molecular biology↗

Digital Reprogramming Decodes Epigenetic Barriers of Cell Fate Changes

The fates of differentiated cells in our body can be induced to change by nuclear reprogramming. In this way, cells valuable for therapeutic purposes and disease modeling can be produced. However, the efficiency of this process is low, partly due to properties of somatic donor nuclei which stabilize their differentiated fate but also act as barriers reprogramming-associated cell fate changes. The identity of these reprogramming barriers is not fully understood. Here, we developed an artificial intelligence-based approach to model nuclear reprogramming and used it to identify the chromatin modification H3K27ac as an epigenetic barrier to reprogramming-induced cell fate changes. Using reprogramming by nuclear transfer (NT) to eggs of Xenopus laevis as a model system, we profiled chromatin modifications in differentiated cell types alongside gene expression patterns before and after reprogramming. Our model integrated the data and by leveraging model predictions, we find that genes resisting inactivation during reprogramming display chromatin modification barcodes. This revealed H3K27ac as a novel candidate barrier to NT reprogramming. Reducing H3K27ac levels using p300/CBP inhibitors before reprogramming led to an improved downregulation of genes linked to H3K27ac-modified enhancers after reprogramming. Importantly, these effects were accompanied by improved embryonic development of the resulting nuclear transfer embryos. In summary, our study identified H3K27ac as a safeguarding mechanism of cellular identities and as a reprogramming barrier during NT. Hence, the here-developed Digital Reprogramming" approach is capable of modelling and improving current cell-fate reprogramming strategies.

genomics↗

Chameau (HBO1) regulates starvation in a temperature-dependent manner in Drosophila melanogaster

The body temperature of Drosophila melanogaster depends on the extrinsic temperature. Numerous studies in fruit flies have shown that environmental temperature has an effect on metabolism, lifespan and starvation resilience. We have previously shown that Chameau (Chm), a MYST-domain acetyltransferase, promotes aging but also increases starvation resilience. As starvation resilience is highly temperature dependent, we explored the effect of temperature on starvation resilience in fruit flies with reduced chm expression. Strikingly, we found that an increase of 2{degrees}C was sufficient to restore starvation resilience in chm mutants. The increase in temperature rescued the dampened expression of genes involved in insulin, hormone and starvation response as well as a reduced rate of weight loss and misregulation of trehalose, which we observed in chm mutants at 23{degrees}C. Our data show that whereas Chm has an important role in regulating starvation at 23{degrees}C and below, it becomes obsolete at higher temperatures. Our finding that a gene plays an important role only under specific environmental conditions has important implications in light of the recent global change of climate conditions.

molecular biology↗

H3K4 methylation-promoted transcriptional memory ensures faithful zygotic genome activation and embryonic development

In the life of a vertebrate embryo, gene expression is initiated for the first time at zygotic genome activation (ZGA). Maternally expressed transcription factors present in the embryo are essential for this process. However, it is unknown if active chromatin modifications established in the gamete are propagated in the embryo as an epigenetic memory to support ZGA and embryonic development. Here, we provide evidence that in Xenopus laevis, H3K4 methylation provides an epigenetic memory of active chromatin states. We show that this is required for faithful zygotic genome activation and successful embryonic development. Chromatin configurations of promoters displaying high H3K4me3 intensity and breadth, alongside DNA hypomethylation and increased GC content, are propagated from the gametes to the embryo across multiple cell divisions and a transcriptionally quiescent phase in early development. We show that this transmission of H3K4 methylation is essential for precise zygotic genome activation and expression of key pioneer ZGA transcription factors Pou5f3.2 and Sox3. Finally, we demonstrate that the H3K4 methyltransferases Kmt2b and Cxxc1 ensure transcription-independent propagation of H3K4me3 and proper zygotic gene expression. In summary, our study establishes the role of H3K4 methylation in maintaining memory of active chromatin states in Xenopus embryos and reveals its importance for successful embryonic development.

developmental biology↗

Context-dependent epigenome rewiring during neuronal differentiation

Transcription factors (TFs) are pivotal in orchestrating lineage decisions and their binding is often accompanied by chromatin remodeling. However, a comprehensive understanding of how cellular and epigenetic contexts influence TF binding and the subsequent activation of lineage-specific programs remains lacking. Here, we dissected the molecular mechanisms underlying the binding of one such TF - Neurog2 and its functional consequences in two cell-types: mouse embryonic stem cells and neural progenitor cells. Our findings reveal that cell-type-specific chromatin accessibility and motif syntax are key determinants for TF binding. While Neurog2 binding primarily leads to chromatin opening, DNA demethylation and increased chromatin interactions, we also uncovered strong indirect, cell-type-specific effects, which ultimately result in vastly different epigenetic landscape. Furthermore, we identify shared and cell-type-specific Neurog2 interactors, including the SWI/SNF and NuRD complexes. Our study shed light on how cellular environment can modulate TF function to establish lineage identity during development.

genomics↗

An aberrant protamine ratio is associated with decreased H4ac levels in murine and human sperm

Protamine 2 (Prm2/PRM2), together with Protamine 1 (Prm1/PRM1), constitute the two protamines found in both murine and human sperm. During spermiogenesis in haploid male germ cells, chromatin undergoes significant condensation, a phase in which most histones are replaced by a species-specific ratio of these two protamines. Altered PRM1/PRM2 ratios are associated with subfertility and infertility in both male mice and men. Notably, during histone-to-protamine exchange a small fraction of histones remains (ranging from 1% to 15%) bound to DNA. The regulatory roles of these residual histones, governed by post-translational modifications (PTMs), play a pivotal role in spermatogenesis, particularly in chromatin remodeling and epigenetic regulation of genes during sperm differentiation or even in early embryogenesis. In this study, utilizing a Prm2-deficient mouse model and conducting an analysis of sperm samples from men exhibiting either normozoospermia or atypical spermiograms, we observed alterations in the methylation and acetylation profiles of histones H3 and H4. Subsequent in-depth analysis revealed that discrepancies in protamine ratios do not significantly influence the post-translational modifications (PTMs) of histones in testicular sperm. In epididymal sperm these altered protamine ratios are associated with a reduction in the acetylation levels of histone H4 (H4ac), a phenomenon consistent across both murine and human samples. In particular, H4K5ac and H4K12ac were identified as the two modifications that appear to decrease as a result of reduced Prm2/PRM2 levels. Our findings reveal that Protamine 2 is necessary for the maintenance of specific histone PTMs, such as acetylation, which is essential for proper spermatogenesis and particularly for chromatin remodeling.

molecular biology↗

The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry

Eukaryotic chromatin is organised in individual domains, which are defined by their location within the nuclear space, the molecular interactions within each other and their distinct proteomic compositions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To identify proteins associated with a specific chromatin domain and to better understand how those domains are established and maintained, we used a new method, which we termed AMPL-MS (Antibody mediated proximity labelling mass spectrometry). This method is based on antibodies and does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize chromatin domains containing specific signature proteins or histone modifications. We used AMPL-MS to characterize the composition of chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. As an outcome of this method, we show the importance of RNA in maintaining the integrity of these domains as treatment with RNAse alters their proteomic composition. Our data demonstrate the power of AMPL-MS to characterize the composition of non-membranous nuclear domains.

biochemistry↗

Decrypting the functional design of unmodified translation elongation factor P

Stalling of ribosomes during polypeptide synthesis due to consecutive proline motifs is a challenge faced by organisms across all kingdoms. To overcome this, bacteria employ translation elongation factor P (EF-P), while archaea and eukaryotes rely on a/eIF5A. Typically, these elongation factors become active only after undergoing post-translational modifications (PTMs) such as {beta}-lysinylation, (deoxy-)hypusinylation, rhamnosylation, or 5-aminopentanolyation. An exception to this rule is found in EF-P members of the PGKGP-subfamily, which remain unmodified. However, the mechanism behind the ability of certain bacteria to avoid metabolically and energetically costly PTMs, while retaining active EF-P, remains unclear. In this study, we investigated the design principles governing the full functionality of unmodified EF-Ps in Escherichia coli. We first screened for naturally unmodified EF-Ps that are active in an E. coli reporter strain. We identified EF-P from Rhodomicrobium vannielii capable of rescuing the growth deficiencies and changes in the proteome of an E. coli {Delta}epmA mutant lacking the gene for the modifying EF-P-(R)-{beta}-lysine ligase. We then identified specific amino acids in domain I of the unmodified EF-P variant that affected its activity. Ultimately, we transferred these functional properties to other marginally active members of the PGKGP EF-P subfamily, resulting in fully functional unmodified variants in E. coli. These results have implications for the improved heterologous expression of polyproline-containing proteins in E. coli and offer applications in other bacterial hosts. Understanding the mechanisms that underlie the functionality of unmodified EF-P provides insights into cellular adaptations to optimize protein synthesis.

microbiology↗

SAMD1 suppresses epithelial-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma

SAMD1 (SAM-domain containing protein 1), a CpG island-binding protein, plays a pivotal role in the repression of its target genes. Despite its significant correlation with outcomes in various tumor types, the role of SAMD1 in cancer has remained largely unexplored. In this study we focused on pancreatic ductal adenocarcinoma (PDAC) and revealed that SAMD1 acts as a repressor of genes associated with epithelial-mesenchymal transition (EMT). Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor of SAMD1. FBXO11 ubiquitinates SAMD1 within its DNA-binding winged helix domain and inhibits SAMD1 chromatin binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide new insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.

molecular biology↗