Biology subjects

Roque, A.

Publications and source records attributed to Roque, A..

9 recordsLinked to original sources

bioRxiv2025

Differential regulation of histone H1 subtypes by N6-methyladenosine RNA methylation.

Histone H1 is involved in the regulation of chromatin structure and gene expression. Up to seven H1 subtypes or variants are expressed in human somatic cells. The H1 complement, defined as the subtype composition and proportions in each cell, is highly variable depending on the cell type, cell cycle stage, developmental context, and several diseases such as cancer. This variability results from the combined action of multiple regulatory processes. Epitranscriptomic modifications have emerged as a new regulatory layer capable of controlling all aspects of mRNA metabolism. In this work, we examined the role of the most prevalent mRNA modification, N6 methyladenosine (m6A), in the regulation of H1 subtypes. MeRIP-seq showed that H1-0 and H1-4 are enriched in m6A, whereas H1-2 displays intermediate levels of this mark. Integration of the functional studies involving m6A inhibition and partial depletion of these m6A readers led us to propose the first model of the differential regulation of H1 subtypes by m6A. In this model, m6A promotes the degradation of H1-0 mRNA mediated by YTHDF2, while it enhances the stability of H1-2 mRNA through IGF2BP1 binding and its translation by the combined action of IGF2BP1 and hnRNPD. In the case of H1-4, m6A promotes its transcription and stimulates its translation via hnRNPD binding. These findings suggest that m6A participates in the subtype-specific regulation of H1 variants, adding another layer to their complex regulation and contributing to the variability of the H1 complement in cancer. KeypointsO_LIHistone transcripts have different levels of m6A. C_LIO_LIWe analyzed the function of m6A in H1-0, H1-2, and H1-4 and identified its readers. C_LIO_LIWe propose the first model of the role of m6A in the subtype-specific regulation of histone H1. C_LI

molecular biology↗

bioRxiv2024

Antimicrobial and antibiofilm activity of human recombinant H1 histones against bacterial infections

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin and gentamicin. Our results demonstrate antimicrobial activity against of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa. ImportanceThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in reduction in biomass of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

microbiology↗

bioRxiv2024

Quantification of histone H1 subtypes using targeted proteomics.

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. Our preliminary data revealed differences in the H1 complement between responders and non-responder CML patients and suggest that the H1 content could help predicting imatinib response.

biochemistry↗

bioRxiv2023

Proteasome-dependent degradation of histone H1 subtypes is mediated by its C-terminal domain.

Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent, whereas the whole protein and its C-terminal domain can be degraded directly by the 20S proteasome. Our study shows that histone H1 protein levels are under tight regulation preventing its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome. StatementHistone H1 subtypes are a family of proteins involved in the regulation of chromatin structure. This work describes the degradation mechanism controlling the levels of histone H1 subtypes and the region within these proteins involved in the initial recognition. This regulatory mechanism protects the cell nucleus from the damaging effects of its accumulation.

molecular biology↗

bioRxiv2022

Emergence and clonal expansion in Europe of Vibrio aestuarianus lineages pathogenic for oysters

Crassostrea gigas oysters represent a significant global food source, with 4.7 million tons harvested per year. In 2001, the bacterium V. aesturianus francensis emerged as a pathogen that causes adult oyster mortality in France and Ireland. Its impact on oyster aquaculture has increased in Europe since its reemergence in 2012. To better understand the evolutionary mechanisms leading to the emergence and persistence over time of this pathogen, we conducted a survey of mollusk diseases through national reference laboratories (NRLs) across Europe. We analyzed 54 new genomes of V. aestuarianus (Va) isolated from multiple environmental compartments since 2001, in areas with and without bivalve mortalities. We used a combination of comparative genomics and population genetics approaches to show that Va francensis lineages have undergone clonal expansion in Europe, likely after a recent selective bottleneck. Low mutation and recombination rates may have selected particular virulent genotypes. Furthermore, we identified a specific cus-cop-containing island conferring copper resistance to Va francensis whose acquisition may have favored the emergence of pathogenic lineages adapted to oysters.

microbiology↗

bioRxiv2021

mRNA-decapping associated DcpS enzyme controls critical steps of neuronal development

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development, and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

neuroscience↗

bioRxiv2020

Microglial synaptic pruning on axon initial segment of dentate granule cells: sexually dimorphic effects on fear response of adult rats subjected to early life stress

Axon initial segments (AIS) of dentate granule cells (GC) in hippocampus exhibit prominent spines during early development that are associated with microglial contacts. Here, we asked if developmental changes in axon initial segment spines (AISS) could be modified by neonatal maternal separation through stress hormones and microglial activation and examined the potential behavioral consequences. We examined AISS densities at postnatal day (PND) 15 and 50, using Golgi-Cox staining and anatomical analysis. Neuron-microglial interaction was assessed using antibodies against ankyrinG, PSD95 and Iba1, for AIS, AISS and microglia, respectively, in normally reared and neonatal maternally separated (MS) male and female rats. We observed a higher density of AISS in MS groups at both PND15 and PND50 compared to control. Effects were more pronounced in female than in male rats. AIS-associated microglia showed a hyper-ramified morphology and less co-localization with PSD95 in MS compared to normally reared animals at PND 15. An MS-like alteration in microglial morphology and synaptic pruning could be produced ex vivo by vasopressin application in acute hippocampal slices from normally reared animals. MS rats exhibited increased freezing behavior during auditory fear memory testing which, like effects on AISS density, was more pronounced in females than males. Freezing behavior was associated with Fos expression in dorsal and ventral dentate GC. In summary, AIS associated microglial activity is altered by MS. Sex differences in the long-term effects of MS on AISS density are penetrant to a behavioral phenotype of increased stimulus reactivity in adult female subjects.

neuroscience↗

bioRxiv2020

Towards understanding the regulation of histone H1 somatic subtypes with OMICs.

BackgroundHistone H1 is involved in the regulation of chromatin higher-order structure and compaction. In humans, histone H1 is a multigene family with seven subtypes differentially expressed in somatic cells. Which are the regulatory mechanisms that determine the variability of the H1 complement is a long-standing biological question regarding histone H1. We have used a new approach based on the integration of OMICs data to address this question. ResultsWe have examined the 3D-chromatin structure, the binding of transcription factors (TFs), and the expression of somatic H1 genes in human cell lines, using data from public repositories, such as ENCODE. Analysis of Hi-C, ChIP-seq, and RNA-seq data, have shown that transcriptional control has a greater impact on H1 regulation than previously thought. Somatic H1 genes located in TADs show higher expression than in boundaries. H1 genes are targeted by a variable number of transcription factors including cell cycle-related TFs, and tissue-specific TFs, suggesting a finetuned, subtype-specific transcriptional control. We describe, for the first time, that all H1 somatic subtypes are under transcriptional co-regulation. The replication-independent subtypes, which are encoded in different chromosomes, isolated from other histone genes are also co-regulated with the rest of the somatic H1 genes, indicating that transcriptional co-regulation extends beyond the histone cluster. ConclusionsTranscriptional control and transcriptional co-regulation explain, at least in part, the variability of H1 complement, the fluctuations of H1 subtypes during development, and also the compensatory effects observed, in model systems, after perturbation of one or more H1 subtypes.

molecular biology↗

bioRxiv2016

THE CREATION OF A NEGATIVE SLOPE CONDUCTANCE REGION BY THE ACTIVATION OF THE PERSISTENT SODIUM CURRENT PROLONGS NEAR-THRESHOLD SYNAPTIC POTENTIALS

A change of the input resistance (Rin) of the neuron involves a change in the membrane conductances by opening and closing of ion channels. In passive membranes, i.e., membranes with only linear leak conductances, the increase or decrease of these conductances leads to a decrease or increase of the Rin and the membrane time constant ({tau}m). However, the presence of subthreshold voltage dependent currents can produce non-linear effects generating deviations from this relationship, especially the contradictory effect of negative conductances, as produced by the sodium-persistent current (INaP), on the Rin. In this work we aimed to analyze experimentally and theoretically the impact of the negative conductance produced by INaP on Rin. Experiments of whole-cell patch-clamp conducted in CA1 hippocampus pyramidal cells from brain slices showed a paradoxical voltage-dependent decrease of the Rin and the {tau}m in subthreshold membrane potentials close to the firing threshold after the perfusion with TTX, which inhibits INaP. This effect is postulated to be a result of the negative slope conductance in the subthreshold region produced by this conductance. The analysis of the experimental data, together with simulations found that the slope conductance of INaP is negative for subthreshold membrane potentials and its magnitude is voltage dependent in the same range observed for the voltage-dependence of Rin and {tau}m. The injection of an artificial INaP using dynamic-clamp in the presence of TTX restored the Rin and {tau}m to its original values. Additionally the injection of an artificial leak current with a negative conductance in the presence of TTX restored the Rin and {tau}m as the artificial Inap did. On the other hand, the injection of an artificial leak current with a positive conductance in the presence of TTX had no effect on the Rin and {tau}m. We conclude that INaP increases the Rin and {tau}m by the negative slope conductance observed in its non-monotonic I-V relationship. These results demonstrate that the effect of Inap on Rin and {tau}m is stronger in potentials near the firing threshold, which could potentiate the temporal summation of the EPSPs increasing their temporal integration and facilitating action potential firing. Because of its negative slope conductance, INaP is more effective in increasing excitability near threshold than a depolarizing leak current.

biophysics↗