Biology subjects

Gallardo, A.

Publications and source records attributed to Gallardo, A..

11 recordsLinked to original sources

bioRxiv2025

OGDHL regulates nucleotide metabolism, tumor growth, and neuroendocrine marker expression in prostate cancer

As cancer cells evade therapeutic pressure and adopt alternate lineage identities not commonly observed in the tissue of origin, they likely adopt alternate metabolic programs to support their evolving demands. Targeting these alternative metabolic programs in distinct molecular subtypes of aggressive prostate cancer may lead to new therapeutic approaches to combat treatment-resistance. We identify the poorly studied metabolic enzyme Oxoglutarate Dehydrogenase-Like (OGDHL), named for its structural similarity to the tricarboxylic acid (TCA) cycle enzyme Oxoglutarate Dehydrogenase (OGDH), as an unexpected regulator of tumor growth, treatment-induced lineage plasticity, and DNA Damage in prostate cancer. While OGDHL has been described as a tumor-suppressor in various cancers, we find that its loss impairs prostate cancer cell proliferation and tumor formation. Loss of OGDHL profoundly alters Androgen Receptor inhibition-induced plasticity, including suppressing the neuroendocrine markers DLL3 and HES6, induces accumulation of the DNA damage response marker {gamma}H2AX, and reduces nucleotide synthesis. Our data suggest that OGDHL has minimal impact on TCA cycle activity, and that mitochondrial localization is not required for its regulation of prostate cancer plasticity and nucleotide metabolism. Finally, we demonstrate that OGDHL expression is tightly correlated with neuroendocrine differentiation in clinical prostate cancer. These findings underscore the importance of investigating poorly characterized metabolic genes as potential regulators of distinct molecular subtypes of aggressive cancer.

cancer biology↗

bioRxiv2024

BMAL1 represses transposable elements independently of CLOCK in pluripotent cells

Circadian oscillations of gene transcripts rely on a negative feedback loop executed by the activating BMAL1-CLOCK heterodimer and its negative regulators PER and CRY. Although circadian rhythms and CLOCK protein are mostly absent during embryogenesis, the lack of BMAL1 during prenatal development causes an early aging phenotype during adulthood, suggesting that BMAL1 carries out an unknown non-circadian function during organism development that is fundamental for healthy adult life. Here, we show that BMAL1 interacts with TRIM28 and represses transcription of totipotency-associated MERVL retrotransposons in mouse pluripotent cells. Deletion of Bmal1 leads to genome-wide upregulation of MERVLs, changes in the three-dimensional organization of the genome, and acquisition of totipotency-associated features. Overall, we demonstrate that in pluripotent cells BMAL1 is redeployed as a transcriptional repressor of transposable elements (TEs) in a CLOCK-independent way. We propose that BMAL1-TRIM28 activity during prenatal life is essential for optimal health and life span in mammals.

molecular biology↗

bioRxiv2023

Binding of Small Molecule Inhibitors to RNA Polymerase-Spt5 Complex Impacts RNA and DNA Stability

Spt5 is an elongation factor that associates with RNA polymerase II (Pol II) during transcription and has important functions in promoter-proximal pausing and elongation processivity. Spt5 was also recognized for its roles in the transcription of expanded-repeat genes that are related to neurodegenerative diseases. Recently, a set of Spt5-Pol II small molecule inhibitors (SPIs) were reported, which selectively inhibit mutant huntingtin gene transcription. Inhibition mechanisms as well as interaction sites of these SPIs with Pol II and Spt5 are not entirely known. In this study, we predicted the binding sites of three selected SPIs at the Pol II-Spt5 interface by docking and molecular dynamics simulations. Two molecules out of three demonstrated strong binding with Spt5 and Pol II, while the other molecule was more loosely bound and sampled multiple binding sites. Strongly bound SPIs indirectly affected RNA and DNA dynamics at the exit site as DNA became more flexible while RNA was stabilized by increased interactions with Spt5. Our results suggest that the transcription inhibition mechanism induced by SPIs can be related to Spt5-nucleic acid interactions, which were altered to some extent with strong binding of SPIs.

biophysics↗

bioRxiv2023

EZH2 represses mesenchymal genes and upholds epithelial identity in breast cancer cells

Emerging studies support that the Polycomb Repressive Complex 2 (PRC2) regulates phenotypic changes of carcinoma cells by modulating their shifts among metastable states within the epithelial and mesenchymal spectrum. This new role of PRC2 in cancer has been recently proposed to stem from the ability of its catalytic subunit EZH2 to bind and modulate the transcription of mesenchymal genes during epithelial-mesenchymal transition (EMT) in lung cancer cells. Here, we asked whether this mechanism is conserved across different types of carcinomas. By combining TGF-{beta}-mediate reversible induction of epithelial to mesenchymal transition and pharmacological inhibition of EZH2 activity we demonstrate that EZH2 represses a large set of mesenchymal genes and favours the residence of breast cancer cells towards the more epithelial spectrum during EMT. In agreement, analysis of human patient samples support that EZH2 is required to efficiently repress mesenchymal genes in breast cancer tumours. Our results indicate that PRC2 operates through similar mechanisms in breast and lung cancer cells. We propose that PRC2-mediated direct transcriptional modulation of the mesenchymal gene expression program is a conserved molecular mechanism underlying cell dissemination across human carcinomas.

cancer biology↗

bioRxiv2022

Polycomb repression during S/G2 phases restrain initiation of cell differentiation to the G1 phase of the cell cycle

The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we asked whether the Polycomb Repressive Complex 1 (PRC1) modulates the ability of mouse embryonic stem cells (ESCs) to differentially respond to developmental cues depending on the phase of the cell cycle in which they are found. We discovered that recruitment of PRC1 complexes and their associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit Ring1b. Importantly, depletion of Ring1b during retinoic acid stimulation interferes with the preference of mESCs to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to developmental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.

molecular biology↗

bioRxiv2022

The global distribution and environmental drivers of the soil antibiotic resistome

BackgroundLittle is known about the global distribution and environmental drivers of key microbial functional traits such as antibiotic resistance genes (ARGs). Soils are one of Earths largest reservoirs of ARGs, which are integral for soil microbial competition, and have potential implications for plant and human health. Yet, their diversity and global patterns remain poorly described. Here, we analyzed 285 ARGs in soils from 1012 sites across all continents, and created the first global atlas with the distributions of topsoil ARGs. ResultsWe show that ARGs peaked in high latitude cold and boreal forests. Climatic seasonality and mobile genetic elements, associated with the transmission of antibiotic resistance, were also key drivers of their global distribution. Dominant ARGs were mainly related to multidrug resistance genes and efflux pump machineries. We further pinpointed the global hotspots of the diversity and proportions of soil ARGs. ConclusionsTogether, our work provides the foundation for a better understanding of the ecology and global distribution of the environmental soil antibiotic resistome.

ecology↗

bioRxiv2022

EZH2 endorses cell plasticity to carcinoma cells facilitating mesenchymal to epithelial transition and tumour colonization

Reversible transition between the epithelial and mesenchymal states are key aspects of carcinoma cell dissemination and the metastatic disease, and thus, characterizing the molecular basis of the epithelial to mesenchymal transition (EMT) is crucial to find druggable targets and more effective therapeutic approaches in cancer. Emerging studies suggest that epigenetic regulators might endorse cancer cells with the cell plasticity required to conduct dynamic changes in cell state during EMT. However, epigenetic mechanisms involved remain mostly unknown. Polycomb Repressive Complexes (PRCs) proteins are well-established epigenetic regulators of development and stem cell differentiation, but their role in different cancer systems is inconsistent and sometimes paradoxical. In this study, we have analysed the role of the PRC2 protein EZH2 in lung carcinoma cells. We found that besides its described role in CDKN2A-dependent cell proliferation, EZH2 upholds the epithelial state of cancer cells by repressing the transcription of hundreds of mesenchymal genes. Chemical inhibition or genetic removal of EZH2 promotes the residence of cancer cells in the mesenchymal state during reversible epithelial-mesenchymal transition. In fitting, analysis of human patient samples and tumour xenograft models indicate that EZH2 is required to efficiently repress mesenchymal genes and facilitate tumour colonization in vivo. Overall, this study discloses a novel role of PRC2 as a master regulator of EMT in carcinoma cells. This finding has important implications for the design of therapies based on EZH2 inhibitors in human cancer patients.

cancer biology↗

bioRxiv2022

Protein-Nucleic Acid Interactions for RNA Polymerase II Elongation Factors by Molecular Dynamics Simulations

RNA polymerase II (Pol II) forms a complex with elongation factors to proceed the elongation stage of the transcription process. In this work, we studied elongation factor SPT5 and explored protein nucleic acid interactions for the isolated systems of KOW1 and KOW4 domains of SPT5 with DNA and RNA, respectively. We performed molecular dynamics (MD) simulations using three commonly used force fields that are CHARMM c36m, AMBER ff14sb and ff19sb. These simulations showed that most of the protein-nucleic acid interactions in the native state were retained with an increased electrostatic binding free energy for all force fields used. RNA was found highly dynamic with all force fields while DNA had relatively more stable conformations with the AMBER force fields compared to CHARMM. Furthermore, we performed MD simulations of the complete elongation complex using CHARMM c36m force field to compare the dynamics and interactions in the isolated systems. Similar strong KOW1 and DNA interactions were observed in the complete elongation complex simulations and DNA was further stabilized by a network of interactions involving SPT5-KOW1, SPT4 and rpb2 of Pol II. Overall, our study showed that the accuracy of force fields and the presence of the entire interaction network are important for elucidating the dynamics of protein-nucleic acid systems.

biophysics↗

bioRxiv2020

Temperature increases soil respiration across ecosystem types and soil development, but soil properties determine the magnitude of this effect

Soil carbon losses to the atmosphere, via soil heterotrophic respiration, are expected to increase in response to global warming, resulting in a positive carbon-climate feedback. Despite the well-known suite of abiotic and biotic factors controlling soil respiration, much less is known about how the magnitude of soil respiration responses to temperature changes over soil development and across contrasting soil properties. Here, we investigated the role of soil development stage and soil properties in driving the responses of soil heterotrophic respiration to increasing temperatures. We incubated soils from eight chronosequences ranging in soil age from hundreds to million years, and encompassing a wide range of vegetation types, climatic conditions, and chronosequences origins, at three assay temperatures (5, 15 and 25{degrees}C). We found a consistent positive effect of assay temperature on soil respiration rates across the eight chronosequences evaluated. However, soil properties such as organic carbon concentration, texture, pH, phosphorus content, and microbial biomass determined the magnitude of temperature effects on soil respiration. Finally, we observed a positive effect of soil development stage on soil respiration that did not alter the magnitude of assay temperature effects. Our work reveals that key soil properties alter the magnitude of the positive effect of temperature on soil respiration found across ecosystem types and soil development stages. This information is essential to better understand the magnitude of the carbon-climate feedback, and thus to establish accurate greenhouse gas emission targets.

ecology↗

bioRxiv2020

Mobilization of a Kluyvera-like arnBCADTEF operon, besides mcr genes, confers colistin resistance to Escherichia coli isolated from healthy animals

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB two-component system and also by some other(s) still unknown mechanism(s). This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine animals, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to control strains carrying mutations that confer colistin resistance. The expression of mcr genes negatively controls arnBCADTEF expression, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 60 Kb encoding an arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, named pArnT1, was cured by ethidum bromide and lost in parallel to colistin resistance. This work reveals that, besides mcr genes and chromosomal mutations, mobilization of arnBCADTEF operon represents a colistin resistance mechanism whose spread and relevance for public health should be carefully surveyed. Abstract ImportanceColistin is an old antibiotic that has returned to first-line fighting against (Gram negative) microorganisms after pandemic rising of antimicrobial resistance. However, low susceptibility to colistin is also becoming spread, mainly by plasmid mobilization of one of the enzymes (encoded by mcr genes) that modify covalently the external layer (the lipid A component of the lipopolysaccharide) of bacterial envelope, interfering antibiotic effectiveness. The second enzymatic system that performs envelope modification and confers colistin resistance when overexpressed is encoded by arnBCADTEF operon, a set of seven genes with location restricted (up to now) to the chromosome of Gram negative bacteria. This work describes plasmid mobilization of this operon between enterobacteria, from Kluyvera to Escherichia coli, where a Kluyvera-like arnBCADTEF operon carried by pArnT1 might represent, besides mcr genes, a potential risk for antimicrobial therapy and might require careful surveillance.

microbiology↗

bioRxiv2020

Polycomb regulation is coupled to cell cycle transition in pluripotent stem cells

When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifiers genes and cell differentiation is confined to the G1-phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are dramatically accumulated at target promoters during S and G2, while the transcriptionally activating subunits EPOP and EloB are enriched during G1. Importantly, fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 -/- mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G1.

molecular biology↗