Biology subjects

Lopes, M.

Publications and source records attributed to Lopes, M..

29 records · Page 2Linked to original sources

bioRxiv2022

Human Satellite 1 (HSAT1) analysis provides novel evidence of pericentromeric transcription

Pericentromeric regions of human chromosomes are composed of tandem-repeated and highly organized sequences named satellite DNAs. Although being known for a long time as the most AT-rich fraction of the human genome, classical satellite HSAT1 has been disregarded in genomic and transcriptional studies, falling behind other human satellites in terms of knowledge. The path followed herein trails with HSAT1 isolation and cloning, followed by in silico analysis. Monomer copy number and expression data was obtained in a wide variety of human cell lines, with greatly varying profiles in tumoral/non-tumoral samples. HSAT1 was mapped in human chromosomes and applied in in situ transcriptional assays. Additionally, it was possible to observe the nuclear organization of HSAT1 transcripts and further characterize them by 3 RACE-Seq. Size-varying polyadenylated HSAT1 transcripts were detected, which possibly accounts for the intricate regulation of alternative polyadenylation. As far as we know, this work pioneers HSAT1 transcription studies. With the emergence of new human genome assemblies, acrocentric pericentromeres are becoming relevant characters in disease and other biological contexts. HSAT1 sequences and associated noncoding RNAs will most certainly prove significant in the future of HSAT research.

genomics↗

bioRxiv2022

Large-scale expansions and replication stalling of Friedreich's ataxia GAA repeats in an experimental mammalian system

1Human disease Friedreichs ataxia (FRDA) is caused by large-scale expansions of (GAA)n repeats in the first intron of the FXN gene. While repeat expansions during intergenerational transmissions are causative for the disease development, somatic expansions additionally contribute to the disease progression. We and others have previously shown that (GAA)n repeats transiently pause the replication fork progression in cultured human cells. However, whether and by which mechanisms fork stalling underlies repeat expansions remained unclear. Here we developed a new genetically tractable experimental system to simultaneously analyze repeat-mediated fork stalling and large-scale repeat expansions in cultured human cells. It is based on a mammalian/yeast shuttle vector that can transiently replicate from the SV40 replication origin in human HEK-293T cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6; it also contains a cassette for selecting repeat expansions in yeast. Repeat expansions accumulate in mammalian cells and are then detected upon plasmid transformation into yeast. We found that large-scale expansions of (GAA)n repeats do occur in this experimental mammalian system. Further, we observed that repeat expansions frequency depends on several previously implicated proteins in replication fork stalling, reversal, and restart. These proteins include SHPRH, RAD52, ZRANB3, DDX11, SMARCAL1, HLTF, RECQ1 and WRN. Therefore, we propose that GAA repeat expansions might occur as a consequence of deregulated replication fork regression and restoration process.

genetics↗

bioRxiv2021

Stable Isotope Tracing In Vivo Reveals A Metabolic Bridge Linking The Microbiota To Host Histone Acetylation

The gut microbiota influences host epigenetics by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-CoA, a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.

biochemistry↗

bioRxiv2021

Fork Slowing and Reversal as an Adaptive Response to Chronic ATR Inhibition

Inhibitors of the replication stress response kinase ATR are currently being explored in anti-cancer therapy. Acute ATR inhibition is known to impair the proper control of origin firing, DNA repair, and cell cycle, resulting in DNA breaks and mitotic catastrophe. Less is understood about the effects of clinically relevant regimes of ATR inhibition, which involve chronic and low doses of ATR inhibitors (cATRi) to cells. Here we report unexpected molecular effects of cATRi on replication dynamics. cATRi strongly reduces fork speed but has minimal effects on the accumulation of DNA breaks or cell survival. cATRi promotes extensive fork reversal and RAD51- and PARP-mediated fork slowing that correlate with the accumulation of DNA-RNA hybrids. Our work shows that fork reversal is a critical adaptive response ensuring cell survival during cATRi and that the manipulation of fork reversal causes hypersensitivity to cATRi, increasing the effectiveness of ATR inhibitors in anti-cancer therapies.

cancer biology↗

bioRxiv2020

Diverse natural variants suppress mutations in hundreds of essential genes

The consequence of a mutation can be influenced by the context in which it operates. For example, loss of gene function may be tolerated in one genetic background, but lead to lethality in another. The extent to which mutant phenotypes are malleable, the complexity of the architecture of modifiers, and the identities of causal genes and pathways remain largely unknown. Here, we measure the fitness effects of ~1,500 temperature sensitive alleles of yeast essential genes in the context of variation from ten different natural genetic backgrounds, and map the modifiers for 19 combinations. Altogether, fitness defects for 183 of the 530 tested genes (35%) could be suppressed by standing genetic variation in at least one wild strain. Suppression was generally driven by gain-of-function of a single, strong modifier gene. The validated causes included both variants in protein interaction partners or pathway members suppressing specific genes, as well as general modifiers altering the effect of many temperature sensitive alleles. The emerging frequency of suppression and range of possible suppression mechanisms suggest that a substantial fraction of monogenic diseases could be repressed by modulating other gene products.

genetics↗

bioRxiv2020

Loss of erythroblasts in acute myeloid leukemia causes iron redistribution with clinical implications

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in AML patients at diagnosis and mechanisms involved using the syngeneic MLL-AF9-induced AML mouse model. We found that AML is a disorder with a unique iron profile not associated with inflammation or transfusion and characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that leukemic blasts take up iron and that the AML-induced loss of erythroblasts is responsible for iron redistribution and an increase in TSAT. We also show that elevated TSAT at diagnosis is independently associated with increased overall survival in AML and suggest that TSAT may be a relevant prognostic marker in AML.

cancer biology↗

bioRxiv2020

PROFILING OF Y1 CELLS TREATED WITH FGF-2 REVEALS PARALLELS WITH ONCOGENE-INDUCED SENESCENCE

Paradoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Along these lines, Y1 cells, which carry an amplification of Ras, become senescent after treatment with the mitogen FGF-2. To understand how FGF-2 promotes senescence, we profiled the epigenome, transcriptome, proteome, and phospho-proteome of Y1 cells stimulated with FGF-2. FGF-2 caused delayed acetylation of histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. In contrast, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. Altogether, the response of Y1 cells to FGF-2 is consistent with oncogene-induced senescence. We propose that Y1 cells enter senescence due to deficient cyclin expression and high levels of p21, which may stem from DNA damage or TGFb signaling.

systems biology↗

bioRxiv2020

PrimPol primase mediates replication traverse of DNA interstrand crosslinks

Interstrand crosslinks (ICLs) are DNA lesions frequently induced by chemotherapy that interfere with essential processes such as replication and transcription. ICL repair may be initiated by the convergence of two replication forks at the crosslink, which results in a termination-like DNA structure recognized and processed by the Fanconi Anemia (FA) pathway. An alternative possibility to generate a suitable substrate for ICL repair involves "ICL traverse", a DNA damage tolerance mechanism in which a single fork arriving at the ICL can skip the lesion and restart DNA synthesis from a downstream point. This reaction requires FANCM translocase, the BLM/TOP3A/RMI1-2 (BTR) complex and other factors. Here we report that PrimPol, the second primase-polymerase identified in mammalian cells after Pol/Primase, interacts with BTR and participates in the ICL traverse reaction. A functional complementation assay reveals that the primase activity of PrimPol is required, confirming the need for re-priming events during ICL traverse. Genetic ablation of PRIMPOL strongly impaired this tolerance mechanism, making cells more dependent on fork convergence to initiate ICL repair. PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs, opening the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

molecular biology↗

bioRxiv2019

THAP11F80L cobalamin disorder-associated mutation reveals normal and pathogenic THAP11 functions in gene expression and cell proliferation

Twelve human THAP proteins share the THAP domain, an evolutionary conserved zinc-finger DNA-binding domain. Studies of different THAP proteins have indicated roles in gene transcription, cell proliferation and development. We have analyzed this protein family, focusing on THAP7 and THAP11. We show that human THAP proteins possess differing homo- and heterodimer formation properties and interaction abilities with the transcriptional co-regulator HCF-1. HEK-293 cells lacking THAP7 were viable but proliferated more slowly. In contrast, HEK-293 cells were very sensitive to THAP11 alteration. Nevertheless, HEK-293 cells bearing a THAP11 mutation identified in a patient suffering from cobalamin disorder (THAP11F80L) were viable although proliferated more slowly. Cobalamin disorder is an inborn vitamin deficiency characterized by neurodevelopmental abnormalities, most often owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin (vitamin B12) metabolic pathway. We show that THAP11F80L selectively affected promoter binding by THAP11, having more deleterious effects on a subset of THAP11 targets, and resulting in altered patterns of gene expression. In particular, THAP11F80L exhibited a strong effect on association with the MMACHC promoter and led to a decrease in MMACHC gene transcription, suggesting that the THAP11F80L mutation is directly responsible for the observed cobalamin disorder.

cell biology↗

bioRxiv2018

Toll-Like Receptor-4 Disruption Suppresses Adipose Tissue Remodeling and Increases Survival During Cancer Cachexia Syndrome

Cancer-induced cachexia, characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling and muscle wasting, is a malignant metabolic syndrome with undefined etiology. Here, we show that Toll-like receptor 4 (TLR4) mediates AT remodeling, in particular, AT browning and inflammatory response in mice bearing Lewis lung carcinoma (LLC). LLC tumor-bearing (TB) TLR4-/- mice were spared from AT remodeling due to a reduced macrophage infiltration and adipocyte atrophy. TLR4-/- mice were also resistant to cold-induced browning of subcutaneous AT (scAT). Importantly, pharmacological inhibition of TLR4 reproduced the main protective effect against AT remodeling found in TLR4-/- TB mice. Moreover, the treatment was effective in prolonging the survival and attenuating tumor mass growth when compared to non-treated-TB animals. Further, tumor-induced elevation of circulating pro-inflammatory cytokines was similarly abolished in both genetic ablation and pharmacological inhibition of TLR4. These data suggest that TLR4 is a critical mediator and a promising therapeutic target for cancer-induced AT remodeling.\n\nHIGHLIGHTSO_LIGenetic ablation and pharmacological inhibition of TLR4 attenuate adipose tissue remodeling during cancer-associated cachexia;\nC_LIO_LITLR4 suppression play an essential role in the browning phenotype induced by cachexia;\nC_LIO_LIAdministration of TLR4 drug inhibitor increase survival and reduces tumor mass growth in tumor bearing mice;\nC_LIO_LITLR4 pathway is a promising target for cancer-cachexia therapeutic intervention.\nC_LI

cancer biology↗

bioRxiv2018

Unraveling rice tolerance mechanisms against Schizotetranychus oryzae mite infestation

Infestation of Schizotetranychus oryzae (Acari: Tetranychidae) causes great losses in rice productivity. Infestation in Puita INTA-CL cultivar reduced the number of seeds/plant, percentage of full seeds, 1,000 seeds weight, and seed length, whereas infestation in IRGA423 increased 1,000 seeds weight and seed length. Reduction in seed weight/plant caused by infestation was higher in Puita INTA-CL than IRGA423. Thus, Puita INTA-CL was established as susceptible, and IRGA423 as tolerant to S. oryzae infestation. Photosynthetic parameters were less affected by infestation in IRGA423 than in Puita INTA-CL. Infestation also caused accumulation of H2O2, decreased cell membrane integrity and accelerated senescence in leaves of Puita INTA-CL, while leaves of IRGA423 presented higher levels of phenolics compounds. Using proteomic analysis, we identified proteins related to plant defense, such as jasmonate synthesis, and related to other mechanisms of tolerance such as oxidative stress, photosynthesis, and DNA structure maintenance, more abundant in IRGA423 after seven days of infestation. We detected higher levels of silicon (as amorphous silica cells) in leaves of infested IRGA423 plants compared to Puita INTA-CL, an element previously linked to plant defense. Our data shows that IRGA423 presents tolerance to S. oryzae infestation, and that multiple mechanisms might be employed by this cultivar.\n\nHighlightThis is the first report evaluating the defense responses (tolerance and susceptiblity) of two contrasting rice cultivars to Schizotetranychus oryzae mite infestation.

plant biology↗