Biology subjects

Salas, M.

Publications and source records attributed to Salas, M..

9 recordsLinked to original sources

bioRxiv2024

Epigenetic changes regulating the epithelial-mesenchymal transition in human trophoblast differentiation

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial- mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome- wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

developmental biology↗

bioRxiv2022

Regulation of Nuclear Transcription by Mitochondrial RNA

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, which we termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

genomics↗

bioRxiv2021

Impact of protein conformational diversity on AlphaFold predictions

After the outstanding breakthrough of AlphaFold in predicting protein 3D models, new questions appeared and remain unanswered. The ensemble nature of proteins, for example, challenges the structural prediction methods because the models should represent a set of conformers instead of single structures. The evolutionary and structural features captured by effective deep learning techniques may unveil the information to generate several diverse conformations from a single sequence. Here we address the performance of AlphaFold2 predictions under this ensemble paradigm. Using a curated collection of apo-holo conformations, we found that AlphaFold2 predicts the holo form of a protein in 70% of the cases, being unable to reproduce the observed conformational diversity with an equivalent error than in the estimation of a single conformation. More importantly, we found that AlphaFold2s performance worsens with the increasing conformational diversity of the studied protein. This impairment is related to the heterogeneity in the degree of conformational diversity found between different members of the homologous family of the protein under study. Finally, we found that main-chain flexibility associated with apo-holo pairs of conformers negatively correlates with the predicted local model quality score plDDT, indicating that plDDT values in a single 3D model could be used to infer local conformational changes linked to ligand binding transitions.

bioinformatics↗

bioRxiv2021

Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach

AO_SCPLOWBSTRACTC_SCPLOWBacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level. AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWMembers of the family Tectiviridae, composed of non-tailed icosahedral, membrane-containing bacteriophages, have been increasingly scrutinized in recent years for their possible role in the origin of dsDNA viruses. In particular, the genus Betatectivirus receives increased attention as these phages can infect clinical strains as well as industrially relevant members of the B. cereus group. However, little is known about the interactions between these temperate viruses and their hosts. Here, we present the first high-throughput study of tectivirus-host protein-protein interactions focusing on Bam35, model virus of betatectiviruses, and its host B. thuringiensis, an important entomopathogenic bacterium. We adapted the well-known technique yeast-two-hybrid and integrated high-throughput sequencing and bioinformatics for the downstream analysis of the results which enables large-scale analysis of protein-protein interactions. In total, 182 detected interactions show an enrichment in host metabolic proteins and peptidases, in contrast with the current knowledge on host-phage PPIs. Specific host-viral protein-protein interactions were also detected enabling us to propose functions for uncharacterized proteins.

microbiology↗

bioRxiv2021

Unlimited cooperativity of Betatectivirus SSB, a novel DNA binding protein related to an atypical group of SSBs from protein-primed replicating bacterial viruses

Bam35 and related betatectiviruses are tail-less bacteriophages that prey on members of the Bacillus cereus group. These temperate viruses replicate their linear genome by a protein-primed mechanism. In this work, we have identified and characterized the product of the viral ORF2 as a single-stranded DNA binding protein (hereafter B35SSB). B35SSB binds ssDNA with great preference over dsDNA or RNA in a sequence-independent, highly cooperative manner that results in a non-specific stimulation of DNA replication. We have also identified several aromatic and basic residues, involved in base-stacking and electrostatic interactions, respectively, that are required for effective protein-ssDNA interaction. Although SSBs are essential for DNA replication in all domains of life as well as many viruses, they are very diverse proteins. However, most SSBs share a common structural domain, named OB-fold. Protein-primed viruses could constitute an exception, as no OB-fold DNA binding protein has been reported. Based on databases searches as well as phylogenetic and structural analyses, we showed that B35SSB belongs to a novel and independent group of SSBs. This group contains proteins encoded by protein-primed viral genomes from unrelated viruses, spanning betatectiviruses and {Phi}29 and close podoviruses, and they share a conserved pattern of secondary structure. Sensitive searches and structural predictions indicate that B35SSB contains a conserved domain resembling a divergent OB-fold, which would constitute the first occurrence of an OB-fold-like domain in a protein-primed genome. HighlightsO_LIBam35 ORF 2 product encodes a viral single-stranded DNA binding protein (B35SSB). C_LIO_LIB35SSB binds ssDNA in a highly cooperative manner but with no sequence specificity. C_LIO_LIB35SSB-ssDNA binding is mediated by base-stacking and ionic interactions. C_LIO_LIBam35 and {Phi}29-related SSBs form a novel group of SSBs from protein-primed viruses. C_LIO_LIThe B35-{Phi}29 SSBs group shares a highly divergent OB-fold-like domain. C_LI

biochemistry↗

bioRxiv2020

The fully resolved genome of Bacillus thuringiensis HER1410 reveals a cry-containing chromosome, two megaplasmids & an integrative plasmidial prophage

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been resolved, while the other draft genomes are incomplete, precluding plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-reads techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368kb), a likely conjugative plasmid involved in virulence, and pLUSID2 potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome, causes the disruption of a flagellar key component. Finally, phylogenetic analysis located this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B.cereus s. s. This highlights the intermingled taxonomy of B. cereus sensu lato group, where genomics alone does not support the present taxonomy between B. cereus s. s. and B. thuringiensis as species designation currently relies solely on the presence of entomocidal genes. ImportanceBacillus cereus group species have been extensively studied due to their economical and clinical relevance. This importance originally set the basis for B. cereus group members classification which are commonly based on phenotypical criteria. Sequencing era has shed light about genomic characterization of these species, showing their chromosomal genomic similarity and highlighting the role of mobile genetic elements, especially megaplasmids, in the classification and characterization of this group. However, only the 5% of the sequenced B. thuringiensis genomes have been fully resolved. Thus, here we addressed efficiently the study B. thuringiensis HER1410 genomic features by the use of a combination of short and long-reads sequencing. This methodology resulted in the high-quality assembly, which led to the identification of an uncommon location of a cry gene close to the chromosomal origin, as well as three fully resolved extrachromosomal elements, two megaplasmids and an integrative plasmidial prophage.

microbiology↗

bioRxiv2020

High diversity and variability of pipolins among a wide range of pathogenic Escherichia coli strains

Self-synthesizing transposons are integrative mobile genetic elements (MGEs) that encode their own B-family DNA polymerase (PolB). Discovered a few years ago, they are proposed as key players in the evolution of several groups of DNA viruses and virus-host interaction machinery. Pipolins are the most recent addition to the group, are integrated in the genomes of bacteria from diverse phyla and also present as circular plasmids in mitochondria. Remarkably, pipolins-encoded PolBs are proficient DNA polymerases endowed with DNA priming capacity, hence the name, primer-independent PolB (piPolB). We have now surveyed the presence of pipolins in a collection of 2238 human and animal pathogenic Escherichia coli strains and found that, although detected in only 25 new isolates (1.1%), they are present in E. coli strains from a wide variety of pathotypes, serotypes, phylogenetic groups and sequence types. Overall, the pangenome of strains carrying pipolins is highly diverse, despite the fact that a considerable number of strains belongs to only three clonal complexes (CC10, CC23 and CC32). Comparative analysis with a set of 67 additional pipolin-harboring strains from GenBank further confirmed these results. The genetic structure of pipolins shows great flexibility and variability, with the piPolB gene and the attachment sites being the only common features. Most pipolins contain one or more recombinases that would be involved in excision/integration of the element in the same conserved tRNA gene. This mobilization mechanism might explain the apparent incompatibility of pipolins with other integrative MGEs such as integrons. In addition, analysis of cophylogeny between pipolins and pipolin-harboring strains showed a lack of congruence between several pipolins and their host strains, in agreement with horizontal transfer between hosts. Overall, these results indicate that pipolins can serve as a vehicle for genetic transfer among circulating E. coli and possibly also among other pathogenic bacteria.

microbiology↗

bioRxiv2019

Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs

BackgroundMapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). However, the advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified. ResultsWe performed whole genome methyl-seq on diverse normal cells and tissues and three types of cancers (multiple myeloma, lymphoma, glioblastoma multiforme). After excluding imprinting, the data pinpointed 15,114 high-confidence ASM differentially methylated regions (DMRs), of which 1,842 contained SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies were increased 5 to 9-fold in cancers vs. matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancers showed increased allele switching at ASM loci, but disruptive SNPs in specific classes of CTCF and transcription factor (TF) binding motifs were similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations affecting these same motif classes tracked with de novo ASM in the cancers. Allele-specific TF binding from ChIP-seq was enriched among ASM loci, but most ASM DMRs lacked such annotations, and some were found in otherwise uninformative "chromatin deserts". ConclusionsASM is increased in cancers but occurs by a shared mechanism involving disruptive SNPs in CTCF and TF binding sites in both normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, neuropsychiatric, and neoplastic diseases. Custom genome browser tracks with annotated ASM loci can be viewed at a UCSC browser session hosted by our laboratory (https://bit.ly/tycko-asm)

genomics↗

bioRxiv2018

Genetic variants influence on the placenta regulatory landscape

BackgroundFrom genomic association studies, quantitative trait loci analysis, and epigenomic mapping, it is evident that significant efforts are necessary to define genetic-epigenetic interactions and understand their role in disease susceptibility and progression. For this reason, an analysis of the effects of genetic variation on gene expression and DNA methylation in human placentas at high resolution and whole-genome coverage will have multiple mechanistic and practical implications.\n\nResultsBy producing and analyzing DNA sequence variation (n=303), DNA methylation (n=303) and mRNA expression data (n=80) from placentas from healthy women, we investigate the regulatory landscape of the human placenta and offer analytical approaches to integrate different types of genomic data and address some potential limitations of current platforms. We distinguish two profiles of interaction between expression and DNA methylation, revealing linear or bimodal effects, reflecting differences in genomic context, transcription factor recruitment, and possibly cell subpopulations.\n\nConclusionsThese findings help to clarify the interactions of genetic, epigenetic, and transcriptional regulatory mechanisms in normal human placentas. They also provide strong evidence for genotype-driven modifications of transcription and DNA methylation in normal placentas. In addition to these mechanistic implications, the data and analytical methods presented here will improve the interpretability of genome-wide and epigenome-wide association studies for human traits and diseases that involve placental functions.\n\nAuthor summaryThe placenta is a critical organ playing multiple roles including oxygen and metabolite transfer from mother to fetus, hormone production, and vascular perfusion. With this study, we aimed to deliver a placenta-specific regulatory map based on a combination of publicly available and newly generated data. To complete this reference, we obtained genotype information (n=303), DNA methylation (n=303) and expression data (n=80) for placentas from healthy women. Our analysis of methylation and expression quantitative trait loci (QTLs) and correlations between methylation and expression data were designed to identify fundamental associations between genome, transcriptome, and epigenome in this key fetal organ. The results provide high-resolution genetic and epigenetic maps specific to the placenta based on a representative ethnically diverse cohort. As interest and efforts are growing to better understand the etiology of placental disease and the impact of the environment on placental function these data will provide a reference and enhance future investigations.

genomics↗