Biology subjects

Gonzalez, I.

Publications and source records attributed to Gonzalez, I..

14 recordsLinked to original sources

bioRxiv2024

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair for somatic benchmarks

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first to be explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from seventeen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, and Hi-C, and karyotyping. In future publications, these data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic mutations. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

genomics↗

bioRxiv2024

Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

Pioneer transcription factors (TF) bind nucleosome-embedded DNA motifs to activate new regulatory elements and promote differentiation. However, the complexity, binding dependencies and temporal effects of their action remain unclear. Here, we dissect how the pioneer TF GATA6 triggers Primitive Endoderm (PrE) differentiation from pluripotent cells. We show that transient GATA6 binding exploits accessible regions to decommission active enhancers and promote pluripotency gene silencing. Simultaneously, GATA6 targets closed chromatin and initiates an extensive remodeling culminating in the establishment of fragile nucleosomes flanked by ordered nucleosome arrays and increased accessibility. This is directly enhanced by rapidly expressed PrE TFs (SOX17) and by pluripotency TFs repurposed for differentiation (OCT4/SOX2). Furthermore, GATA6 mediates the replacement of essential nuclear receptors for PrE differentiation, from ESRRB to ESRRA. Therefore, pioneer TFs orchestrate a complex gene regulatory network involving many if not all available pioneer TFs, including those required to support the original identity of differentiating cells.

molecular biology↗

bioRxiv2023

Mitotic chromosomes harbor cell type and species-specific structural features within a universal looping architecture

The architecture of mammalian mitotic chromosomes is considered to be universal across species and cell types. However, some studies suggest that features of mitotic chromosomes might be cell type or species specific. We previously reported that CTCF binding in human differentiated cell lines is lost in mitosis, whereas mouse embryonic stem cells (mESC) display prominent binding at a subset of CTCF sites in mitosis. Here, we perform parallel footprint ATAC-seq data analyses of mESCs and somatic mouse and human cells to further explore these differences. We then investigate roles of mitotically bound (bookmarked) CTCF in prometaphase chromosome organization by Hi-C. We do not find any remaining interphase structures such as TADs or CTCF loops at mitotically bookmarked CTCF sites in mESCs. This suggests that mitotic loop extruders condensin I and II are not blocked by bound CTCF, and thus that any remaining CTCF binding does not alter mitotic chromosome folding. Lastly, we compare mitotic Hi-C data generated in this study in mouse with publicly available data from human and chicken cell lines. We do not find any cell type specific differences; however, we do find a difference between species. The average genomic size of mitotic loops is much smaller in chicken (200-350 kb), compared to human (500-750 kb) and mouse (1-2 mb). Interestingly, we find that this difference in loop size is correlated with the average genomic length of the q-arm in these species, a finding we confirm by microscopy measurements of chromosome compaction. This suggests that the dimensions of mitotic chromosomes can be modulated through control of sizes of loops generated by condensins to facilitate species-appropriate shortening of chromosome arms.

genomics↗

bioRxiv2023

MYC and MAX drive the reactivation of the genome after mitosis

Shortly after cell division, a robust wave of hyper-transcription reactivates the genome1-3. This phenomenon is particularly pronounced in pluripotent cells4, which necessitate rapid transcrip-tome reactivation to maintain their undifferentiated state and prevent premature differentiation. While recent work has illuminated how specific groups of genes are reactivated4-8, the mechanisms enabling the global, efficient and accurate post-mitotic reactivation of the genome remain unknown. Here we elucidate the direct involvement of the MYC/MAX transcription factors in the post-mitotic reactivation of pluripotent mouse embryonic stem cells. While MYC undergoes extensive phosphorylation and largely dissociates from its DNA binding sites during mitosis, we report that MAX remains bound to its targets, preferentially at promoters, and facilitates early recruitment of MYC following mitosis. Through the application of MYC/MAX heterodimerization inhibitors, we demonstrate their indispensable role in sustaining hyper-transcription in ES cells, including during the critical transition from mitosis to G1 phase. Our findings uncover a novel role for MAX in mitotic book-marking, highlighting its pivotal role in post-mitotic MYC recruitment and the re-establishment of high global transcription levels. These findings hold significant implications for medically relevant contexts, particularly when cell proliferation is of paramount importance9. We anticipate that the study of mitotic bookmarking by MYC and MAX and of the effects of anticancer drugs targeting MYC/MAX interactions in such process10-12 will be relevant for our understanding of cancer and its potential treatments.

molecular biology↗

bioRxiv2023

Nr5a2 is essential for morula development

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. While the TFs specifying the blastocysts lineages are well characterised, those playing earlier roles are ill-defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit overt phenotypical problems such as altered lineage specification, frequent mitotic failure and substantial chromosome segregation defects. Transcriptomic profiling shows that NR5A2 is a master regulator required for appropriate expression of thousands of genes at the 8-cells stage, including lineage-specifying TFs and genes involved in mitosis, telomere maintenance and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability and lineage specification to ensure proper morula development.

developmental biology↗

bioRxiv2022

Drug Resistance Mutations In Transplant Recipients With Suspected Resistance

Resistant CMV infections are challenging complications after SOT and HSCT. Prompt recognition of ARMs is imperative for appropriate therapy. 108 plasma samples from 96 CMV+ transplant recipients with suspected resistance were analysed in CNM in a retrospective nationwide study from January 2018 to July 2022 for resistance genotyping. ARMs in UL97 and UL54 were found in 26.87 % (18/67) and 10.60 % (7/66) of patients, respectively. Patients ARM distribution in UL97 was as follows: L595S n=3; L595S/M460I n=1; L595S/N510S n=1; L595W n=1; C603W n=4; A594V n=3; A594E n=1; C607Y n=1; L397R/T409M/H411L/M460I n=1; L397I n=1; H520Q n=1; four patients showed ARMs in UL54 as well (F412C n=1; T503I n=2; P522S n=1), whereas three patients exhibited ARMs in UL54 only (L501I/T503I/L516R/A834P n=1; A987G n=2). L516R in UL54 and L397R/I and H411L in UL97 have been found for the first time in a clinical sample. L595S/W was the most prevalent ARM found to lend resistance to GCV. In UL54 all ARMs lent resistance to GCV and CDV. In addition, A834P, found in one patient, also lent resistance to FOS. CMV load did not differ significantly in patients with or without ARMs, and no differences were found either between patients with ARMs in UL97 or in UL97 and UL54. Despite extensive use of classical antivirals for the treatment of CMV infection after HSCT and SOT, ARMs occurred mainly in viral UL97 kinase, which suggests that CDV and mostly FOS continue to be useful alternatives to nucleoside analogues after genotypic detection of ARMs.

microbiology↗

bioRxiv2022

Y chromosome sequence and epigenomic reconstruction across human populations

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for 7 major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications amongst the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.

genomics↗

bioRxiv2021

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage ([≥] 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

biochemistry↗

bioRxiv2020

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9

Autophagy is a cellular degradation pathway essential for neuronal health and function. Autophagosome biogenesis occurs at synapses, is locally regulated and increases in response to neuronal activity. The mechanisms that couple autophagosome biogenesis to synaptic activity remain unknown. In this study we determine that trafficking of ATG-9, the only transmembrane protein in the core autophagy pathway, links the synaptic vesicle cycle with autophagy. ATG-9 positive vesicles in C. elegans are generated from the trans-Golgi network via AP3-dependent budding, and delivered to presynaptic sites. At presynaptic sites, ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt endocytosis, including one associated with Parkinsons disease, result in abnormal ATG-9 accumulation at clathrin-rich synaptic foci and defects in activity-dependent presynaptic autophagy. Our findings uncover regulated key steps of ATG-9 trafficking at presynaptic sites, and provide evidence that ATG-9 exo-endocytosis couples autophagosome biogenesis at presynaptic sites with the activity-dependent synaptic vesicle cycle. HighlightsO_LIIn C. elegans, ATG-9 is delivered to presynaptic sites in vesicles generated from the trans-Golgi network via AP-3-dependent budding C_LIO_LIATG-9 vesicles undergo activity-dependent exo-endocytosis at presynaptic sites C_LIO_LIMutations in endocytic proteins, including a mutation associated with Parkinsons disease, result in abnormal ATG-9 accumulation at clathrin-rich foci C_LIO_LIAbnormal accumulation of ATG-9 at clathrin-rich foci is associated with defects in activity-dependent presynaptic autophagy C_LI

neuroscience↗

bioRxiv2019

Finding differentially expressed sRNA-Seq regions with srnadiff

Small RNAs (sRNAs) encompass a great variety of different molecules of different kinds, such as micro RNAs, small interfering RNAs, Piwi-associated RNA, among other. These sRNA have a wide range of activities, which include gene regulation, protection against virus, transposable element silencing, and have been identified as a key actor to study and understand the development of the cell. Small RNA sequencing is thus routinely used to assess the expression of the diversity of sRNAs, usually in the context of differentially expression, where two conditions are compared. Many tools have been presented to detect differentially expressed micro RNAs, because they are well documented, and the associated genes are well defined. However, tools are lacking to detect other types of sRNAs, which are less studied, and have an imprecise \"gene\" structure. We present here a new method, called srnadiff, to find all kinds of differentially expressed sRNAs. To the extent of our knowledge, srnadiff is the first tool that detects differentially expressed sRNAs without the use of external information, such as genomic annotation or reference sequence of sRNAs.\n\nAuthor summaryWe present here a new method for the ab initio discovery of differentially expressed small RNAs. The standard method, sometimes named annotate-then-identify, first finds possible genes, and tests for differential expression. In contrast, our method skips the first step and scans the genome for potential differentially expressed regions (the identify-then-annotate strategy). Since our method is the first one to use the identify-then-annotate strategy on sRNAs, we compared our method against a similar method, developed for long RNAs (derfinder), and to the annotate-then-identify strategy, where the sRNAs have been identified beforehand using a segmentation tool, on three published datasets, and a simulated one. Results show that srnadiff gives much better results than derfinder, and is also better than the annotate-then-identify strategy on many aspects. srnadiff is available as a Bioconductor package, together with a detailed manual: https://bioconductor.org/packages/release/bioc/html/srnadiff.html

bioinformatics↗

bioRxiv2019

The Glycolytic Protein Phosphofructokinase Dynamically Relocalizes into Subcellular Compartments with Liquid-like Properties in vivo

While much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. Here we built a hybrid microfluidic-hydrogel device for use in Caenorhabditis elegans to systematically examine and quantify the dynamic subcellular localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1/PFK-1.1. We determine that endogenous PFK-1.1 localizes to distinct, tissue-specific subcellular compartments in vivo. In neurons, PFK-1.1 is diffusely localized in the cytosol, but capable of dynamically forming phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in the dispersion of PFK-1.1 in the cytosol, indicating that PFK-1.1 reversibly organizes into biomolecular condensates in response to cues within the cellular environment. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Prolonged conditions of energy stress during sustained hypoxia alter the biophysical properties of PFK-1.1 in vivo, affecting its viscosity and mobility within phase-separated condensates. PFK-1.1s ability to form tetramers is critical for its capacity to form condensates in vivo, and heterologous self-association domain such as cryptochrome 2 (CRY2) is sufficient to constitutively induce the formation of PFK-1.1 condensates. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically compartmentalize in vivo to specific subcellular compartments in response to acute energy stress via multivalency as phase-separated condensates.

cell biology↗

bioRxiv2019

CTCF confers local nucleosome resiliency after DNA replication and during mitosis

The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome posi-tioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nu-cleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. While regions bound by other TFs, such as Oct4 and Sox2, display major rearrangement, the post-replication and mitotic nucleosome organization activity of CTCF is not likely to be unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, we propose that selected TFs, such as CTCF and Esrrb, govern the inheritance of nucleosome positioning at gene regulatory regions through-out the ES cell-cycle.

genomics↗

bioRxiv2018

Transcription factor activity and nucleosome organisation in mitosis

Mitotic bookmarking transcription factors (BFs) maintain the capacity to bind to their targets during mitosis, despite major rearrangements of the chromatin. While they were thought to propagate gene regulatory information through mitosis by statically occupying their DNA targets, it has recently become clear that BFs are highly dynamic in mitotic cells. This represents both a technical and a conceptual challenge to study and understand the function of BFs: first, formaldehyde has been suggested to be unable to efficiently capture these transient interactions, leading to profound contradictions in the literature; second, if BFs are not permanently bound to their targets during mitosis, it becomes unclear how they convey regulatory information to daughter cells. Here, comparing formaldehyde to alternative fixatives we clarify the nature of the chromosomal association of previously proposed BFs in embryonic stem cells: while Esrrb can be considered as a canonical BF that binds at selected regulatory regions in mitosis, Sox2 and Oct4 establish DNA sequence independent interactions with the mitotic chromosomes, either throughout the chromosomal arms (Sox2) or at pericentromeric regions (Oct4). Moreover, we show that ordered nucleosomal arrays are retained during mitosis at Esrrb book-marked sites, whereas regions losing transcription factor binding display a profound loss of order. By maintaining nucleosome positioning during mitosis, Esrrb might ensure the rapid post-mitotic re-establishment of functional regulatory complexes at selected enhancers and promoters. Our results provide a mechanistic framework that reconciles dynamic mitotic binding with the transmission of gene regulatory information across cell division.

genomics↗

bioRxiv2018

The molecular logic of Nanog-induced self-renewal

Transcription factor networks, together with histone modifications and signalling pathways, underlie the establishment and maintenance of gene regulatory architectures associated with the molecular identity of each cell type. However, how master transcription factors individually impact the epigenomic landscape and orchestrate the behaviour of regulatory networks under different environmental constraints is only very partially understood. Here, we show that the transcription factor Nanog deploys multiple distinct mechanisms to enhance embryonic stem cell self-renewal. In the presence of LIF, which fosters self-renewal, Nanog rewires the pluripotency network by promoting chromatin accessibility and binding of other pluripotency factors to thousands of enhancers. In the absence of LIF, Nanog blocks differentiation by sustaining H3K27me3, a repressive histone mark, at developmental regulators. Among those, we show that the repression of Otx2 plays a preponderant role. Our results underscore the versatility of master transcription factors, such as Nanog, to globally influence gene regulation during developmental processes.

genomics↗