Biology subjects

Gutierrez, C.

Publications and source records attributed to Gutierrez, C..

25 records · Page 2Linked to original sources

bioRxiv2019

Expressed barcodes enable clonal characterization of chemotherapeutic responses in chronic lymphocytic leukemia

The remarkable evolutionary capacity of cancer is a major challenge to current therapeutic efforts. Fueling this evolution is its vast clonal heterogeneity and ability to adapt to diverse selective pressures. Although the genetic and transcriptional mechanisms underlying these responses have been independently evaluated, the ability to couple genetic alterations present within individual clones to their respective transcriptional or functional outputs has been lacking in the field. To this end, we developed a high-complexity expressed barcode library that integrates DNA barcoding with single-cell RNA sequencing through use of the CROP-seq sgRNA expression/capture system, and which is compatible with the COLBERT clonal isolation workflow for subsequent genomic and epigenomic characterization of specific clones of interest. We applied this approach to study chronic lymphocytic leukemia (CLL), a mature B cell malignancy notable for its genetic and transcriptomic heterogeneity and variable disease course. Here, we demonstrate the clonal composition and gene expression states of HG3, a CLL cell line harboring the common alteration del(13q), in response to front-line cytotoxic therapy of fludarabine and mafosfamide (an analog of the clinically used cyclophosphamide). Analysis of clonal abundance and clonally-resolved single-cell RNA sequencing revealed that only a small fraction of clones consistently survived therapy. These rare highly drug tolerant clones comprise 94% of the post-treatment population and share a stable, pre-existing gene expression state characterized by upregulation of CXCR4 and WNT signaling and a number of DNA damage and cell survival genes. Taken together, these data demonstrate at unprecedented resolution the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population. Further, this approach provides a template for the high-resolution study of thousands of clones and the respective gene expression states underlying their response to therapy.

cancer biology↗

bioRxiv2019

Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types

Early in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retina progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist. Here, we describe a CRISPR/Cas9 gene editing strategy to generate a triple transgenic reporter hiPSC line (PGP1) that utilizes the endogenous VSX2, BRN3b, and RCVRN promoters to specifically express fluorescent proteins (Cerulean in NRPs, eGFP in RGCs and mCherry in PRs) without disrupting the function of the endogenous alleles. Retinal organoid formation from the PGP1 line demonstrated the ability of the edited cells to undergo normal retina development while exhibiting appropriate fluorescent protein expression consistent with the onset of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types. The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.

developmental biology↗

bioRxiv2018

In vivo assembly and trafficking of olfactory Ionotropic Receptors

lonotropic Receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors(iGluRs), with roles in chemosensation, thermosensation and hygrosensation. Analogous to the synaptic targeting mechanisms of their iGluR ancestors, IRs are thought to form complexes of broadly-expressed co-receptors and selectively-expressed tuning receptors to localise to sensory cilia. While tuning receptors extracellular ligand-binding domain (LBD) defines sensory specificity, the role of this domain in co-receptors is unclear. We identify a coreceptor-specific sequence in the LBD, which contains a single N-glycosylation site. Combining molecular genetic and cell biological analyses, we show that this site is dispensable for assembly of IR complexes in olfactory sensory neurons, but essential for endoplasmic reticulum exit of some,but not all, IR complexes. Our data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes, and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.

neuroscience↗

bioRxiv2018

BioModelos: a collaborative online system to map species distributions

Information on species distribution is recognized as a crucial input for biodiversity conservation and management. To that end, considerable resources have been dedicated towards increasing the quantity and availability of species occurrence data, boosting their use in species distribution modeling and online platforms for their dissemination. Currently, those platforms face the challenge of bringing biology into modeling by making informed decisions that result in meaningful models. Here we describe BioModelos, a modeling approach supported by an online system and a core team, whereby a network of experts contributes to the development of species distribution models by assessing the quality of occurrence data, identifying potentially limiting environmental variables, establishing species accessible areas and validating qualitatively modeling predictions. Models developed through BioModelos become publicly available once validated by experts, furthering their use in conservation applications. This approach has been implemented in Colombia since 2013 and it currently consist of a network of nearly 500 experts that collaboratively contribute to enhance the knowledge on the distribution of a growing number of species and where it has aided the development of several decision support products such as national risk assessments and biodiversity compensation manuals. BioModelos is an example of operationalization of an essential biodiversity variable at a national level through the implementation of a research infrastructure that enhances the value of open access species data.

ecology↗

bioRxiv2018

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

biochemistry↗

bioRxiv2018

Differences in firing efficiency, chromatin and transcription underlie the developmental plasticity of Arabidopsis originome

Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs) that constitute the originome. A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have determined the originome and chromatin landscape of Arabidopsis thaliana at two stages of postembryonic development. ORIs associate with multiple chromatin signatures including TSS but also regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin whereas at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the originome of an organism at the postembryo stage that should allow us to study ORI biology in response to development, environment and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems.

plant biology↗

bioRxiv2016

Retrotransposons are specified as DNA replication origins in the gene-poor regions of Arabidopsis heterochromatin

Genomic stability depends on faithful genome replication. This is achieved by the concerted activity of thousands of DNA replication origins (ORIs) scattered throughout the genome. In spite of multiple efforts, the DNA and chromatin features that determine ORI specification are not presently known. We have generated a high-resolution genome-wide map of ORIs in cultured Arabidopsis thaliana cells that rendered a collection of 3230 ORIs. In this study we focused on defining the features associated with ORIs in heterochromatin. We found that while ORIs tend to colocalize with genes in euchromatic gene-rich regions, they frequently colocalize with transposable elements (TEs) in pericentromeric gene-poor domains. Interestingly, ORIs in TEs associate almost exclusively with retrotransposons, in particular, of the Gypsy family. ORI activity in retrotransposons occurs independently of TE expression and while maintaining high levels of H3K9me2 and H3K27me1, typical marks of repressed heterochromatin. ORI-TEs largely colocalize with chromatin signatures defining GC-rich heterochromatin. Importantly, TEs with active ORIs contain a local GC content higher than the TEs lacking them. Our results lead us to conclude that ORI colocalization with TEs is largely limited to retrotransposons, which are defined by their transposition mechanisms based on transcription, and they occur in a specific chromatin landscape. Our detailed analysis of ORIs responsible for heterochromatin replication has also implications on the mechanisms of ORI specification in other multicellular organisms in which retrotransposons are major components of heterochromatin as well as of the entire genome.

genomics↗