Publications by authors named "José L McFaline-Figueroa"

13 Publications

  • Page 1 of 1

The promise of single-cell genomics in plants.

Curr Opin Plant Biol 2020 04 5;54:114-121. Epub 2020 May 5.

Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address:

Single-cell genomic approaches have the potential to revolutionize the study of plant systems. Here, we highlight newly developed techniques to analyze transcriptomes at single-cell resolution. We focus on the rigorous standards necessary to generate and compare these data sets introducing analysis methods that can be applied to interpret their results. Lastly, we discuss the inherent limitations of single-cell studies and address future directions for plant single-cell genomics.
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http://dx.doi.org/10.1016/j.pbi.2020.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971421PMC
April 2020

Inferring Causal Gene Regulatory Networks from Coupled Single-Cell Expression Dynamics Using Scribe.

Cell Syst 2020 03 4;10(3):265-274.e11. Epub 2020 Mar 4.

Department of Electrical Engineering, University of Washington, Seattle, WA, USA. Electronic address:

Here, we present Scribe (https://github.com/aristoteleo/Scribe-py), a toolkit for detecting and visualizing causal regulatory interactions between genes and explore the potential for single-cell experiments to power network reconstruction. Scribe employs restricted directed information to determine causality by estimating the strength of information transferred from a potential regulator to its downstream target. We apply Scribe and other leading approaches for causal network reconstruction to several types of single-cell measurements and show that there is a dramatic drop in performance for "pseudotime"-ordered single-cell data compared with true time-series data. We demonstrate that performing causal inference requires temporal coupling between measurements. We show that methods such as "RNA velocity" restore some degree of coupling through an analysis of chromaffin cell fate commitment. These analyses highlight a shortcoming in experimental and computational methods for analyzing gene regulation at single-cell resolution and suggest ways of overcoming it.
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http://dx.doi.org/10.1016/j.cels.2020.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223477PMC
March 2020

Massively multiplex chemical transcriptomics at single-cell resolution.

Science 2020 01 5;367(6473):45-51. Epub 2019 Dec 5.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we introduce "sci-Plex," which uses "nuclear hashing" to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with histone deacetylase inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.
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http://dx.doi.org/10.1126/science.aax6234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289078PMC
January 2020

A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition.

Nat Genet 2019 09 2;51(9):1389-1398. Epub 2019 Sep 2.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-β. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.
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http://dx.doi.org/10.1038/s41588-019-0489-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756480PMC
September 2019

Thyroid hormone regulates distinct paths to maturation in pigment cell lineages.

Elife 2019 05 29;8. Epub 2019 May 29.

Department of Biology, University of Virginia, Charlottesville, United States.

Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form.
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http://dx.doi.org/10.7554/eLife.45181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588384PMC
May 2019

Dynamics of Gene Expression in Single Root Cells of .

Plant Cell 2019 05 28;31(5):993-1011. Epub 2019 Mar 28.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195

Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to Arabidopsis () root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.
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http://dx.doi.org/10.1105/tpc.18.00785DOI Listing
May 2019

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.

Cell 2019 01 3;176(1-2):377-390.e19. Epub 2019 Jan 3.

Department of Genome Sciences, University of Washington, Seattle, WA 98105, USA; Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA 98105, USA; Howard Hughes Medical Institute, Seattle, WA 98105, USA. Electronic address:

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.
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http://dx.doi.org/10.1016/j.cell.2018.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690346PMC
January 2019

Joint profiling of chromatin accessibility and gene expression in thousands of single cells.

Science 2018 09 30;361(6409):1380-1385. Epub 2018 Aug 30.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing-based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney. With the resulting data, we compare the pseudotemporal dynamics of chromatin accessibility and gene expression, reconstruct the chromatin accessibility profiles of cell types defined by RNA profiles, and link cis-regulatory sites to their target genes on the basis of the covariance of chromatin accessibility and transcription across large numbers of single cells.
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http://dx.doi.org/10.1126/science.aau0730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571013PMC
September 2018

Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data.

Mol Cell 2018 09 2;71(5):858-871.e8. Epub 2018 Aug 2.

Department of Genome Sciences, University of Washington, Seattle, WA, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA, USA. Electronic address:

Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.
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http://dx.doi.org/10.1016/j.molcel.2018.06.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582963PMC
September 2018

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma.

Mol Oncol 2018 06 29;12(7):1188-1202. Epub 2018 May 29.

Department of Neurosurgery, Houston Methodist Hospital and Research Institute, Houston, TX, USA.

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial-to-mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self-renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss-of-function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW-associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.
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http://dx.doi.org/10.1002/1878-0261.12320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026950PMC
June 2018

On the design of CRISPR-based single-cell molecular screens.

Nat Methods 2018 04 19;15(4):271-274. Epub 2018 Feb 19.

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.
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http://dx.doi.org/10.1038/nmeth.4604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882576PMC
April 2018

Minor Changes in Expression of the Mismatch Repair Protein MSH2 Exert a Major Impact on Glioblastoma Response to Temozolomide.

Cancer Res 2015 Aug 29;75(15):3127-38. Epub 2015 May 29.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts. Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide, but the disease inevitably recurs in a drug-resistant form after initial treatment. Here, we report that in GBM cells, even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on temozolomide sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of temozolomide resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress temozolomide-induced tumor regression. Using The Cancer Genome Atlas to analyze mRNA expression patterns in tumors from temozolomide-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial temozolomide therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades temozolomide sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of temozolomide resistance and argue that MMR activity offers a predictive marker for initial therapeutic response to temozolomide treatment.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-3616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526337PMC
August 2015