Publications by authors named "Charles G Danko"

52 Publications

Chromosome fusion affects genetic diversity and evolutionary turnover of functional loci, but consistently depends on chromosome size.

Mol Biol Evol 2021 Jun 19. Epub 2021 Jun 19.

School of Biological Sciences, University of Bristol Bristol - Life Sciences Building, Bristol, UK.

Major changes in chromosome number and structure are linked to a series of evolutionary phenomena, including intrinsic barriers to gene flow or suppression of recombination due to chromosomal rearrangements. However, chromosome rearrangements can also affect the fundamental dynamics of molecular evolution within populations by changing relationships between linked loci and altering rates of recombination. Here, we build chromosome-level assembly Eueides isabella and, together with a recent chromosome-level assembly of Dryas iulia, examine the evolutionary consequences of multiple chromosome fusions in Heliconius butterflies. These assemblies pinpoint fusion points on 10 of the 20 autosomal chromosomes and reveal striking differences in the characteristics of fused and unfused chromosomes. The ten smallest autosomes in D. iulia and E. isabella, which have each fused to a longer chromosome in Heliconius, have higher repeat and GC content, and longer introns than predicted by their chromosome length. When fused, these characteristics change to become more in line with chromosome length. The fusions also led to reduced diversity, which likely reflects increased background selection and selection against introgression between diverging populations, following a reduction in per-base recombination rate. We further show that chromosome size and fusion impact turnover rates of functional loci at a macroevolutionary scale. Together these results provide further evidence that chromosome fusion in Heliconius likely had dramatic effects on population level processes shaping rates of neutral and adaptive divergence. These effects may have impacted patterns of diversification in Heliconius, a classic example of an adaptive radiation.
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http://dx.doi.org/10.1093/molbev/msab185DOI Listing
June 2021

The Dryas iulia Genome Supports Multiple Gains of a W Chromosome from a B Chromosome in Butterflies.

Genome Biol Evol 2021 Jul;13(7)

School of Biological Sciences, University of Bristol, United Kingdom.

In butterflies and moths, which exhibit highly variable sex determination mechanisms, the homogametic Z chromosome is deeply conserved and is featured in many genome assemblies. The evolution and origin of the female W sex chromosome, however, remains mostly unknown. Previous studies have proposed that a ZZ/Z0 sex determination system is ancestral to Lepidoptera, and that W chromosomes may originate from sex-linked B chromosomes. Here, we sequence and assemble the female Dryas iulia genome into 32 highly contiguous ordered and oriented chromosomes, including the Z and W sex chromosomes. We then use sex-specific Hi-C, ATAC-seq, PRO-seq, and whole-genome DNA sequence data sets to test if features of the D. iulia W chromosome are consistent with a hypothesized B chromosome origin. We show that the putative W chromosome displays female-associated DNA sequence, gene expression, and chromatin accessibility to confirm the sex-linked function of the W sequence. In contrast with expectations from studies of homologous sex chromosomes, highly repetitive DNA content on the W chromosome, the sole presence of domesticated repetitive elements in functional DNA, and lack of sequence homology with the Z chromosome or autosomes is most consistent with a B chromosome origin for the W, although it remains challenging to rule out extensive sequence divergence. Synteny analysis of the D. iulia W chromosome with other female lepidopteran genome assemblies shows no homology between W chromosomes and suggests multiple, independent origins of the W chromosome from a B chromosome likely occurred in butterflies.
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http://dx.doi.org/10.1093/gbe/evab128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290107PMC
July 2021

Uncovering transcriptional dark matter via gene annotation independent single-cell RNA sequencing analysis.

Nat Commun 2021 04 12;12(1):2158. Epub 2021 Apr 12.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, 14853, USA.

Conventional scRNA-seq expression analyses rely on the availability of a high quality genome annotation. Yet, as we show here with scRNA-seq experiments and analyses spanning human, mouse, chicken, mole rat, lemur and sea urchin, genome annotations are often incomplete, in particular for organisms that are not routinely studied. To overcome this hurdle, we created a scRNA-seq analysis routine that recovers biologically relevant transcriptional activity beyond the scope of the best available genome annotation by performing scRNA-seq analysis on any region in the genome for which transcriptional products are detected. Our tool generates a single-cell expression matrix for all transcriptionally active regions (TARs), performs single-cell TAR expression analysis to identify biologically significant TARs, and then annotates TARs using gene homology analysis. This procedure uses single-cell expression analyses as a filter to direct annotation efforts to biologically significant transcripts and thereby uncovers biology to which scRNA-seq would otherwise be in the dark.
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http://dx.doi.org/10.1038/s41467-021-22496-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042062PMC
April 2021

Characterizing RNA stability genome-wide through combined analysis of PRO-seq and RNA-seq data.

BMC Biol 2021 02 15;19(1):30. Epub 2021 Feb 15.

Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.

Background: The concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq).

Results: Our method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability.

Conclusion: We introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.
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http://dx.doi.org/10.1186/s12915-021-00949-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885420PMC
February 2021

GLP-1 receptor signaling increases PCSK1 and β cell features in human α cells.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Department of Biomedical Sciences and.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates glucose-stimulated insulin secretion. GLP-1 is classically produced by gut L cells; however, under certain circumstances α cells can express the prohormone convertase required for proglucagon processing to GLP-1, prohormone convertase 1/3 (PC1/3), and can produce GLP-1. However, the mechanisms through which this occurs are poorly defined. Understanding the mechanisms by which α cell PC1/3 expression can be activated may reveal new targets for diabetes treatment. Here, we demonstrate that the GLP-1 receptor (GLP-1R) agonist, liraglutide, increased α cell GLP-1 expression in a β cell GLP-1R-dependent manner. We demonstrate that this effect of liraglutide was translationally relevant in human islets through application of a new scRNA-seq technology, DART-Seq. We found that the effect of liraglutide to increase α cell PC1/3 mRNA expression occurred in a subcluster of α cells and was associated with increased expression of other β cell-like genes, which we confirmed by IHC. Finally, we found that the effect of liraglutide to increase bihormonal insulin+ glucagon+ cells was mediated by the β cell GLP-1R in mice. Together, our data validate a high-sensitivity method for scRNA-seq in human islets and identify a potentially novel GLP-1-mediated pathway regulating human α cell function.
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http://dx.doi.org/10.1172/jci.insight.141851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934853PMC
February 2021

Many functionally connected loci foster adaptive diversification along a neotropical hybrid zone.

Sci Adv 2020 Sep 25;6(39). Epub 2020 Sep 25.

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, USA.

Characterizing the genetic complexity of adaptation and trait evolution is a major emphasis of evolutionary biology and genetics. Incongruent findings from genetic studies have resulted in conceptual models ranging from a few large-effect loci to massively polygenic architectures. Here, we combine chromatin immunoprecipitation sequencing, Hi-C, RNA sequencing, and 40 whole-genome sequences from butterflies to show that red color pattern diversification occurred via many genomic loci. We find that the red wing pattern master regulatory transcription factor Optix binds dozens of loci also under selection, which frequently form three-dimensional adaptive hubs with selection acting on multiple physically interacting genes. Many Optix-bound genes under selection are tied to pigmentation and wing development, and these loci collectively maintain separation between adaptive red color pattern phenotypes in natural populations. We propose a model of trait evolution where functional connections between loci may resolve much of the disparity between large-effect and polygenic evolutionary models.
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http://dx.doi.org/10.1126/sciadv.abb8617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518860PMC
September 2020

Chromatin run-on sequencing analysis finds that ECM remodeling plays an important role in canine hemangiosarcoma pathogenesis.

BMC Vet Res 2020 Jun 22;16(1):206. Epub 2020 Jun 22.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

Background: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression.

Results: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions.

Conclusion: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.
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http://dx.doi.org/10.1186/s12917-020-02395-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310061PMC
June 2020

Hotspots of Aberrant Enhancer Activity in Fibrolamellar Carcinoma Reveal Candidate Oncogenic Pathways and Therapeutic Vulnerabilities.

Cell Rep 2020 04;31(2):107509

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Fibrolamellar carcinoma (FLC) is a rare, therapeutically intractable liver cancer that disproportionately affects youth. Although FLC tumors exhibit a distinct gene expression profile, the chromatin regulatory landscape and the genes most critical for tumor cell survival remain unclear. Here, we use chromatin run-on sequencing to discover ∼7,000 enhancers and 141 enhancer hotspots activated in FLC relative to nonmalignant liver. Bioinformatic analyses reveal aberrant ERK/MEK signaling and candidate master transcriptional regulators. We also define the genes most strongly associated with hotspots of FLC enhancer activity, including CA12 and SLC16A14. Treatment of FLC cell models with inhibitors of CA12 or SLC16A14 independently reduce cell viability and/or significantly enhance the effect of the MEK inhibitor cobimetinib. These findings highlight molecular targets for drug development, as well as drug combination approaches.
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http://dx.doi.org/10.1016/j.celrep.2020.03.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474926PMC
April 2020

Elevated circulating Th2 but not group 2 innate lymphoid cell responses characterize canine atopic dermatitis.

Vet Immunol Immunopathol 2020 Mar 24;221:110015. Epub 2020 Jan 24.

Baker Institute for Animal Health and Department of Microbiology and Immunology, Ithaca, NY 14853, USA; Department of Immunology, University of Washington, Seattle, WA 98109, USA. Electronic address:

Atopic dermatitis (AD) is an allergic skin disease that causes significant morbidity and affects multiple species. AD is highly prevalent in companion dogs, and the clinical management of the disease remains challenging. An improved understanding of the immunologic and genetic pathways that lead to disease could inform the development of novel treatments. In allergic humans and mouse models of AD, the disease is associated with Th2 and group 2 innate lymphoid cell (ILC2) activation that drives type 2 inflammation. Type 2 inflammation also appears to be associated with AD in dogs, but gaps remain in our understanding of how key type 2-associated cell types such as canine Th2 cells and ILC2s contribute to the pathogenesis of canine AD. Here, we describe previously uncharacterized canine ILC2-like cells and Th2 cells ex vivo that produced type 2 cytokines and expressed the transcription factor Gata3. Increased circulating Th2 cells were associated with chronic canine AD. Single-cell RNA sequencing revealed a unique gene expression signature in T cells in dogs with AD. These findings underline the importance of pro-allergic Th2 cells in orchestrating AD and provide new methods and pathways that can inform the development of improved therapies.
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http://dx.doi.org/10.1016/j.vetimm.2020.110015DOI Listing
March 2020

Parallel evolution of ancient, pleiotropic enhancers underlies butterfly wing pattern mimicry.

Proc Natl Acad Sci U S A 2019 11 11;116(48):24174-24183. Epub 2019 Nov 11.

Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853.

Color pattern mimicry in butterflies is a classic case study of complex trait adaptation via selection on a few large effect genes. Association studies have linked color pattern variation to a handful of noncoding regions, yet the presumptive cis-regulatory elements (CREs) that control color patterning remain unknown. Here we combine chromatin assays, DNA sequence associations, and genome editing to functionally characterize 5 cis-regulatory elements of the color pattern gene We were surprised to find that the cis-regulatory architecture of is characterized by pleiotropy and regulatory fragility, where deletion of individual cis-regulatory elements has broad effects on both color pattern and wing vein development. Remarkably, we found orthologous cis-regulatory elements associate with wing pattern convergence of distantly related comimics, suggesting that parallel coevolution of ancestral elements facilitated pattern mimicry. Our results support a model of color pattern evolution in where changes to ancient, multifunctional cis-regulatory elements underlie adaptive radiation.
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http://dx.doi.org/10.1073/pnas.1907068116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883815PMC
November 2019

Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock.

Proc Natl Acad Sci U S A 2019 09 10;116(39):19431-19439. Epub 2019 Sep 10.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853;

Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.
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http://dx.doi.org/10.1073/pnas.1901244116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765289PMC
September 2019

Physical confinement induces malignant transformation in mammary epithelial cells.

Biomaterials 2019 10 26;217:119307. Epub 2019 Jun 26.

Department of Biological and Environmental Engineering, Cornell University, 111 Wing Drive, Ithaca, NY, 14853, USA. Electronic address:

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336132PMC
October 2019

Contrasting Roles of Transcription Factors Spineless and EcR in the Highly Dynamic Chromatin Landscape of Butterfly Wing Metamorphosis.

Cell Rep 2019 04;27(4):1027-1038.e3

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA.

Development requires highly coordinated changes in chromatin accessibility in order for proper gene regulation to occur. Here, we identify factors associated with major, discrete changes in chromatin accessibility during butterfly wing metamorphosis. By combining mRNA sequencing (mRNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and machine learning analysis of motifs, we show that distinct sets of transcription factors are predictive of chromatin opening at different developmental stages. Our data suggest an important role for nuclear hormone receptors early in metamorphosis, whereas PAS-domain transcription factors are strongly associated with later chromatin opening. Chromatin immunoprecipitation sequencing (ChIP-seq) validation of select candidate factors showed spineless binding to be a major predictor of opening chromatin. Surprisingly, binding of ecdysone receptor (EcR), a candidate accessibility factor in Drosophila, was not predictive of opening but instead marked persistent sites. This work characterizes the chromatin dynamics of insect wing metamorphosis, identifies candidate chromatin remodeling factors in insects, and presents a genome assembly of the model butterfly Junonia coenia.
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http://dx.doi.org/10.1016/j.celrep.2019.03.092DOI Listing
April 2019

The Notch signaling pathway promotes basophil responses during helminth-induced type 2 inflammation.

J Exp Med 2019 06 11;216(6):1268-1279. Epub 2019 Apr 11.

Baker Institute for Animal Health and Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY

Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 inflammation during murine infection with the helminth However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for -elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4 T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.
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http://dx.doi.org/10.1084/jem.20180131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547860PMC
June 2019

AlleleHMM: a data-driven method to identify allele specific differences in distributed functional genomic marks.

Nucleic Acids Res 2019 06;47(11):e64

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

How DNA sequence variation influences gene expression remains poorly understood. Diploid organisms have two homologous copies of their DNA sequence in the same nucleus, providing a rich source of information about how genetic variation affects a wealth of biochemical processes. However, few computational methods have been developed to discover allele specific differences in functional genomic data. Existing methods either treat each SNP independently, limiting statistical power, or combine SNPs across gene annotations, preventing the discovery of allele specific differences in unexpected genomic regions. Here we introduce AlleleHMM, a new computational method to identify blocks of neighboring SNPs that share similar allele specific differences in mark abundance. AlleleHMM uses a hidden Markov model to divide the genome into three hidden states based on allele frequencies in genomic data: a symmetric state (state S) which shows no difference between alleles, and regions with a higher signal on the maternal (state M) or paternal (state P) allele. AlleleHMM substantially outperformed naive methods using both simulated and real genomic data, particularly when input data had realistic levels of overdispersion. Using global run-on sequencing (GRO-seq) data, AlleleHMM identified thousands of allele specific blocks of transcription in both coding and non-coding genomic regions. AlleleHMM is a powerful tool for discovering allele specific regions in functional genomic datasets.
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http://dx.doi.org/10.1093/nar/gkz176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582321PMC
June 2019

Discovering Transcriptional Regulatory Elements From Run-On and Sequencing Data Using the Web-Based dREG Gateway.

Curr Protoc Bioinformatics 2019 06 27;66(1):e70. Epub 2018 Dec 27.

Baker Institute for Animal Health, Cornell University, Ithaca, New York.

Transcription is a chromatin mark that can be used effectively to identify the location of active enhancers and promoters, collectively known as transcriptional regulatory elements (TREs). We recently introduced dREG, a tool for the identification of TREs using run-on and sequencing (RO-seq) assays, including global run-on and sequencing (GRO-seq), precision run-on and sequencing (PRO-seq), and chromatin run-on and sequencing (ChRO-seq). In this protocol, we present step-by-step instructions for running dREG on an arbitrary run-on and sequencing dataset. Users provide dREG with bigWig files (in which each read is represented by a single base) representing the location of RNA polymerase in a cell or tissue sample of interest, and dREG returns a list of genomic regions that are predicted to be active TREs. Finally, we demonstrate the use of dREG regions in discovering transcription factors controlling response to a stimulus and predicting their target genes. Together, this protocol provides detailed instructions for running dREG on arbitrary run-on and sequencing data. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpbi.70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584046PMC
June 2019

Identification of regulatory elements from nascent transcription using dREG.

Genome Res 2019 02 20;29(2):293-303. Epub 2018 Dec 20.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.

Our genomes encode a wealth of transcription initiation regions (TIRs) that can be identified by their distinctive patterns of actively elongating RNA polymerase. We previously introduced dREG to identify TIRs using PRO-seq data. Here, we introduce an efficient new implementation of dREG that uses PRO-seq data to identify both uni- and bidirectionally transcribed TIRs with 70% improvement in accuracy, three- to fourfold higher resolution, and >100-fold increases in computational efficiency. Using a novel strategy to identify TIRs based on their statistical confidence reveals extensive overlap with orthogonal assays, yet also reveals thousands of additional weakly transcribed TIRs that were not identified by H3K27ac ChIP-seq or DNase-seq. Novel TIRs discovered by dREG were often associated with RNA polymerase III initiation, bound by pioneer transcription factors, or located in broad domains marked by repressive chromatin modifications. Our results suggest that transcription initiation can be a powerful tool for expanding the catalog of functional elements.
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http://dx.doi.org/10.1101/gr.238279.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360809PMC
February 2019

Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells.

Nat Methods 2019 01 17;16(1):59-62. Epub 2018 Dec 17.

Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

We describe droplet-assisted RNA targeting by single-cell sequencing (DART-seq), a versatile technology that enables multiplexed amplicon sequencing and transcriptome profiling in single cells. We applied DART-seq to simultaneously characterize the non-A-tailed transcripts of a segmented dsRNA virus and the transcriptome of the infected cell. In addition, we used DART-seq to simultaneously determine the natively paired, variable region heavy and light chain amplicons and the transcriptome of B lymphocytes.
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http://dx.doi.org/10.1038/s41592-018-0259-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378878PMC
January 2019

Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme.

Nat Genet 2018 11 22;50(11):1553-1564. Epub 2018 Oct 22.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.
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http://dx.doi.org/10.1038/s41588-018-0244-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204104PMC
November 2018

A common pattern of DNase I footprinting throughout the human mtDNA unveils clues for a chromatin-like organization.

Genome Res 2018 08 12;28(8):1158-1168. Epub 2018 Jul 12.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105 Israel.

Human mitochondrial DNA (mtDNA) is believed to lack chromatin and histones. Instead, it is coated solely by the transcription factor TFAM. We asked whether mtDNA packaging is more regulated than once thought. To address this, we analyzed DNase-seq experiments in 324 human cell types and found, for the first time, a pattern of 29 mtDNA Genomic footprinting (mt-DGF) sites shared by ∼90% of the samples. Their syntenic conservation in mouse DNase-seq experiments reflect selective constraints. Colocalization with known mtDNA regulatory elements, with G-quadruplex structures, in TFAM-poor sites (in HeLa cells) and with transcription pausing sites, suggest a functional regulatory role for such mt-DGFs. Altered mt-DGF pattern in interleukin 3-treated CD34 cells, certain tissue differences, and significant prevalence change in fetal versus nonfetal samples, offer first clues to their physiological importance. Taken together, human mtDNA has a conserved protein-DNA organization, which is likely involved in mtDNA regulation.
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http://dx.doi.org/10.1101/gr.230409.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071632PMC
August 2018

Building a Science Gateway For Processing and Modeling Sequencing Data Via Apache Airavata.

Pract Exp Adv Res Comput 2018 (2018) 2018 Jul;2018

Baker Institute for Animal Health and Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY,

The amount of DNA sequencing data has been exponentially growing during the past decade due to advances in sequencing technology. Processing and modeling large amounts of sequencing data can be computationally intractable for desktop computing platforms. High performance computing (HPC) resources offer advantages in terms of computing power, and can be a general solution to these problems. Using HPCs directly for computational needs requires skilled users who know their way around HPCs and acquiring such skills take time. Science gateways acts as the middle layer between users and HPCs, providing users with the resources to accomplish compute-intensive tasks without requiring specialized expertise. We developed a web-based computing platform for genome biologists by customizing the PHP Gateway for Airavata (PGA) framework that accesses publicly accessible HPC resources via Apache Airavata. This web computing platform takes advantage of the Extreme Science and Engineering Discovery Environment (XSEDE) which provides the resources for gateway development, including access to CPU, GPU, and storage resources. We used this platform to develop a gateway for the dREG algorithm, an online computing tool for finding functional regions in mammalian genomes using nascent RNA sequencing data. The dREG gateway provides its users a free, powerful and user-friendly GPU computing resource based on XSEDE, circumventing the need of specialized knowledge about installation, configuration, and execution on an HPC for biologists. The dREG gateway is available at: https://dREG.dnasequence.org/.
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http://dx.doi.org/10.1145/3219104.3219141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785190PMC
July 2018

A bi-stable feedback loop between GDNF, EGR1, and ERα contribute to endocrine resistant breast cancer.

PLoS One 2018 3;13(4):e0194522. Epub 2018 Apr 3.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America.

Discovering regulatory interactions between genes that specify the behavioral properties of cells remains an important challenge. We used the dynamics of transcriptional changes resolved by PRO-seq to identify a regulatory network responsible for endocrine resistance in breast cancer. We show that GDNF leads to endocrine resistance by switching the active state in a bi-stable feedback loop between GDNF, EGR1, and the master transcription factor ERα. GDNF stimulates MAP kinase, activating the transcription factors SRF and AP-1. SRF initiates an immediate transcriptional response, activating EGR1 and suppressing ERα. Newly translated EGR1 protein activates endogenous GDNF, leading to constitutive GDNF and EGR1 up-regulation, and the sustained down-regulation of ERα. Endocrine resistant MCF-7 cells are constitutively in the GDNF-high/ ERα-low state, suggesting that the state in the bi-stable feedback loop may provide a 'memory' of endocrine resistance. Thus, we identified a regulatory network switch that contributes to drug resistance in breast cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194522PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882141PMC
July 2018

ER-positive breast cancer cells are poised for RET-mediated endocrine resistance.

PLoS One 2018 2;13(4):e0194023. Epub 2018 Apr 2.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America.

The RET tyrosine kinase signaling pathway is involved in the development of endocrine resistant ER+ breast cancer. However, we know little about how ER+ cells activate RET signaling and initiate an endocrine resistant phenotype. Here we show that both ER+ endocrine resistant and sensitive breast cancers have a functional RET tyrosine kinase signaling pathway, but that endocrine sensitive breast cancer cells lack RET ligands that are necessary to drive endocrine resistance. Transcription of one RET ligand, GDNF, is necessary and sufficient to confer resistance in the ER+ MCF-7 cell line. Endogenous GDNF produced by endocrine resistant cells is translated, secreted into the media, and activates RET signaling in nearby cells. In patients, RET ligand expression predicts responsiveness to endocrine therapies and correlates with survival. Collectively, our findings show that ER+ tumor cells are "poised" for RET mediated endocrine resistance, expressing all components of the RET signaling pathway, but endocrine sensitive cells lack high expression of RET ligands that are necessary to initiate the resistance phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194023PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880349PMC
July 2018

Herpes Simplex Virus 1 Dramatically Alters Loading and Positioning of RNA Polymerase II on Host Genes Early in Infection.

J Virol 2018 04 28;92(8). Epub 2018 Mar 28.

Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana, USA

Herpes simplex virus 1 (HSV-1) transcription is mediated by cellular RNA polymerase II (Pol II). Recent studies investigating how Pol II transcription of host genes is altered after HSV-1 are conflicting. Chromatin immunoprecipitation sequencing (ChIP-seq) studies suggest that Pol II is almost completely removed from host genes at 4 h postinfection (hpi), while 4-thiouridine (4SU) labeling experiments show that host transcription termination is extended at 7 hpi, implying that a significant amount of Pol II remains associated with host genes in infected cells. To address this discrepancy, we used precision nuclear run-on analysis (PRO-seq) to determine the location of Pol II to single-base-pair resolution in combination with quantitative reverse transcription-PCR (qRT-PCR) analysis at 3 hpi. HSV-1 decreased Pol II on approximately two-thirds of cellular genes but increased Pol II on others. For more than 85% of genes for which transcriptional termination could be statistically assessed, Pol II was displaced to positions downstream of the normal termination zone, suggesting extensive termination defects. Pol II amounts at the promoter, promoter-proximal pause site, and gene body were also modulated in a gene-specific manner. qRT-PCR of selected RNAs showed that HSV-1-induced extension of the termination zone strongly correlated with decreased RNA and mRNA accumulation. However, HSV-1-induced increases of Pol II occupancy on genes without termination zone extension correlated with increased cytoplasmic mRNA. Functional grouping of genes with increased Pol II occupancy suggested an upregulation of exosome secretion and downregulation of apoptosis, both of which are potentially beneficial to virus production. This study provides a map of RNA polymerase II location on host genes after infection with HSV-1 with greater detail than previous ChIP-seq studies and rectifies discrepancies between ChIP-seq data and 4SU labeling experiments with HSV-1. The data show the effects that a given change in RNA Pol II location on host genes has on the abundance of different RNA types, including nuclear, polyadenylated mRNA and cytoplasmic, polyadenylated mRNA. It gives a clearer understanding of how HSV-1 augments host transcription of some genes to provide an environment favorable to HSV-1 replication.
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http://dx.doi.org/10.1128/JVI.02184-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874419PMC
April 2018

Dynamic evolution of regulatory element ensembles in primate CD4 T cells.

Nat Ecol Evol 2018 03 29;2(3):537-548. Epub 2018 Jan 29.

Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.

How evolutionary changes at enhancers affect the transcription of target genes remains an important open question. Previous comparative studies of gene expression have largely measured the abundance of messenger RNA, which is affected by post-transcriptional regulatory processes, hence limiting inferences about the mechanisms underlying expression differences. Here, we directly measured nascent transcription in primate species, allowing us to separate transcription from post-transcriptional regulation. We used precision run-on and sequencing to map RNA polymerases in resting and activated CD4 T cells in multiple human, chimpanzee and rhesus macaque individuals, with rodents as outgroups. We observed general conservation in coding and non-coding transcription, punctuated by numerous differences between species, particularly at distal enhancers and non-coding RNAs. Genes regulated by larger numbers of enhancers are more frequently transcribed at evolutionarily stable levels, despite reduced conservation at individual enhancers. Adaptive nucleotide substitutions are associated with lineage-specific transcription and at one locus, SGPP2, we predict and experimentally validate that multiple substitutions contribute to human-specific transcription. Collectively, our findings suggest a pervasive role for evolutionary compensation across ensembles of enhancers that jointly regulate target genes.
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http://dx.doi.org/10.1038/s41559-017-0447-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957490PMC
March 2018

XX Disorder of Sex Development is associated with an insertion on chromosome 9 and downregulation of RSPO1 in dogs (Canis lupus familiaris).

PLoS One 2017 20;12(10):e0186331. Epub 2017 Oct 20.

Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC, Australia.

Remarkable progress has been achieved in understanding the mechanisms controlling sex determination, yet the cause for many Disorders of Sex Development (DSD) remains unknown. Of particular interest is a rare XX DSD subtype in which individuals are negative for SRY, the testis determining factor on the Y chromosome, yet develop testes or ovotestes, and both of these phenotypes occur in the same family. This is a naturally occurring disorder in humans (Homo sapiens) and dogs (C. familiaris). Phenotypes in the canine XX DSD model are strikingly similar to those of the human XX DSD subtype. The purposes of this study were to identify 1) a variant associated with XX DSD in the canine model and 2) gene expression alterations in canine embryonic gonads that could be informative to causation. Using a genome wide association study (GWAS) and whole genome sequencing (WGS), we identified a variant on C. familiaris autosome 9 (CFA9) that is associated with XX DSD in the canine model and in affected purebred dogs. This is the first marker identified for inherited canine XX DSD. It lies upstream of SOX9 within the canine ortholog for the human disorder, which resides on 17q24. Inheritance of this variant indicates that XX DSD is a complex trait in which breed genetic background affects penetrance. Furthermore, the homozygous variant genotype is associated with embryonic lethality in at least one breed. Our analysis of gene expression studies (RNA-seq and PRO-seq) in embryonic gonads at risk of XX DSD from the canine model identified significant RSPO1 downregulation in comparison to XX controls, without significant upregulation of SOX9 or other known testis pathway genes. Based on these data, a novel mechanism is proposed in which molecular lesions acting upstream of RSPO1 induce epigenomic gonadal mosaicism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186331PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650465PMC
November 2017

Nascent RNA sequencing reveals a dynamic global transcriptional response at genes and enhancers to the natural medicinal compound celastrol.

Genome Res 2017 11 12;27(11):1816-1829. Epub 2017 Oct 12.

Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Most studies of responses to transcriptional stimuli measure changes in cellular mRNA concentrations. By sequencing nascent RNA instead, it is possible to detect changes in transcription in minutes rather than hours and thereby distinguish primary from secondary responses to regulatory signals. Here, we describe the use of PRO-seq to characterize the immediate transcriptional response in human cells to celastrol, a compound derived from traditional Chinese medicine that has potent anti-inflammatory, tumor-inhibitory, and obesity-controlling effects. Celastrol is known to elicit a cellular stress response resembling the response to heat shock, but the transcriptional basis of this response remains unclear. Our analysis of PRO-seq data for K562 cells reveals dramatic transcriptional effects soon after celastrol treatment at a broad collection of both coding and noncoding transcription units. This transcriptional response occurred in two major waves, one within 10 min, and a second 40-60 min after treatment. Transcriptional activity was generally repressed by celastrol, but one distinct group of genes, enriched for roles in the heat shock response, displayed strong activation. Using a regression approach, we identified key transcription factors that appear to drive these transcriptional responses, including members of the E2F and RFX families. We also found sequence-based evidence that particular transcription factors drive the activation of enhancers. We observed increased polymerase pausing at both genes and enhancers, suggesting that pause release may be widely inhibited during the celastrol response. Our study demonstrates that a careful analysis of PRO-seq time-course data can disentangle key aspects of a complex transcriptional response, and it provides new insights into the activity of a powerful pharmacological agent.
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http://dx.doi.org/10.1101/gr.222935.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668940PMC
November 2017

Transcriptional response to stress is pre-wired by promoter and enhancer architecture.

Nat Commun 2017 08 15;8(1):255. Epub 2017 Aug 15.

Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Turku, 20520, Finland.

Programs of gene expression are executed by a battery of transcription factors that coordinate divergent transcription from a pair of tightly linked core initiation regions of promoters and enhancers. Here, to investigate how divergent transcription is reprogrammed upon stress, we measured nascent RNA synthesis at nucleotide-resolution, and profiled histone H4 acetylation in human cells. Our results globally show that the release of promoter-proximal paused RNA polymerase into elongation functions as a critical switch at which a gene's response to stress is determined. Highly transcribed and highly inducible genes display strong transcriptional directionality and selective assembly of general transcription factors on the core sense promoter. Heat-induced transcription at enhancers, instead, correlates with prior binding of cell-type, sequence-specific transcription factors. Activated Heat Shock Factor 1 (HSF1) binds to transcription-primed promoters and enhancers, and CTCF-occupied, non-transcribed chromatin. These results reveal chromatin architectural features that orient transcription at divergent regulatory elements and prime transcriptional responses genome-wide.Heat Shock Factor 1 (HSF1) is a regulator of stress-induced transcription. Here, the authors investigate changes to transcription and chromatin organization upon stress and find that activated HSF1 binds to transcription-primed promoters and enhancers, and to CTCF occupied, untranscribed chromatin.
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http://dx.doi.org/10.1038/s41467-017-00151-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557961PMC
August 2017
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