Publications by authors named "Austin E Gillen"

30 Publications

  • Page 1 of 1

Dynamic RNA Regulation in the Brain Underlies Physiological Plasticity in a Hibernating Mammal.

Front Physiol 2020 18;11:624677. Epub 2021 Jan 18.

RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, United States.

Hibernation is a physiological and behavioral phenotype that minimizes energy expenditure. Hibernators cycle between profound depression and rapid hyperactivation of multiple physiological processes, challenging our concept of mammalian homeostasis. How the hibernator orchestrates and survives these extremes while maintaining cell to organismal viability is unknown. Here, we enhance the genome integrity and annotation of a model hibernator, the 13-lined ground squirrel. Our new assembly brings this genome to near chromosome-level contiguity and adds thousands of previously unannotated genes. These new genomic resources were used to identify 6,505 hibernation-related, differentially-expressed and processed transcripts using RNA-seq data from three brain regions in animals whose physiological status was precisely defined using body temperature telemetry. A software tool, squirrelBox, was developed to foster further data analyses and visualization. SquirrelBox includes a comprehensive toolset for rapid visualization of gene level and cluster group dynamics, sequence scanning of -mer and domains, and interactive exploration of gene lists. Using these new tools and data, we deconvolute seasonal from temperature-dependent effects on the brain transcriptome during hibernation for the first time, highlighting the importance of carefully timed samples for studies of differential gene expression in hibernation. The identified genes include a regulatory network of RNA binding proteins that are dynamic in hibernation along with the composition of the RNA pool. In addition to passive effects of temperature, we provide evidence for regulated transcription and RNA turnover during hibernation. Significant alternative splicing, largely temperature dependent, also occurs during hibernation. These findings form a crucial first step and provide a roadmap for future work toward defining novel mechanisms of tissue protection and metabolic depression that may 1 day be applied toward improving human health.
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http://dx.doi.org/10.3389/fphys.2020.624677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848201PMC
January 2021

Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System.

Cell Mol Gastroenterol Hepatol 2021 19;11(2):573-595. Epub 2020 Sep 19.

Division of Gastroenterology and Hepatology, University of Colorado Anschutz Medical Campus, Aurora, Colorado; RNA Biosciences Initiative, University of Colorado Anschutz Medical Campus, Aurora, Colorado; Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Electronic address:

Background And Aims: As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease.

Methods: To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo.

Results: Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries.

Conclusions: We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.
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http://dx.doi.org/10.1016/j.jcmgh.2020.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803659PMC
September 2020

Analysis of circulating breast cancer cell heterogeneity and interactions with peripheral blood mononuclear cells.

Mol Carcinog 2020 10 21;59(10):1129-1139. Epub 2020 Aug 21.

Department of Medicine, Division of Medical Oncology, University of Colorado, Aurora, Colorado.

For solid tumors, extravasation of cancer cells and their survival in circulation represents a critical stage of the metastatic process that lacks complete understanding. Gaining insight into interactions between circulating tumor cells (CTCs) and other peripheral blood mononuclear cells (PBMCs) may provide valuable prognostic information. The purpose of this study was to use single-cell RNA-sequencing (scRNA-seq) of liquid biopsies from breast cancer patients to begin defining intravascular interactions. We captured CTCs from the peripheral blood of breast cancer patients using size-exclusion membranes followed by scRNA-seq of enriched CTCs and carry-over PBMCs. Transcriptome analysis identified two populations of CTCs: one enriched for transcripts indicative of estrogen responsiveness and increased proliferation and another enriched for transcripts characteristic of reduced proliferation and epithelial-mesenchymal transition (EMT). We applied interactome and pathway analysis to determine interactions between CTCs and other captured cells. Our analysis predicted for enhanced immune evasion in the CTC population with EMT characteristics. In addition, PD-1/PD-L1 pathway activation and T cell exhaustion were predicted in T cells isolated from breast cancer patients compared with normal T cells. We conclude that scRNA-seq of breast cancer CTCs generally stratifies them into two types based on their proliferative and epithelial state and differential potential to interact with PBMCs. Better understanding of CTC subtypes and their intravascular interactions may help design treatments directed against CTCs with high metastatic and immune-evasive competence.
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http://dx.doi.org/10.1002/mc.23242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895311PMC
October 2020

Single-Cell RNA Sequencing of Childhood Ependymoma Reveals Neoplastic Cell Subpopulations That Impact Molecular Classification and Etiology.

Cell Rep 2020 08;32(6):108023

Morgan Adams Foundation Pediatric Brain Tumor Research Program, Children's Hospital Colorado, Aurora, CO 80045, USA; Department of Pediatrics, University of Colorado Denver, Aurora, CO 80045, USA. Electronic address:

Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in most children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We, therefore, use single-cell RNA sequencing, histology, and deconvolution to catalog cellular heterogeneity of the major childhood EPN subgroups. Analysis of PFA subgroup EPN reveals evidence of an undifferentiated progenitor subpopulation that either differentiates into subpopulations with ependymal cell characteristics or transitions into a mesenchymal subpopulation. Histological analysis reveals that progenitor and mesenchymal subpopulations co-localize in peri-necrotic zones. In conflict with current classification paradigms, relative PFA subpopulation proportions are shown to determine bulk-tumor-assigned subgroups. We provide an interactive online resource that facilitates exploration of the EPN single-cell dataset. This atlas of EPN cellular heterogeneity increases understanding of EPN biology.
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http://dx.doi.org/10.1016/j.celrep.2020.108023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452755PMC
August 2020

clustifyr: an R package for automated single-cell RNA sequencing cluster classification.

F1000Res 2020 1;9:223. Epub 2020 Apr 1.

RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora,, CO, 80045, USA.

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr.
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http://dx.doi.org/10.12688/f1000research.22969.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383722PMC
February 2021

Fibroblast subtypes define a metastatic matrisome in breast cancer.

JCI Insight 2020 02 27;5(4). Epub 2020 Feb 27.

Division of Medical Oncology, Department of Medicine.

Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision-making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this analysis is insufficient - that the stromal microenvironment of the primary tumor plays a key role in tumor cell dissemination and implantation at distant sites. We previously described 2 cancer-associated fibroblasts (CAFs) that either express (CD146+) or lack (CD146-) CD146 (official symbol MCAM, alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer cells: CD146- fibroblasts promoted increased metastasis compared with CD146+ fibroblasts. Potentially novel quantitative and qualitative proteomic analyses showed that CD146+ CAFs produced an environment rich in basement membrane proteins, while CD146- CAFs exhibited increases in fibronectin 1, lysyl oxidase, and tenascin C, all overexpressed in aggressive disease. We also show clinically that CD146- CAFs predicted for likelihood of lymph node involvement even in small primary tumors (<5 cm). Clearly small tumors enriched for CD146- CAFs require aggressive treatments.
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http://dx.doi.org/10.1172/jci.insight.130751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101155PMC
February 2020

Monocytic Subclones Confer Resistance to Venetoclax-Based Therapy in Patients with Acute Myeloid Leukemia.

Cancer Discov 2020 04 23;10(4):536-551. Epub 2020 Jan 23.

Division of Hematology, University of Colorado School of Medicine, Aurora, Colorado.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. SIGNIFICANCE: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.
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http://dx.doi.org/10.1158/2159-8290.CD-19-0710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124979PMC
April 2020

Enhancing Hematopoiesis from Murine Embryonic Stem Cells through MLL1-Induced Activation of a Rac/Rho/Integrin Signaling Axis.

Stem Cell Reports 2020 02 16;14(2):285-299. Epub 2020 Jan 16.

Department of Pediatrics, Section of Hematology/Oncology/BMT University of Colorado, Denver/Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Pharmacology, University of Colorado, Denver/Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

The Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for development and maintenance of hematopoietic stem cells (HSCs), however, whether this protein is limiting for HSC development is unknown due to lack of physiologic model systems. Here, we develop an MLL1-inducible embryonic stem cell (ESC) system and show that induction of wild-type MLL1 during ESC differentiation selectively increases hematopoietic potential from a transitional c-Kit/Cd41 population in the embryoid body and also at sites of hematopoiesis in embryos. Single-cell sequencing analysis illustrates inherent heterogeneity of the c-Kit/Cd41 population and demonstrates that MLL1 induction shifts its composition toward multilineage hematopoietic identities. Surprisingly, this does not occur through increasing Hox or other canonical MLL1 targets but through an enhanced Rac/Rho/integrin signaling state, which increases responsiveness to Vla4 ligands and enhances hematopoietic commitment. Together, our data implicate a Rac/Rho/integrin signaling axis in the endothelial to hematopoietic transition and demonstrate that MLL1 actives this axis.
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http://dx.doi.org/10.1016/j.stemcr.2019.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013201PMC
February 2020

A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice.

Sci Rep 2019 08 14;9(1):11828. Epub 2019 Aug 14.

Dalton Cardiovascular Research Center, University of Missouri, 134 Research Park Dr, Columbia, Missouri, 65211-3300, USA.

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5' and 25 kb of DNA 3' to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr- (H+/m-) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (I) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔI responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.
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http://dx.doi.org/10.1038/s41598-019-48105-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694137PMC
August 2019

Chronic Liver Disease in Humans Causes Expansion and Differentiation of Liver Lymphatic Endothelial Cells.

Front Immunol 2019 15;10:1036. Epub 2019 May 15.

Division of Gastroenterology and Hepatology, Department of Medicine, School of Medicine, University of Colorado, Aurora, CO, United States.

Liver lymphatic vessels support liver function by draining interstitial fluid, cholesterol, fat, and immune cells for surveillance in the liver draining lymph node. Chronic liver disease is associated with increased inflammation and immune cell infiltrate. However, it is currently unknown if or how lymphatic vessels respond to increased inflammation and immune cell infiltrate in the liver during chronic disease. Here we demonstrate that lymphatic vessel abundance increases in patients with chronic liver disease and is associated with areas of fibrosis and immune cell infiltration. Using single-cell mRNA sequencing and multi-spectral immunofluorescence analysis we identified liver lymphatic endothelial cells and found that chronic liver disease results in lymphatic endothelial cells (LECs) that are in active cell cycle with increased expression of CCL21. Additionally, we found that LECs from patients with NASH adopt a transcriptional program associated with increased IL13 signaling. Moreover, we found that oxidized low density lipoprotein, associated with NASH pathogenesis, induced the transcription and protein production of IL13 in LECs both and in a mouse model. Finally, we show that oxidized low density lipoprotein reduced the transcription of and decreased lymphatic stability. Together these data indicate that LECs are active participants in the liver, expanding in an attempt to maintain tissue homeostasis. However, when inflammatory signals, such as oxidized low density lipoprotein are increased, as in NASH, lymphatic function declines and liver homeostasis is impeded.
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http://dx.doi.org/10.3389/fimmu.2019.01036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530422PMC
September 2020

Single cell RNA sequencing identifies TGFβ as a key regenerative cue following LPS-induced lung injury.

JCI Insight 2019 03 26;5. Epub 2019 Mar 26.

Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, National Jewish Health, Denver, Colorado, USA.

Many lung diseases result from a failure of efficient regeneration of damaged alveolar epithelial cells (AECs) after lung injury. During regeneration, AEC2s proliferate to replace lost cells, after which proliferation halts and some AEC2s transdifferentiate into AEC1s to restore normal alveolar structure and function. Although the mechanisms underlying AEC2 proliferation have been studied, the mechanisms responsible for halting proliferation and inducing transdifferentiation are poorly understood. To identify candidate signaling pathways responsible for halting proliferation and inducing transdifferentiation, we performed single cell RNA sequencing on AEC2s during regeneration in a murine model of lung injury induced by intratracheal LPS. Unsupervised clustering revealed distinct subpopulations of regenerating AEC2s: proliferating, cell cycle arrest, and transdifferentiating. Gene expression analysis of these transitional subpopulations revealed that TGFβ signaling was highly upregulated in the cell cycle arrest subpopulation and relatively downregulated in transdifferentiating cells. In cultured AEC2s, TGFβ was necessary for cell cycle arrest but impeded transdifferentiation. We conclude that during regeneration after LPS-induced lung injury, TGFβ is a critical signal halting AEC2 proliferation but must be inactivated to allow transdifferentiation. This study provides insight into the molecular mechanisms regulating alveolar regeneration and the pathogenesis of diseases resulting from a failure of regeneration.
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http://dx.doi.org/10.1172/jci.insight.123637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538357PMC
March 2019

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Nucleic Acids Res 2019 02;47(4):e20

RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.
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http://dx.doi.org/10.1093/nar/gky1204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393243PMC
February 2019

Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in patients with acute myeloid leukemia.

Nat Med 2018 12 12;24(12):1859-1866. Epub 2018 Nov 12.

Division of Hematology, University of Colorado School of Medicine, Aurora, CO, USA.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse. In this report, we show that treatment of older patients with AML with the B cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the tricarboxylic acid (TCA) cycle manifested by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in patients with AML by disrupting the metabolic machinery driving energy metabolism, resulting in promising clinical activity in a patient population with historically poor outcomes.
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http://dx.doi.org/10.1038/s41591-018-0233-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001730PMC
December 2018

Dynamic temperature-sensitive A-to-I RNA editing in the brain of a heterothermic mammal during hibernation.

RNA 2018 11 31;24(11):1481-1495. Epub 2018 Jul 31.

RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA.

RNA editing diversifies genomically encoded information to expand the complexity of the transcriptome. In ectothermic organisms, including and , where body temperature mirrors ambient temperature, decreases in environmental temperature lead to increases in A-to-I RNA editing and cause amino acid recoding events that are thought to be adaptive responses to temperature fluctuations. In contrast, endothermic mammals, including humans and mice, typically maintain a constant body temperature despite environmental changes. Here, A-to-I editing primarily targets repeat elements, rarely results in the recoding of amino acids, and plays a critical role in innate immune tolerance. Hibernating ground squirrels provide a unique opportunity to examine RNA editing in a heterothermic mammal whose body temperature varies over 30°C and can be maintained at 5°C for many days during torpor. We profiled the transcriptome in three brain regions at six physiological states to quantify RNA editing and determine whether cold-induced RNA editing modifies the transcriptome as a potential mechanism for neuroprotection at low temperature during hibernation. We identified 5165 A-to-I editing sites in 1205 genes with dynamically increased editing after prolonged cold exposure. The majority (99.6%) of the cold-increased editing sites are outside of previously annotated coding regions, 82.7% lie in SINE-derived repeats, and 12 sites are predicted to recode amino acids. Additionally, A-to-I editing frequencies increase with increasing cold-exposure, demonstrating that ADAR remains active during torpor. Our findings suggest that dynamic A-to-I editing at low body temperature may provide a neuroprotective mechanism to limit aberrant dsRNA accumulation during torpor in the mammalian hibernator.
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http://dx.doi.org/10.1261/rna.066522.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191720PMC
November 2018

FGFR1 underlies obesity-associated progression of estrogen receptor-positive breast cancer after estrogen deprivation.

JCI Insight 2018 07 26;3(14). Epub 2018 Jul 26.

Division of Endocrinology, Metabolism, & Diabetes, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA.

Obesity increases breast cancer mortality by promoting resistance to therapy. Here, we identified regulatory pathways in estrogen receptor-positive (ER-positive) tumors that were shared between patients with obesity and those with resistance to neoadjuvant aromatase inhibition. Among these was fibroblast growth factor receptor 1 (FGFR1), a known mediator of endocrine therapy resistance. In a preclinical model with patient-derived ER-positive tumors, diet-induced obesity promoted a similar gene expression signature and sustained the growth of FGFR1-overexpressing tumors after estrogen deprivation. Tumor FGFR1 phosphorylation was elevated with obesity and predicted a shorter disease-free and disease-specific survival for patients treated with tamoxifen. In both human and mouse mammary adipose tissue, FGF1 ligand expression was associated with metabolic dysfunction, weight gain, and adipocyte hypertrophy, implicating the impaired response to a positive energy balance in growth factor production within the tumor niche. In conjunction with these studies, we describe a potentially novel graft-competent model that can be used with patient-derived tissue to elucidate factors specific to extrinsic (host) and intrinsic (tumor) tissue that are critical for obesity-associated tumor promotion. Taken together, we demonstrate that obesity and excess energy establish a tumor environment with features of endocrine therapy resistance and identify a role for ligand-dependent FGFR1 signaling in obesity-associated breast cancer progression.
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http://dx.doi.org/10.1172/jci.insight.120594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124402PMC
July 2018

Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells.

J Cyst Fibros 2018 07 17;17(4):444-453. Epub 2018 Feb 17.

Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL, 60614, United States; Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, United States; Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, United States. Electronic address:

Background: Robust methods to culture primary airway epithelial cells were developed several decades ago and these cells provide the model of choice to investigate many diseases of the human lung. However, the molecular signature of cells from different regions of the airway epithelium has not been well characterized.

Methods: We utilize DNase-seq and RNA-seq to examine the molecular signatures of primary cells derived from human tracheal and bronchial tissues, as well as healthy and diseased (cystic fibrosis (CF)) donor lung tissue.

Results: Our data reveal an airway cell signature that is divergent from other epithelial cell types and from common airway epithelial cell lines. The differences between tracheal and bronchial cells are clearly evident as are common regulatory features. Only minor variation is seen between bronchial cells from healthy or CF donors.

Conclusions: These data are a valuable resource for functional genomics analysis of airway epithelial tissues in human disease.
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http://dx.doi.org/10.1016/j.jcf.2018.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026051PMC
July 2018

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases.

Front Oncol 2017 2;7:252. Epub 2017 Nov 2.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2 and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2 BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression , their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER), and 5/9 (55.5%) HER2 clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8-12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.
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http://dx.doi.org/10.3389/fonc.2017.00252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673842PMC
November 2017

Alternative Polyadenylation of Regulates Mitochondrial ROS Signaling and Cancer Outcomes.

Mol Cancer Res 2017 12 14;15(12):1741-1751. Epub 2017 Sep 14.

University of Colorado School of Medicine, RNA Bioscience Initiative, Aurora, Colorado.

Disruption of posttranscriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the mRNA that enhances its steady-state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it has been demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell type-specific manner. Modulation of PRELID1 expression, including its posttranscriptional control, appears to be a common stress response across different cancer types. These data reveal that mRNA processing is an important regulator of cell type-specific responses to stress used by multiple cancers and is associated with patient outcomes. This study suggests that the regulation of PRELID1 expression, by APA and other mechanisms, plays a role in mitochondrial ROS signaling and represents a novel prognostic factor and therapeutic target in cancer. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791535PMC
December 2017

Breast Cancer Suppression by Progesterone Receptors Is Mediated by Their Modulation of Estrogen Receptors and RNA Polymerase III.

Cancer Res 2017 09 20;77(18):4934-4946. Epub 2017 Jul 20.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-3541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600857PMC
September 2017

Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen.

Clin Cancer Res 2017 Apr 4;23(7):1710-1721. Epub 2016 Oct 4.

Department of Medicine, Division of Medical Oncology, University of Colorado Denver, Aurora, Colorado.

Antiendocrine therapy remains the most effective treatment for estrogen receptor-positive (ER) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Tissues from patients with ER breast cancer were analyzed for the presence of CD146-positive (CD146) and CD146-negative (CD146) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER tumor cells cocultured with CD146 or CD146 fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER breast cancer. We demonstrate that ER breast cancers contain two CAF subtypes defined by CD146 expression. CD146 CAFs suppress ER expression in ER breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146 CAFs maintains ER expression in ER breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146 CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146 CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER breast cancer and should be considered a target for drug development. .
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378660PMC
April 2017

Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.

BMC Genomics 2016 05 5;17:338. Epub 2016 May 5.

Department of Medicine, University of Colorado Denver, Aurora, CO, USA.

Background: High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins. Hence, the accuracy and sensitivity of binding site identification is critical.

Results: We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks.

Conclusions: Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.
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http://dx.doi.org/10.1186/s12864-016-2675-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858895PMC
May 2016

The Six1 oncoprotein downregulates p53 via concomitant regulation of RPL26 and microRNA-27a-3p.

Nat Commun 2015 Dec 21;6:10077. Epub 2015 Dec 21.

Program in Molecular Biology, University of Colorado, Denver, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, Colorado 80045, USA.

TP53 is mutated in 50% of all cancers, and its function is often compromised in cancers where it is not mutated. Here we demonstrate that the pro-tumorigenic/metastatic Six1 homeoprotein decreases p53 levels through a mechanism that does not involve the negative regulator of p53, MDM2. Instead, Six1 regulates p53 via a dual mechanism involving upregulation of microRNA-27a and downregulation of ribosomal protein L26 (RPL26). Mutation analysis confirms that RPL26 inhibits miR-27a binding and prevents microRNA-mediated downregulation of p53. The clinical relevance of this interaction is underscored by the finding that Six1 expression strongly correlates with decreased RPL26 across numerous tumour types. Importantly, we find that Six1 expression leads to marked resistance to therapies targeting the p53-MDM2 interaction. Thus, we identify a competitive mechanism of p53 regulation, which may have consequences for drugs aimed at reinstating p53 function in tumours.
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http://dx.doi.org/10.1038/ncomms10077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703841PMC
December 2015

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer.

Breast Cancer Res Treat 2014 Jul 7;146(1):85-97. Epub 2014 Jun 7.

Section of Hematology, Yale Cancer Center, New Haven, CT, USA.

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.
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http://dx.doi.org/10.1007/s10549-014-3004-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115274PMC
July 2014

A genome-wide analysis of open chromatin in human epididymis epithelial cells reveals candidate regulatory elements for genes coordinating epididymal function.

Biol Reprod 2013 Oct 31;89(4):104. Epub 2013 Oct 31.

Human Molecular Genetics Program, Lurie Children's Research Center, and Department of Pediatrics, Northwestern University Feinberg School of Medicine Chicago, Illinois.

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.
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http://dx.doi.org/10.1095/biolreprod.113.110403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076396PMC
October 2013

Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome.

Chromosoma 2013 Oct 9;122(5):351-61. Epub 2013 Jun 9.

Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL, USA.

Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.
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http://dx.doi.org/10.1007/s00412-013-0418-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779492PMC
October 2013

Transcriptional regulation of CFTR gene expression.

Front Biosci (Elite Ed) 2012 Jan 1;4:587-92. Epub 2012 Jan 1.

Human Molecular Genetics Program, Children's Memorial Research Center, and Department of Pediatrics, Northwestern University, Feinberg School of Medicine, Chicago IL, USA.

Cystic Fibrosis results from mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene was identified in 1989, but more than 20 years later, the regulatory mechanisms controlling its complex expression are still not fully understood. Though the promoter binds transcription factors and drives some aspects of CFTR gene expression, it cannot alone account for tissue specific control. This implicates other distal cis-acting elements in cell-type-specific regulation of CFTR expression. Several of these elements, including intronic enhancers and flanking insulators, were identified within or near the CFTR locus. Here we summarize the known regulatory mechanisms controlling CFTR transcription, including those acting through elements in the promoter and others elsewhere in the locus. A special focus will be elements that contribute to tissue specific regulation of expression.
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http://dx.doi.org/10.2741/401DOI Listing
January 2012

Nucleosome occupancy reveals regulatory elements of the CFTR promoter.

Nucleic Acids Res 2012 Jan 24;40(2):625-37. Epub 2011 Sep 24.

Human Molecular Genetics Program, Children's Memorial Research Center, and Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA.

Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the ∼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally.
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http://dx.doi.org/10.1093/nar/gkr754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258145PMC
January 2012

The role of CTCF in coordinating the expression of single gene loci.

Biochem Cell Biol 2011 Oct 14;89(5):489-94. Epub 2011 Sep 14.

Human Molecular Genetics Program, Children's Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine Chicago, IL 60614, USA.

The CCCTC-binding factor (CTCF), which binds insulator elements in vertebrates, also facilitates coordinated gene expression at several gene clusters, including the β-globin, Igf2/H19 (insulin like growth factor 2/H19 noncoding RNA), and major histocompatibility complex (MHC) class II loci. CTCF controls expression of these genes both by enabling insulator function and facilitating higher order chromatin interactions. While the role of CTCF in gene regulation is best studied at these multi-gene loci, there is also evidence that CTCF contributes to the regulated expression of single genes. Here, we discuss how CTCF participates in coordinating gene expression at the CFTR (cystic fibrosis transmembrane conductance regulator) and IFNG (interferon-gamma) loci. We consider the structural similarities between the loci with regard to CTCF-binding elements, the possible interaction between nuclear receptors and CTCF, and the role of CTCF in chromatin looping at these genes. These comparisons reveal a functional model that may be applicable to other single-gene loci that require CTCF for coordinated gene expression.
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http://dx.doi.org/10.1139/o11-040DOI Listing
October 2011

MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene.

Biochem J 2011 Aug;438(1):25-32

Human Molecular Genetics Program, Children's Memorial Research Center, Chicago, IL 60614, USA.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3' UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3' UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsa-miR-1246, also inhibit expression of a reporter carrying the Na(+)-K(+)-Cl(-) co-transporter SLC12A2 [solute carrier family 12 (Na(+)-K(+)-Cl(-) transporters), member 2] 3' UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.
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http://dx.doi.org/10.1042/BJ20110672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323381PMC
August 2011

An insulator element 3' to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells.

Nucleic Acids Res 2009 Mar 7;37(4):1086-94. Epub 2009 Jan 7.

Human Molecular Genetics Program, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA.

Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3'-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.
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http://dx.doi.org/10.1093/nar/gkn1056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651798PMC
March 2009