Publications by authors named "Tamas Ordog"

83 Publications

Epigenetic alteration contributes to the transcriptional reprogramming in T-cell prolymphocytic leukemia.

Sci Rep 2021 Apr 15;11(1):8318. Epub 2021 Apr 15.

Division of Hematology, Mayo Clinic, Rochester, MN, USA.

T cell prolymphocytic leukemia (T-PLL) is a rare disease with aggressive clinical course. Cytogenetic analysis, whole-exome and whole-genome sequencing have identified primary structural alterations in T-PLL, including inversion, translocation and copy number variation. Recurrent somatic mutations were also identified in genes encoding chromatin regulators and those in the JAK-STAT signaling pathway. Epigenetic alterations are the hallmark of many cancers. However, genome-wide epigenomic profiles have not been reported in T-PLL, limiting the mechanistic study of its carcinogenesis. We hypothesize epigenetic mechanisms also play a key role in T-PLL pathogenesis. To systematically test this hypothesis, we generated genome-wide maps of regulatory regions using H3K4me3 and H3K27ac ChIP-seq, as well as RNA-seq data in both T-PLL patients and healthy individuals. We found that genes down-regulated in T-PLL are mainly associated with defense response, immune system or adaptive immune response, while up-regulated genes are enriched in developmental process, as well as WNT signaling pathway with crucial roles in cell fate decision. In particular, our analysis revealed a global alteration of regulatory landscape in T-PLL, with differential peaks highly enriched for binding motifs of immune related transcription factors, supporting the epigenetic regulation of oncogenes and genes involved in DNA damage response and T-cell activation. Together, our work reveals a causal role of epigenetic dysregulation in T-PLL.
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http://dx.doi.org/10.1038/s41598-021-87890-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050249PMC
April 2021

ZNF416 is a pivotal transcriptional regulator of fibroblast mechanoactivation.

J Cell Biol 2021 May;220(5)

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN.

Matrix stiffness is a central regulator of fibroblast function. However, the transcriptional mechanisms linking matrix stiffness to changes in fibroblast phenotype are incompletely understood. Here, we evaluated the effect of matrix stiffness on genome-wide chromatin accessibility in freshly isolated lung fibroblasts using ATAC-seq. We found higher matrix stiffness profoundly increased global chromatin accessibility relative to lower matrix stiffness, and these alterations were in close genomic proximity to known profibrotic gene programs. Motif analysis of these regulated genomic loci identified ZNF416 as a putative mediator of fibroblast stiffness responses. Genome occupancy analysis using ChIP-seq confirmed that ZNF416 occupies a broad range of genes implicated in fibroblast activation and tissue fibrosis, with relatively little overlap in genomic occupancy with other mechanoresponsive and profibrotic transcriptional regulators. Using loss- and gain-of-function studies, we demonstrated that ZNF416 plays a critical role in fibroblast proliferation, extracellular matrix synthesis, and contractile function. Together, these observations identify ZNF416 as novel mechano-activated transcriptional regulator of fibroblast biology.
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http://dx.doi.org/10.1083/jcb.202007152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918622PMC
May 2021

Duodenal mucosal mitochondrial gene expression is associated with delayed gastric emptying in diabetic gastroenteropathy.

JCI Insight 2021 01 25;6(2). Epub 2021 Jan 25.

Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.

Hindered by a limited understanding of the mechanisms responsible for diabetic gastroenteropathy (DGE), management is symptomatic. We investigated the duodenal mucosal expression of protein-coding genes and microRNAs (miRNA) in DGE and related them to clinical features. The diabetic phenotype, gastric emptying, mRNA, and miRNA expression and ultrastructure of duodenal mucosal biopsies were compared in 39 DGE patients and 21 controls. Among 3175 differentially expressed genes (FDR < 0.05), several mitochondrial DNA-encoded (mtDNA-encoded) genes (12 of 13 protein coding genes involved in oxidative phosphorylation [OXPHOS], both rRNAs and 9 of 22 transfer RNAs) were downregulated; conversely, nuclear DNA-encoded (nDNA-encoded) mitochondrial genes (OXPHOS) were upregulated in DGE. The promoters of differentially expressed genes were enriched in motifs for transcription factors (e.g., NRF1), which regulate mitochondrial biogenesis. Seventeen of 30 differentially expressed miRNAs targeted differentially expressed mitochondrial genes. Mitochondrial density was reduced and correlated with expression of 9 mtDNA OXPHOS genes. Uncovered by principal component (PC) analysis of 70 OXPHOS genes, PC1 was associated with neuropathy (P = 0.01) and delayed gastric emptying (P < 0.05). In DGE, mtDNA- and nDNA-encoded mitochondrial genes are reduced and increased - associated with reduced mitochondrial density, neuropathy, and delayed gastric emptying - and correlated with cognate miRNAs. These findings suggest that mitochondrial disturbances may contribute to delayed gastric emptying in DGE.
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http://dx.doi.org/10.1172/jci.insight.143596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934845PMC
January 2021

Genomic and Epigenomic Landscaping Defines New Therapeutic Targets for Adenosquamous Carcinoma of the Pancreas.

Cancer Res 2020 10 14;80(20):4324-4334. Epub 2020 Sep 14.

Division of Hematology/Oncology, Department of Internal Medicine, Mayo Clinic, Scottsdale, Arizona.

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in (87%) and (73%), amplification of (47%), and homozygous deletion of (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase and the pancreatic cancer stem cell regulator in all three ASCPs, and a fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the fusion-positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. SIGNIFICANCE: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906529PMC
October 2020

Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

Cell 2020 09 10;182(6):1460-1473.e17. Epub 2020 Sep 10.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA. Electronic address:

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2020.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109273PMC
September 2020

Wnt-induced, TRP53-mediated Cell Cycle Arrest of Precursors Underlies Interstitial Cell of Cajal Depletion During Aging.

Cell Mol Gastroenterol Hepatol 2021 7;11(1):117-145. Epub 2020 Aug 7.

Enteric Neuroscience Program and Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota. Electronic address:

Background & Aims: Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC).

Methods: Mice aged 1-107 weeks, klotho mice, APC mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription-polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference.

Results: The klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G/S and G/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity.

Conclusions: Increased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers.
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http://dx.doi.org/10.1016/j.jcmgh.2020.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672319PMC
August 2020

Translational Opportunities for Microfluidic Technologies to Enable Precision Epigenomics.

Anal Chem 2020 06 4;92(12):7989-7997. Epub 2020 Jun 4.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.

Personalizing health care by taking genetic, environmental, and lifestyle factors into account is central to modern medicine. The crucial and pervasive roles epigenetic factors play in shaping gene-environment interactions are now well recognized. However, identifying robust epigenetic biomarkers and translating them to clinical tests has been difficult due in part to limitations of available platforms to detect epigenetic features genome-wide (epigenomic assays). This Feature introduces several important prospects for precision epigenomics, highlights capabilities and limitations of current laboratory technologies, and emphasizes opportunities for microfluidic tools to facilitate translation of epigenetic analyses to the clinic, with a particular focus on methods to profile gene-associated histone modifications and their impacts on chromatin structure and gene expression.
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http://dx.doi.org/10.1021/acs.analchem.0c01288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053002PMC
June 2020

Inferring multimodal latent topics from electronic health records.

Nat Commun 2020 05 21;11(1):2536. Epub 2020 May 21.

Computer Science and Artificial Intelligence Lab, Massachusetts Institute of Technology, 32 Vassar St, Cambridge, MA, 02139, USA.

Electronic health records (EHR) are rich heterogeneous collections of patient health information, whose broad adoption provides clinicians and researchers unprecedented opportunities for health informatics, disease-risk prediction, actionable clinical recommendations, and precision medicine. However, EHRs present several modeling challenges, including highly sparse data matrices, noisy irregular clinical notes, arbitrary biases in billing code assignment, diagnosis-driven lab tests, and heterogeneous data types. To address these challenges, we present MixEHR, a multi-view Bayesian topic model. We demonstrate MixEHR on MIMIC-III, Mayo Clinic Bipolar Disorder, and Quebec Congenital Heart Disease EHR datasets. Qualitatively, MixEHR disease topics reveal meaningful combinations of clinical features across heterogeneous data types. Quantitatively, we observe superior prediction accuracy of diagnostic codes and lab test imputations compared to the state-of-art methods. We leverage the inferred patient topic mixtures to classify target diseases and predict mortality of patients in critical conditions. In all comparison, MixEHR confers competitive performance and reveals meaningful disease-related topics.
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http://dx.doi.org/10.1038/s41467-020-16378-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242436PMC
May 2020

Epigenetic Alterations Are Associated With Gastric Emptying Disturbances in Diabetes Mellitus.

Clin Transl Gastroenterol 2020 03;11(3):e00136

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA.

Introduction: Epigenetic modifications have been implicated to mediate several complications of diabetes mellitus (DM), especially nephropathy and retinopathy. Our aim was to ascertain whether epigenetic alterations in whole blood discriminate among patients with DM with normal, delayed, and rapid gastric emptying (GE).

Methods: Using the ChIP-seq (chromatin immunoprecipitation combined with next-generation sequencing) assays, we compared the genome-wide enrichment of 3 histone modifications (i.e., H3K4me3, H3K9ac, and H3K27ac) in buffy coats from 20 diabetic patients with gastrointestinal symptoms and normal (n = 6), delayed (n = 8), or rapid (n = 6) GE.

Results: Between patients with DM with delayed vs normal GE, there were 108 and 54 genes that were differentially bound (false discovery rate < 0.05) with H3K27ac and H3K9ac, respectively; 100 genes were differentially bound with H3K9ac in patients with rapid vs normal GE. The differentially bound genes with H3K27ac were functionally linked to the type 2 immune response, particularly Th2 cell activation and function (e.g., CCR3, CRLF2, CXCR4, IL5RA, and IL1RL1) and glucose homeostasis (FBP-1, PDE4A, and CMKLR1). For H3K9ac, the differentially occupied genes were related to T-cell development and function (e.g., ICOS and CCR3) and innate immunity (RELB, CD300LB, and CLEC2D). Compared with normal GE, rapid GE had differential H3K9ac peaks at the promoter site of diverse immunity-related genes (e.g., TNFRSF25 and CXCR4) and genes related to insulin resistance and glucose metabolism. Motif analysis disclosed enrichment of binding sites for transcription factors relevant to the pathogenesis and complications of DM.

Discussion: GE disturbances in DM are associated with epigenetic alterations that pertain to dysimmunity, glucose metabolism, and other complications of DM.
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http://dx.doi.org/10.14309/ctg.0000000000000136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145053PMC
March 2020

microRNA overexpression in slow transit constipation leads to reduced Na1.5 current and altered smooth muscle contractility.

Gut 2020 05 22;69(5):868-876. Epub 2019 Nov 22.

Enteric NeuroScience Program (ENSP), Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA

Objective: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC).

Design: All human tissue samples were from the of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat organotypic cultures.

Results: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target , the gene that encodes the Na channel Na1.5. The expression of mRNA was decreased in STC samples. Let-7f significantly decreased Na current density in vitro in human smooth muscle cells. In rat organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction.

Conclusions: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na channel Na1.5. Within this set, a novel Na1.5 regulator, let-7f, resulted in decreased Na1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.
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http://dx.doi.org/10.1136/gutjnl-2019-318747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147984PMC
May 2020

Chromatin Assembly Factor 1 (CAF-1) facilitates the establishment of facultative heterochromatin during pluripotency exit.

Nucleic Acids Res 2019 12;47(21):11114-11131

Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.

Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.
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http://dx.doi.org/10.1093/nar/gkz858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868363PMC
December 2019

TGFβ-induced fibroblast activation requires persistent and targeted HDAC-mediated gene repression.

J Cell Sci 2019 10 18;132(20). Epub 2019 Oct 18.

Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA

Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβ-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene () in response to TGFβ stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFβ-mediated fibroblast activation via targeted gene repression.
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http://dx.doi.org/10.1242/jcs.233486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826010PMC
October 2019

Single Nucleotide Polymorphisms at a Distance from Aryl Hydrocarbon Receptor (AHR) Binding Sites Influence AHR Ligand-Dependent Gene Expression.

Drug Metab Dispos 2019 09 10;47(9):983-994. Epub 2019 Jul 10.

Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (D.R.N., D.L., H.L., L.W., R.M.W.), Epigenomics Program, Center for Individualized Medicine (J.-H.L., T.O.), Division of Experimental Pathology and Laboratory Medicine, Department of Laboratory Medicine and Pathology (J.-H.L.), Division of Biomedical Statistics and Informatics (Z.Y.), Department of Physiology and Biomedical Engineering (T.O.), and Division of Gastroenterology and Hepatology, Department of Medicine (T.O.), Mayo Clinic, Rochester, Minnesota

Greater than 90% of significant genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) are in noncoding regions of the genome, but only 25.6% are known expression quantitative trait loci (eQTLs). Therefore, the function of many significant GWAS SNPs remains unclear. We have identified a novel type of eQTL for which SNPs distant from ligand-activated transcription factor (TF) binding sites can alter target gene expression in a SNP genotype-by-ligand-dependent fashion that we refer to as pharmacogenomic eQTLs (PGx-eQTLs)-loci that may have important pharmacotherapeutic implications. In the present study, we integrated chromatin immunoprecipitation-seq with RNA-seq and SNP genotype data for a panel of lymphoblastoid cell lines to identify 10 novel PGx-eQTLs dependent on the ligand-activated TF aryl hydrocarbon receptor (AHR)-a critical environmental sensor for xenobiotic (drug) and immune response. Those 10 PGx-eQTLs were eQTLs only after AHR ligand treatment, even though the SNPs did not create/destroy an AHR response element-the DNA sequence motif recognized and bound by AHR. Additional functional studies in multiple cell lines demonstrated that some PGx-eQTLs are functional in multiple cell types, whereas others displayed SNP-by-ligand-dependent effects in just one cell type. Furthermore, four of those PGx-eQTLs had previously been associated with clinical phenotypes, indicating that those loci might have the potential to inform clinical decisions. Therefore, SNPs across the genome that are distant from TF binding sites for ligand-activated TFs might function as PGx-eQTLs and, as a result, might have important clinical implications for interindividual variation in drug response. SIGNIFICANCE STATEMENT: More than 90% of single-nucleotide polymorphisms (SNPs) that are associated with clinical phenotypes are located in noncoding regions of the genome. However, the mechanisms of action of many of those SNPs have not been elucidated, and drugs may unmask functional expression quantitative trail loci (eQTLs). In the current study, we used drugs that bind to the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and identified SNPs that were associated with interindividual variation in gene expression following drug exposure-termed pharmacogenomic (PGx)-eQTLs. Possibly of greater significance, those PGx-eQTL SNPs were outside of AHR binding sites, indicating that they do not interrupt AHR DNA recognition. PGx-eQTLs such as those described in this work may have crucial implications for interindividual variation in drug.
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http://dx.doi.org/10.1124/dmd.119.087312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184190PMC
September 2019

Recurrent mutations in ALK-negative anaplastic large cell lymphoma.

Blood 2019 06 17;133(26):2776-2789. Epub 2019 May 17.

Division of Hematology, Mayo Clinic, Rochester, MN.

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK) or ALK, based on the presence or absence of rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, , exclusively in ALK ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of for ALK ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSC acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene as a direct transcriptional target of musculin. MSC reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the promoter. Finally, ALCL cells expressing were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.
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http://dx.doi.org/10.1182/blood.2019000626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598380PMC
June 2019

Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq.

BMC Genomics 2019 Mar 29;20(1):249. Epub 2019 Mar 29.

Epigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, Stabile building 12-04, 200 First Street SW, Rochester, MN, 55905, USA.

Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.

Results: During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45-60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays.

Conclusions: This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.
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http://dx.doi.org/10.1186/s12864-019-5639-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440302PMC
March 2019

Genomic aberrations in cell cycle genes predict progression of -mutant gastrointestinal stromal tumors (GISTs).

Clin Sarcoma Res 2019 5;9. Epub 2019 Mar 5.

Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany.

Background: Activating mutations of the receptor tyrosine kinase are early events in the development of most gastrointestinal stromal tumors (GISTs). Although GISTs generally remain dependent on oncogenic during tumor progression, mutations alone are insufficient to induce malignant behavior. This is evidenced by -mutant micro-GISTs, which are present in up to one-third of normal individuals, but virtually never progress to malignancy.

Methods: We performed whole exome sequencing on 29 tumors obtained from 21 patients with high grade or metastatic -mutant GIST (discovery set). We further validated the frequency and potential prognostic significance of aberrations in and in an independent series of 71 patients with primary GIST (validation set).

Results: Using whole exome sequencing we found significant enrichment of genomic aberrations in cell cycle-associated genes (Fisher's Exact p = 0.001), most commonly affecting in our discovery set. We found a low mutational tumor burden in these 29 advanced GIST samples, a finding with significant implications for the development of immunotherapy for GIST. In addition, we found mutation of spliceosome genes in a minority of cases, implicating dysregulation of splicing as a potential cancer promoting mechanism in GIST. We next assessed the prognostic significance of or mutation/copy loss in an independent cohort of 71 patients with primary GIST. Genetic events (mutation, deletion, and/or LOH) involving at least one of the three genes examined were found in 17% of the very low-risk, 36% of the low-risk, 42% of the intermediate risk, 67% of the high-risk/low mitotic-count, and in 86% of the high-risk/high mitotic-count group. The presence of cell cycle-related events was associated with a significantly shorter relapse-free survival (median 67 months versus not reached;  < 0.0001) and overall survival (Log Rank, = 0.042).

Conclusion: Our results demonstrate that genomic events targeting cell cycle-related genes are associated with GIST progression to malignant disease. Based on this data, we propose a model for molecular pathogenesis of malignant GIST.
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http://dx.doi.org/10.1186/s13569-019-0112-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399846PMC
March 2019

Direct repression of anoctamin 1 () gene transcription by Gli proteins.

FASEB J 2019 05 25;33(5):6632-6642. Epub 2019 Feb 25.

Enteric NeuroSciences, Mayo Clinic, Rochester, Minnesota, USA.

The Ca-activated Cl channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human promoter contains binding sites for the glioma-associated oncogene (Gli) proteins. We investigated regulation of transcription by Gli. promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca-activated Cl currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome-wide association studies were used to test promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed transcription. Repression depended on Gli binding to a site close to the transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of RNA and Ca-activated Cl currents in HEK293 cells. A single-nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.-Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez-Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 () gene transcription by Gli proteins.
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http://dx.doi.org/10.1096/fj.201802373RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463924PMC
May 2019

HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4CD8 thymocytes for CD4-lineage commitment.

Elife 2019 01 18;8. Epub 2019 Jan 18.

Department of Immunology, Mayo Clinic, Rochester, United States.

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.
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http://dx.doi.org/10.7554/eLife.43821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338460PMC
January 2019

Association Between Renal Cell Carcinoma and Myelodysplastic Syndromes: Epigenetic Underpinning?

Clin Genitourin Cancer 2018 12 28;16(6):e1117-e1122. Epub 2018 Jun 28.

Division of Hematology, Mayo Clinic, Rochester, MN. Electronic address:

Background: Renal cell carcinoma (RCC) and certain myeloid malignancies are both characterized by widespread aberrant DNA hypermethylation. After clinical observations of patients with a personal history of both malignancies, we sought to explore a potential association, and to describe the clinical characteristics of these patients.

Patients And Methods: Mayo Clinic's 'Advanced Cohort Explorer' database was used to identify patients with a history of both malignancies. Clinical features and long-term outcome were abstracted. Prevalence of myelodysplastic syndromes (MDSs) in patients ≥ 65 years with a personal history of nephrectomy for RCC was then compared with the prevalence of MDSs in the Dusseldorf MDS registry and the general patient population at Mayo Clinic, using 1-sample test of proportions.

Results: A total of 59 patients with a diagnosis of both RCC and myeloid malignancy were identified. The myeloid malignancies included 38 MDSs, 12 acute myelogenous leukemia, and 9 myeloproliferative neoplasms. The cohort was characterized by marked male predominance (4.4:1). The median age at RCC diagnosis was 64 years (range, 37-87 years), and for myeloid malignancy was 75 years (range, 44-90 years). Prevalence of MDS in patients > 65 years with a personal history of nephrectomy for RCC was ≈ 8.4 times that of the age-concordant general population based on the Dusseldorf registry (28/6490 or 395/100,000 vs. ≈ 47/100,000; P < .001), and 3.07 times that of the age-concordant patient population at Mayo Clinic (28/6490 or 395/100,000 vs. 128.4/100,000; P < .001).

Conclusions: We observed a strong association between RCC and MDS. Patients with a history of RCC appear to have a substantially increased risk of developing MDS compared with the general population.
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http://dx.doi.org/10.1016/j.clgc.2018.06.008DOI Listing
December 2018

Epigenetics of gastrointestinal diseases: notes from a workshop.

Epigenetics 2018 30;13(4):449-457. Epub 2018 Jul 30.

a Schulze Center for Novel Therapeutics, Division of Oncology Research , Mayo Clinic , Rochester , MN , USA.

International experts gathered at the Mayo Clinic (Rochester MN, USA) on February 27th-28th, 2017 for a meeting entitled 'Basic and Translational Facets of the Epigenetics of GI Diseases'. This workshop summarized recent advances on the role of epigenetics in the pathobiology of gastrointestinal (GI) diseases. Highlights of the meeting included recent advances on the involvement of different epigenetic mechanisms in malignant and nonmalignant GI disorders and the epigenetic heterogeneity exhibited in these diseases. The translational value of epigenetic drugs, as well as the current and future use of epigenetic changes (i.e., DNA methylation patterns) as biomarkers for early detection tools or disease stratification were also important topics of discussion.
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http://dx.doi.org/10.1080/15592294.2018.1464351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140811PMC
February 2019

A droplet microfluidic platform for efficient enzymatic chromatin digestion enables robust determination of nucleosome positioning.

Lab Chip 2018 08;18(17):2583-2592

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

The first step in chromatin-based epigenetic assays involves the fragmentation of chromatin to facilitate precise genomic localization of the associated DNA. Here, we report the development of a droplet microfluidic device that can rapidly and efficiently digest chromatin into single nucleosomes starting from whole-cell input material offering simplified and automated processing compared to conventional manual preparation. We demonstrate the digestion of chromatin from 2500-125 000 Jurkat cells using micrococcal nuclease for enzymatic processing. We show that the yield of mononucleosomal DNA can be optimized by controlling enzyme concentration and incubation time, with resulting mononucleosome yields exceeding 80%. Bioinformatic analysis of sequenced mononucleosomal DNA (MNase-seq) indicated a high degree of reproducibility and concordance (97-99%) compared with conventionally processed preparations. Our results demonstrate the feasibility of robust and automated nucleosome preparation using a droplet microfluidic platform for nucleosome positioning and downstream epigenomic assays.
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http://dx.doi.org/10.1039/c8lc00599kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103843PMC
August 2018

Distinct epigenetic landscapes underlie the pathobiology of pancreatic cancer subtypes.

Nat Commun 2018 05 17;9(1):1978. Epub 2018 May 17.

Programme Cartes d'Identité des Tumeurs (CIT), Ligue Nationale Contre Le Cancer, 14 rue Corvisart, Paris, 75013, France.

Recent studies have offered ample insight into genome-wide expression patterns to define pancreatic ductal adenocarcinoma (PDAC) subtypes, although there remains a lack of knowledge regarding the underlying epigenomics of PDAC. Here we perform multi-parametric integrative analyses of chromatin immunoprecipitation-sequencing (ChIP-seq) on multiple histone modifications, RNA-sequencing (RNA-seq), and DNA methylation to define epigenomic landscapes for PDAC subtypes, which can predict their relative aggressiveness and survival. Moreover, we describe the state of promoters, enhancers, super-enhancers, euchromatic, and heterochromatic regions for each subtype. Further analyses indicate that the distinct epigenomic landscapes are regulated by different membrane-to-nucleus pathways. Inactivation of a basal-specific super-enhancer associated pathway reveals the existence of plasticity between subtypes. Thus, our study provides new insight into the epigenetic landscapes associated with the heterogeneity of PDAC, thereby increasing our mechanistic understanding of this disease, as well as offering potential new markers and therapeutic targets.
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http://dx.doi.org/10.1038/s41467-018-04383-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958058PMC
May 2018

Change in Populations of Macrophages Promotes Development of Delayed Gastric Emptying in Mice.

Gastroenterology 2018 06 6;154(8):2122-2136.e12. Epub 2018 Mar 6.

Enteric Neuroscience Program, Mayo Clinic, Rochester, Minnesota. Electronic address:

Background & Aims: Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying.

Methods: Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45, CD11b, F4/80 macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow-derived macrophages from wild-type mice.

Results: Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45, CD11b, and F4/80 macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying.

Conclusions: In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.
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http://dx.doi.org/10.1053/j.gastro.2018.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985210PMC
June 2018

CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity.

Nat Commun 2018 02 12;9(1):631. Epub 2018 Feb 12.

Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, 19104, USA.

CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 is often overexpressed in human cancers. However, clinically applicable cancer therapeutic strategies based on CARM1 expression remain to be explored. Here, we report that EZH2 inhibition is effective in CARM1-expressing epithelial ovarian cancer. Inhibition of EZH2 activity using a clinically applicable small molecule inhibitor significantly suppresses the growth of CARM1-expressing, but not CARM1-deficient, ovarian tumors in two xenograft models and improves the survival of mice bearing CARM1-expressing ovarian tumors. The observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes in a CARM1-dependent manner. Mechanistically, CARM1 promotes EZH2-mediated silencing of EZH2/BAF155 target tumor suppressor genes by methylating BAF155, which leads to the displacement of BAF155 by EZH2. Together, these results indicate that pharmacological inhibition of EZH2 represents a novel therapeutic strategy for CARM1-expressing cancers.
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http://dx.doi.org/10.1038/s41467-018-03031-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809368PMC
February 2018

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

BMC Genomics 2017 12 21;18(1):985. Epub 2017 Dec 21.

Epigenomics Development Laboratory, Epigenomics Program, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA.

Background: Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored.

Results: We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents.

Conclusions: This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.
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http://dx.doi.org/10.1186/s12864-017-4371-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740926PMC
December 2017

Conserved DNA methylation combined with differential frontal cortex and cerebellar expression distinguishes C9orf72-associated and sporadic ALS, and implicates SERPINA1 in disease.

Acta Neuropathol 2017 11 14;134(5):715-728. Epub 2017 Aug 14.

Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.

We previously found C9orf72-associated (c9ALS) and sporadic amyotrophic lateral sclerosis (sALS) brain transcriptomes comprise thousands of defects, among which, some are likely key contributors to ALS pathogenesis. We have now generated complementary methylome data and combine these two data sets to perform a comprehensive "multi-omic" analysis to clarify the molecular mechanisms initiating RNA misregulation in ALS. We found that c9ALS and sALS patients have generally distinct but overlapping methylome profiles, and that the c9ALS- and sALS-affected genes and pathways have similar biological functions, indicating conserved pathobiology in disease. Our results strongly implicate SERPINA1 in both C9orf72 repeat expansion carriers and non-carriers, where expression levels are greatly increased in both patient groups across the frontal cortex and cerebellum. SERPINA1 expression is particularly pronounced in C9orf72 repeat expansion carriers for both brain regions, where SERPINA1 levels are strictly down regulated across most human tissues, including the brain, except liver and blood, and are not measurable in E18 mouse brain. The altered biological networks we identified contain critical molecular players known to contribute to ALS pathology, which also interact with SERPINA1. Our comprehensive combined methylation and transcription study identifies new genes and highlights that direct genetic and epigenetic changes contribute to c9ALS and sALS pathogenesis.
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http://dx.doi.org/10.1007/s00401-017-1760-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647251PMC
November 2017

Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying.

Gastroenterology 2017 08 21;153(2):521-535.e20. Epub 2017 Apr 21.

Enteric Neuroscience Program, Mayo Clinic, Rochester, Minnesota; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota. Electronic address:

Background & Aims: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Lepr), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc).

Methods: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase.

Results: In obese, hyperglycemic, hyperinsulinemic female Lepr mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female Kit mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc.

Conclusions: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.
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http://dx.doi.org/10.1053/j.gastro.2017.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526732PMC
August 2017

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix.

Proc Natl Acad Sci U S A 2017 04 27;114(15):3933-3938. Epub 2017 Mar 27.

Department of Dermatology, Mayo Clinic, Rochester, MN 55905;

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)-kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.
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http://dx.doi.org/10.1073/pnas.1614894114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393228PMC
April 2017