Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Epigenetics and cell fate in JIA and pulmonary fibrosis by Jim Hagood

1,416 views

Published on

Epigenetics and cell fate in JIA and pulmonary fibrosis by Jim Hagood

Published in: Health & Medicine
  • How I Cured My Uterine Fibroids? Reverse And Eliminate Uterine Fibroids, Safe & Natural With Fast Results.. ■■■ http://t.cn/Aig7c6mX
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Be the first to like this

Epigenetics and cell fate in JIA and pulmonary fibrosis by Jim Hagood

  1. 1. Epigenetics and cell fate in JIA and pulmonary fibrosis Jim Hagood UCSD/RCHSD Division of Respiratory Medicine Caring, Curing, Discovering
  2. 2. Outline • Lung remodeling in fibrosis • Possible role of epigenetic mechanisms in IPF and autoimmunity, JIA • What can we learn from epigenomics? • miRNA and other non-coding RNA will not be covered • Promise and pitfalls of epigenetics targeted therapy
  3. 3. Adults-G. Cosgrove U Colorado Kids-G. Kurland U Pittsburgh ILD: Big Picture
  4. 4. IPF: Impact • Affects more than 120,000 people in the U.S., with about 48,000 new cases diagnosed annually. 40,000 people die each year to IPF, the same as to breast cancer • IPF is five times more common than cystic fibrosis and Lou Gehrig’s Disease (ALS), yet the disease remains virtually unknown to general public. • IPF receives a fraction of the research funding (IPF: approx. $18 million per year; Cystic Fibrosis and ALS: $85 million and $48 million per year respectively. • There is no known cause, no cure. New FDA-approved treatments slow progression but no impact on mortality. www.coalitionforpf.org
  5. 5. Pathogenesis of IPF Predisposition • Genetic factors • Unknown predisposition • Aging Injury • TGF-β activation • gene- environment interactions • Oxidative damage • Epigenetic changes Disrepair • IPF myofibroblast phenotypes • Pathologic matrix remodeling Combined, prolonged, recurrent insult: injury/inflammation • Infection • Tobacco smoke • Pollutants • Radiation • Gastroesophageal reflux Individual Risk Annu. Rev. Pathol. Mech. Dis. 2014. 9:157–79 Homeostasis shifts from normal to lost: compromised/aberrant repair
  6. 6. Lung Cell Phenotype Regulation • Lung development begins as a simple epithelial tube invading a mesenchymal matrix • Subsequently there is a marked increase in structural complexity, accompanied by cellular differentiation, that persist into adolescence • In addition to genetic influences, interaction with the environment (e.g., infection, toxicants, oxyradicals, mechanical environment) can have major effects on cell phenotype, lung development, and remodeling • Most diffuse/interstitial lung disease is characterized by marked alteration in cellular phenotypes
  7. 7. Epigenetics • study of heritable changes in gene function that occur without a change in the DNA sequence • “The structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states” • DNA methylation, histone acetylation, and RNA interference, and their effects in gene activation and inactivation • DNA is not just a string of bases Bird A, Nature 2007, 447:396–398
  8. 8. Why Epigenetics? • From single cell to 50-75 x 1012 cells, >200 cell types; genome remains the same, for the most part • Disease phenotype variability within single genomic abnormalities • Genetic variants collectively account for a small fraction of the heritability of complex phenotypes • Epigenetic modifications (DNA methylation, histone tail modifications, chromatin remodeling and noncoding RNA expression) have major influence on gene expression, which drives cell phenotype alteration • All disease paradigms (inflammation, wound repair, etc.) relevant to CTD and ILD involve changes in cell phenotype
  9. 9. DNA Methylation • Covalent modification in the 5’ position of cytosine at CpG dinucleotides; catalyzed by DNA methyltransferases (DNMTs); plays a role in the long- term silencing of transcription and in heterochromatin formation. • Non-mutational gene inactivation that can be faithfully propagated from precursor cells to clones of daughter cells. • Genome-wide CpG content is low; CpG islands in gene promoter regions are unmethylated in housekeeping genes, methylated in certain imprinted genes, tissue-restricted genes and inactive X chromosomes in females. Methylation silences transposons and other parasitic elements; correct pattern of genomic methylation essential for healthy tissues and organs • In many cancers there is global hypomethylation (genomic instability) and hypermethylation of specific genes (e.g., tumor suppressors) Hypomethylated Hypermethylated X
  10. 10. Histone Modifications Barnes P, Proc Am Thorac Soc. 2009 Dec;6(8):693-6.
  11. 11. Non-histone effects of histone modifiers Barnes P, Proc Am Thorac Soc. 2009 Dec;6(8):693-6.
  12. 12. Non-coding RNA Costa F. Bioessays 32: 599–608, 2010
  13. 13. Chromatin and Nuclear Architecture • Chromatin: highly ordered structure that contains DNA, RNA, histones and other chromosomal proteins. • Originally classified into two domains, euchromatin and heterochromatin, based on the density of staining in micrographs • Euchromatin is gene-rich, transcriptionally active, hyperacetylated, hypomethylated chromatin. • Heterochromatin is transcriptionally inactive, gene-poor, hypoacetylated and hypermethylated • Lamins (A, B1, B2 and C3) interact with chromatin and each other to create a specific three-dimensional nuclear architecture, disruption of which leads to deformed nuclei, genome instability, age-related diseases and cancer Black JC Epigenetics 6:1, 9-15; January 2011
  14. 14. IPF and epigenetics • IP-10 expression is decreased in F-IPF due to histone modifications and altered recruitment of HATs and HDAC- containing repressor complexes to the IP-10 promoter; expression is restored by HDACand G9a inhibitors • Suberoylanilide hydroxamic acid (SAHA, an HDACi) abrogates TGF-β1 effects on IPF and normal lung fibroblasts by preventing transdifferentiation into α-SMA positive myofibroblasts and increased collagen deposition • THY1 is silenced in IPF fibroblasts; DNMT and HDAC inhibitors restore expression and suppress myofibroblast phenotype • Interaction between DNMT-1 and miR-17~92 regulates multiple profibrotic pathways in IPF lung tissue Coward WR, Mol Cell Biol. 30(12):2874, 2010; Wang Z, Eur Respir J. 34(1):145, 2009; Sanders Y, Am J Respir Cell Mol Biol 39:610, 2008; Marsh CB
  15. 15. Other diseases • Rheumatoid arthritis synovial fibroblasts (RASF): hypermethylation of DR3, hypomethylation of IL6, reversible histone acetylation and apoptosis; altered methylation in mononuclear cells, T cells • Myofibroblastic activation of hepatic stellate cells by epigenetic mechanisms; methylation silencing of SOCS-1 in hepatic fibrosis, hepatocellular carcinoma • HDAC4 required for TGF-b-induced myofibroblastic differentiation of skin fibroblasts • Methylation of FLI1 associated with increased collagen expression in scleroderma fibroblasts Sánchez-Pernaute O, J Autoimmunity 30: 12, 2008; Ellis et al. Clinical Epigenetics 2012, 4:20; Mann DA J Gastroenterol Hepatol 23: S108, 2008; Ogata H, Oncogene 25: 2520, 2006; Glenisson W, BBA-MCR 1773: 1572, 2007; Wang Y, Arthritis Rheum 54: 2271, 2006
  16. 16. Methylation Pattern of miR-17~92 CpG Islands in Control and IPF Human Lung Tissue P=0.0025, N=3 Dakhlallah D, Am J Respir Crit Care Med. 2013 Feb 15;187(4):397-405
  17. 17. Epigenomics: the “methylome”: searching for new targets • Sanders YY, Am J Respir Crit Care Med 2012;186:525–535 – Lung tissue IPF (12, severe, explant, 60.3y) v. normal (7, failed donor, 39y) – Illumina human Methylation27 BeadChip (bisulfite modification, identifies known CpG sites) and human HT-12 BeadChip (RNA) – Validation of selected genes with RT-PCR, methylation-specific PCR, WB, IHC
  18. 18. IPF Normal Up-RegulatedDown-Regulated 16 DNA methylation array RNA expression array RNA Expression Array-IPF
  19. 19. -0.150 -0.100 -0.050 0.000 0.050 0.100 0.150 -8.000 -6.000 -4.000 -2.000 0.000 2.000 4.000 6.000 8.000 10.000 12.000 ΔBeta FoldChange Fold Change (IPF vs Normal) IPF Delta Beta Overlap: Methylation/Expression
  20. 20. Rabinovich Sanders Yang Huang Samples N = 12, lung tissue, severe IPF, mean age 60 N = 12, lung tissue, severe IPF, mean age 60.3 N=94, lung tissue from subjects with IPF, mean age 64.8 N=6, lung fibroblast from IPF patients, mean age 58.4 Controls N = 10, adenoCa and uninvolved lung, mean age 71 N = 7, lung tissue, failed donors, mean age 39 N=67, lung tissue, mean age 64 N=3, lung fibroblasts, nonfibrotic patients, mean age 56.5; N=3 commercial non fibrotic cell lines Transcriptome Not done Illumina human HT-12 BeadChip Agilent human gene expression microarrays (GE 4 × 44 k v2 or G3 Sure print 8 × 60 k formats) Not done Methylome Agilent human CGI oligonucleotide microarrays Illumina human Methylation27 BeadChip Array Nimblegen CHARM array design Illumina HumanMethylation27 BeadChip Array Genes N/A 373 at > 2-fold difference 738 at > 2-fold difference N/A DMRs 625 at FDR < 5% 870 at p < 0.05 2,130 at p < 0.05 787 at p < 0.05 Validation RT-PCR, EpiTYPER RT-PCR, MSP, WB, IHC EpiTYPER, pyrosequencing, siRNA treatment and IHC Pyrosequencing, RT-PCR, WB Methylation Studies: Characteristics
  21. 21. Rabinovich Sanders Yang Cellular Assembly and Organization Humoral Immune Response Gene Expression Cellular Growth and Proliferation Energy Production Cellular Development Cell Morphology Cellular Assembly and Organization Cellular Growth and Proliferation Cancer Molecular Transport Hematological System Development and Function Cell Signaling DNA Replication Cardiovascular System Development and Function Gene Expression Cellular Growth and Proliferation Organismal Development Cell Death Protein Trafficking Hematopoiesis Methylation Studies: Functional Analysis
  22. 22. “Methylome” Studies: Key Points Limitations Key Insights Based on whole tissue (signals from mixtures of cells) Differential methylation at CpG sites across the genome; confirmed by alternate techniques Different platforms may yield different results Many of the DMRs are outside promoters Omit hydroxymethylcytosine and N6- methyladenine Can be used to identify novel mediators and pathways Confirmation and biological plausibility of differentially methylated genes
  23. 23. Differential Expression, Epigenetic Suppression Fibroblastic focus: Myofibroblasts Predisposition Exacerbation Circulating precursors 2 Interstitial fibroblasts Precursor or Fibroblast Recruitment, EMT 1 3 gene x Me 4 B Scenario 1: Epigenetic Predisposition 5 EMT
  24. 24. Interstitial fibroblasts Thy-1 Selection, Ongoing Recruitment, Epigenetic Changes Fibroblast or Precursor Recruitment Circulating precursors Fibroblastic focus: Myofibroblasts Initiation Progression gene x Me 1 2 3 4 A Scenario 2: Epigenetic Response EMT 5 miR Histone
  25. 25. European Respiratory Society Monographs, Vol. 56. 2012.P.97-114; www.smm.org Genome Development Environment Aging Fibroblast Myeloid cell Stem cell Epithelial cell
  26. 26. Epigenetics and JIA • T cell differentiation is in part epigenetically controlled • T cell methylation different at 145 loci vs. controls (11 after adjusting for methotrexate) • Top networks with differentially methylated loci included ‘immunological disease’ (21), ‘cellular growth and proliferation’ (16), ‘antigen presentation’ (15) and ‘cell-to-cell signalling and interaction’ (15) • Differential IL32 methylation and expression confirmed Ellis et al. Clinical Epigenetics 2012, 4:20
  27. 27. Epigenetics and Autoimmunity • Gender bias in some autoimmune diseases, modest concordance in MZ twins suggest epigenetic contribution • Demethylation of inflammatory loci in SLE T cells and B Cells • Neutrophil “methylome” in SLE has significant demethylation in “interferon signature” loci; similar to prior findings in CD4+ T cells • Multiple alterations in histone acetylation and histone lysine methylation in SLE monocytes • Significant alterations in DNA methylation in RA monocytes and RASF • HDACi block inflammatory cytokine production by RA macrophages Mau T, Front Genet. 2014 Dec 19; Coit P, J Autoimmun. 2015 Jan 28; Grabiec et al, J. Immunol. 184, 2718–2728 ; Jeffries MA, Expert Rev Clin Immunol. 2015 Jan
  28. 28. Epigenetic therapies • DNA methyltransferase (DNMT) inhibitors • Histone deacetylase (HDAC) inhibitors • Many are already in clinical trials for a number of malignancies; many have been tested in animal models of systemic inflammatory disorders or in vitro • Many other chromatin modifications can be targeted by small molecule inhibitors • miRNA-based therapeutics in development • Specificity and targeting are critical • Ongoing study of critical “nodes” controlling epigenetic modifications
  29. 29. The epigenetic therapy pipeline Dawson, MA. Cell 150: 12, 2012
  30. 30. Morrow, KJ. BioMarket Trends, Oct 15, 2012 (Vol. 32, No. 18); The Economist, Apr. 7 2012
  31. 31. Normal Saline Bleomycin Bleo+SAHA (d. 10-28 qod) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Resistance 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 Compliance * * * * * * † †
  32. 32. Next Steps: Sequence-Based Approaches-Potential and Challenges • Non-CpG methylation, hydroxymethylcytosine (5hMC), 5-methyladenine • Chromatin modifications (ChIP-Seq) yield much larger datasets • Limitations of tissue-based studies; dynamic nature of epigenetic alterations • Understanding hierarchy of epigenetic alterations and “epigenome code”
  33. 33. What is needed: JIA • Analysis of DNA methylation, histone modifications, miRs, chromatin organization in well-defined samples • Temporal variation; response to “biologics” • Interaction of epigenetic paradigms, interaction with genome variants, response to environment • Mechanisms of epigenomic alteration and targetable “nodes” • Epigenome as biomarker; especially circulating RNA • Preclinical models and clinical trials of epigenetic- targeted therapies • Funding for additional research!

×