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

1. Epigenetics and cell fate in JIA and
pulmonary fibrosis
Jim Hagood
UCSD/RCHSD Division of Respiratory Medicine
Caring, Curing, Discovering

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. Adults-G. Cosgrove U Colorado Kids-G. Kurland U Pittsburgh
ILD: Big Picture

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. 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. 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. 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. 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

10. 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

11. Histone Modifications
Barnes P, Proc Am Thorac Soc. 2009 Dec;6(8):693-6.

12. Non-histone effects of histone
modifiers
Barnes P, Proc Am Thorac Soc. 2009 Dec;6(8):693-6.

13. Non-coding RNA
Costa F. Bioessays 32: 599–608, 2010

14. 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

15. 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

16. 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

17. 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

18. 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

19. IPF Normal
Up-RegulatedDown-Regulated
16
DNA
methylation
array
RNA
expression
array
RNA Expression Array-IPF

20. -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

21. 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

22. 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

23. “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

24. 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

25. 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

26. European Respiratory Society Monographs, Vol. 56. 2012.P.97-114; www.smm.org
Genome
Development
Environment
Aging
Fibroblast
Myeloid cell Stem cell
Epithelial cell

27. 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

28. 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

29. 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

30. The epigenetic therapy pipeline
Dawson, MA. Cell 150: 12, 2012

31. Morrow, KJ. BioMarket Trends, Oct 15, 2012 (Vol. 32, No. 18); The Economist, Apr. 7 2012

32. 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
* *
*
* *
*
†
†

33. 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”

34. 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!