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Integrative Analysis of Epigenomics and Expression Data in an Immune Cell Proliferation System
1. Integrative Analysis of Epigenomics and Expression
data in an Immune Cell Proliferation System
Esteban Ballestar
Chromatin and Disease Group
Cancer Epigenetics and Biology Programme (PEBC)
Bellvitge Medical Research Institute (IDIBELL)
Barcelona,
Barcelona Spain
eballestar@idibell.org
PEBC
COST‐STATEGRA Workshop
2. DNA methylation is the most studied epigenetic modification
Methyl group introduced in the 5’ position of cytosine
In CG dinucleotides
Methylation of promoter CpG islands leads to transcriptional silencing
y p p p g
Gene DNA repeats
Promoter &
CpG is land Body of the gene
HDAC HDAC HDAC
MBD MBD MBD
E1 E2 E3
x SILENCING
GENE EXPRESSION
Inactive X‐chromosome, imprinted and tissue‐specific genes
Maintained by
M i t i d b DNA methyltransferases. Id tit of active d
th lt f Identity f ti demethylases
th l
controversial
5. Interplay between epigenetic modifications
and miRNAs in gene regulation
Transcriptional control
T i i l l
Epigenetics + transcription factors TF
p
promoter miRNA gene
g
Post‐transcriptional control
miRNAs + RNA binding proteins
TF mature miRNA
promoter protein gene
mature mRNA
6. DNA methylation in changes in cancer
Normal cell
Normal cell
Gene DNA repeats
Promoter &
CpG island Body of the gene
•Unmethylated CpG
•Methylated CpG
E1 E2 E3
GENE EXPRESSION
Cancer cell
C ll
E1 E2 E3
x GENE SILENCING
Aberrant DNA Global DNA
hypermethylation of tumor
hypermethylation of tumor hypomethylation
suppressor genes
Chromosomal
Chromosomal
Gene repression instability
7. DNA methylation changes in different models of immune
disease‐related disease: predominance of DNA
hypomethylation
h th l ti
• ICF syndrome is a rare autosomal recessive disease characterized by a variable
immunodeficiency, mild facial anomalies, and centromeric decondensation—
chromosomal instability involving chromosomes 1, 9, and 16, (1, 2). Hypomethylation of
the satellite 2 and satellite 3 regions of chromosomes 1, 9, and 16 (3).
• Autoimmune diseases are characterized by the breakdown of immune tolerance to
specific self‐antigens. Two basic types: systemic (systemic lupus erythematosus,
rheumatoid arthritis and psoriasis) and organ‐specific (Sjögren’s syndrome, type 1
diabetes and multiple sclerosis). Analysis of different lymphocyte subsets have revealed a
predominance of DNA hypomethylation/overexpression in key genes for immune
function.
8. ICF syndrome: mutations in DNMT3b and hypomethylation
PNAS 96, 14412–14417 (1999)
Decrease of DNA methylation level of 42%, profound changes occurring in
inactive heterochromatic regions, satellite repeats and transposons.
Transcriptional active loci and ribosomal RNA repeats escape global
hypomethylation. Despite a genome‐wide loss of DNA methylation the
epigenetic landscape and crucial regulatory structures are conserved.
[Heyn et al (2012) Epigenetics]
10. MZ twins discordant for autoimmune diseases to investigate the
role of DNA methylation in pathogenesis
y p g
Collection of MZ twins discordant for several AI diseases: SLE, RA, DM
PBMC
Clinically caracterized samples: age, activity, tissue damage
Fred Miller, Environmental Autoimmunity Group, NIEHS, NIH
Methylation Arrays
807 CpG‐containing gene promoter probes
Selected genes fall into various classes:
tumor suppressor genes
oncogenes
genes involved in DNA repair
cell cycle control
differentiation
differentiation
apoptosis
X‐linked
imprinted genes
11. A set of genes display DNA hypomethylation in SLE with respect to healthy twins
MATCHED CONTROLS HEALTHY TWINS SLE TWINS
TRIP6
TM7SF3
LCN2
IL10
ERCC3
MMP8
THPO
MAP3K8
CSF3
MST1R
AGXT
SOD3
LCN2
PI3
CSF1R
TNFRSF1AM
PO
NOTCH4
RARA
EMR3
GRB7
GRB10
CARD15
IFNGR2
CD82
CARD15
STAT5A
GFI1
SEPT9
LTB4R
HGF
SPI1
PECAM1
PADI4
MMP9
PECAM1
TIE1
SLC5A5
MPL
SYK
SLC22A18
S100A2
CD9
CSF3R
LMO2
SPI1
LMO2
DCHR24
HOXB2
MMP14
EPHA2
VAMP8
AIM2
SPDEF
‐6.0 ‐5.4 ‐4.7 ‐4.1 ‐3.5 ‐2.8 ‐2.2 ‐1.6 ‐0.32 0.95 1.6 2.2 2.8 3.5 4.1 4.7 5.4 6.0
Javierre et al (2010) Genome Res
12. DNA methylation changes associated with conversion of resting B
cells to proliferating lymphoblasts
p g y p
Resting B cell EBV LCLs
Primary Infection continuous B cell proliferation (naïve hosts, immunocompromised)
type III latency
l
Latency Cancer: Burkitt Lymphoma, Hodking Lymphoma, Diffuse large‐cell lymphoma (DLBCL),
Nasopharyngeal C i
N h l Carcinoma
Autoimmune Diseases: Systemic Lupus Erithematosus, Rheumatoid Arthritis, Multiple
Sclerosis
13. EBV‐mediated B cell to LCL transformation associates with promoter hypomethylation
RBL LCL
M F M F
CCL3L1 1.0 1.0
Beta Value LCL F1
FCER2
Ls
SLAMF7
Beta Value LCL
0.8
08 0.8
BLNK
IL25 0.6 0.6
IRS2
0.4 0.4
TRAF1 0.2 0.2
TAP1
CD19 0.0 0.0
IL21 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Beta Value RBLs Beta Value RBL F1
COLEC12
1.0 1.0
Beta Value LC L Male
Beta Value LC F2
0.8 0.8
CL
MAP3K7IP1
BLK 0.6 0.6
CCR7
0.4 0.4
0.2 0.2
TCL1A
0.0 0.0
CD1C
0.0 0.2 0.4 0.6 0.8 1.0
00 02 04 06 08 10 0.0 0.2 0.4 0.6 0.8 1.0
00 02 04 06 08 10
CD80 Beta Value RBL Male Beta Value RBL F2
CD79A
Beta Value LC Female
1.0 1.0
Beta Value LCL F3
0.8 0.8
CL
LCK 0.6 0.6
e
0.4 0.4
0.2 0.2
DOK3 0.0 0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
-3 0 3
27K Beta V l
B Value RBL F
RB Female
l Beta Value
B t V l RBL F3
256 genes hypomethylated in LCLs
(FDR ≤ 0.05 & Fold‐change ≥ 2)
Hernando et al (2013) Genome Biol.
14. No changes in DNA methylation in repeats in EBV‐mediated
B cell to LCL transformation
Hernando et al (2013) Genome Biol.
16. Potential pathways to DNA demethylation
• DNA hypomethylation associated with inefficient/defective maintenance of DNA
methylation throughout replication cycles
• Active DNA demethylation
19. AID not involved in demethylation in RBL to LCL conversion
-LMB +LMB
DAPI Anti-HA MERGE DAPI Anti-HA MERGE
OCK
MO
AID WT
A
BLNK CCL3L1 CD19
TSS TSS TSS
+149 bp +119 bp +87 bp
+122 bp +122 bp +122 bp
3 2 2
1,5 1,5
2
1 1
1
0,5 0,5
0 0 0
28. Demethylating agents promote transformation and
proliferation
29. Conclusions
• Transformation of resting B cells into proliferating lymphoblasts involves
hypomethylation of around 250 genes. No hypermethylation is detected.
• A significant group of those 250 hypomethylated genes are already highly expressed in
B cells, are bound by NFkB RELA and REL and other B cell specific transcription factors
and their expression levels do not change during this process.
• Hypomethylation does not appear to occur through an active process and it is likely
that is associated with the inefficient maintenance of DNA methylation at active regions
(it does not occur at repetitive heterochromatic regions)
• Demethylation may contribute to the efficiency of the process by further enhancing
gene upregulation of certain genes
30. Chromatin and Disease Group, IDIBELL, Barcelona Spain Environmental Autoimmunity, NIEHS, NIH, Bethesda
Laura Ciudad Terry O’Hanlon
Henar Hernando Lisa G. Rider
Virginia Rodríguez Fred Miller
Roser Vento
Lorenzo de la Rica University of Oklahoma
José Urquiza Amr Sawalha (U Michigan)
Lluís Pons John Harley (CCHMC)
Javier Rodríguez‐Ubreva
Computational Medicine Unit, Karolinska Institutet, Stokholm, Sweden
Leiden University Medical Center David Gómez‐Cabrero
René Toes Jesper Tegnér
University of Birmingham
Claire Shannon‐Lowe
Claire Shannon‐Lowe
Broad Institute
Fatima Al‐Shahrour
INNPACTO, SAF
FUNDACIÓN
RAMÓN ARECES
PEBC