Integrative Analysis of Epigenomics and Expression data in an Immune Cell Proliferation Systemdata in an Immune Cell Proliferation System
Esteban BallestarEsteban Ballestar
Chromatin and Disease GroupChromatin and Disease GroupCancer Epigenetics and Biology Programme (PEBC)
Bellvitge Medical Research Institute (IDIBELL)Barcelona SpainBarcelona, Spain
PPEEBCBCPPEEBCBC
COST‐STATEGRA Workshop
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
Gene
Promoter &
DNA repeatsGene
Promoter &
DNA repeatsGene
Promoter &
DNA repeats
y p p p g
CpG is land Body of the geneCpG is land Body of the geneCpG is land Body of the gene
HDACMBDHDACMBDMBD
HDACMBDHDACMBDMBD
HDACMBDHDACMBDMBD
GENE EXPRESSION
E1 E2 E3
GENE EXPRESSION
E1 E2 E3
GENE EXPRESSION
E1E1 E2 E3
GENE SILENCINGx GENE SILENCINGxE1 E2 E3E1E1 E2 E3
Inactive X‐chromosome, imprinted and tissue‐specific genes
M i t i d b DNA th lt f Id tit f ti d th lMaintained by DNA methyltransferases. Identity of active demethylasescontroversial
Molecular anatomy of CpG sites in chromatin and their roles in gene expression
Jones (2012) Nat. Rev. Genet
Histone post‐translational modifications
H3
K9 K27K4 K36R17
H3
K20R3
K14K18 K23K9
H4
K20
K79R i
Activation
Acetylation
Phosphorylation
K8 K12 K16K5 Repression
Phosphorylation
Methylation
Ubiquitylation
Interplay between epigenetic modifications
T i i l l
Interplay between epigenetic modificationsand miRNAs in gene regulation
Transcriptional controlEpigenetics + transcription factors
miRNA genemiRNA genepromoterpromoter
TFTF
Post‐transcriptional controlmiRNAs + RNA binding proteins
ggpp
mature miRNA
protein geneprotein genepromoterpromoter
TFTF
mature mRNA
DNA methylation in changes in cancer
Normal cellGene
Promoter & CpG island Body of the gene
DNA repeats
•Unmethylated CpG
Normal cell
C ll
E1 E2 E3 •Methylated CpG
GENE EXPRESSION
Cancer cell
GENE SILENCING
E1 E2 E3
x
Aberrant DNA hypermethylation of tumor
Global DNA hypomethylation
x
hypermethylation of tumor suppressor genes
hypomethylation
ChromosomalGene repression
Chromosomal instability
DNA methylation changes in different models of immunedisease‐related disease: predominance of DNAh th l tihypomethylation
• ICF syndrome is a rare autosomal recessive disease characterized by a variableimmunodeficiency, mild facial anomalies, and centromeric decondensation—chromosomal instability involving chromosomes 1, 9, and 16, (1, 2). Hypomethylation ofthe satellite 2 and satellite 3 regions of chromosomes 1, 9, and 16 (3).
• Autoimmune diseases are characterized by the breakdown of immune tolerance tospecific self‐antigens. Two basic types: systemic (systemic lupus erythematosus,rheumatoid arthritis and psoriasis) and organ‐specific (Sjögren’s syndrome, type 1diabetes and multiple sclerosis). Analysis of different lymphocyte subsets have revealed apredominance of DNA hypomethylation/overexpression in key genes for immunefunction.
ICF syndrome: mutations in DNMT3b and hypomethylation
PNAS 96, 14412–14417 (1999)
Decrease of DNA methylation level of 42%, profound changes occurring ininactive heterochromatic regions, satellite repeats and transposons.Transcriptional active loci and ribosomal RNA repeats escape globalhypomethylation. Despite a genome‐wide loss of DNA methylation theepigenetic landscape and crucial regulatory structures are conserved.[Heyn et al (2012) Epigenetics]
Genetic Elements Hypomethylated in autoimmune diseases
Ballestar (2011) Nat. Rev. Rheumatol
MZ twins discordant for autoimmune diseases to investigate the role of DNA methylation in pathogenesis
Collection of MZ twins discordant for several AI diseases: SLE, RA, DMPBMC
y p g
PBMCClinically caracterized samples: age, activity, tissue damageFred 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 differentiationdifferentiation apoptosis X‐linked imprinted genes
A set of genes display DNA hypomethylation in SLE with respect to healthy twins
TRIP6TM7SF3LCN2IL10ERCC3MMP8THPO
MATCHED CONTROLS HEALTHY TWINS SLE TWINS
THPOMAP3K8CSF3MST1RAGXTSOD3LCN2PI3CSF1RTNFRSF1AMPONOTCH4RARAEMR3GRB7GRB10CARD15IFNGR2CD82CARD15STAT5AGFI1SEPT9LTB4RHGFSPI1PECAM1PADI4MMP9MMP9PECAM1TIE1SLC5A5MPLSYKSLC22A18S100A2CD9CSF3RLMO2SPI1LMO2DCHR24HOXB2MMP14EPHA2VAMP8AIM2SPDEFSPDEF
‐6.0 ‐5.4 ‐4.7 ‐4.1 ‐2.8‐3.5 ‐1.6‐2.2 0.95 1.6 2.2 2.8 4.13.5 5.44.7 6.0‐0.32
Javierre et al (2010) Genome Res
DNA methylation changes associated with conversion of resting B cells to proliferating lymphoblastsp g y p
Resting B cell LCLsEBV
Primary Infection continuous B cell proliferation (naïve hosts, immunocompromised)
ltype III latency
Latency Cancer: Burkitt Lymphoma, Hodking Lymphoma, Diffuse large‐cell lymphoma (DLBCL), N h l C iNasopharyngeal Carcinoma
Autoimmune Diseases: Systemic Lupus Erithematosus, Rheumatoid Arthritis, MultipleSclerosis
EBV‐mediated B cell to LCL transformation associates with promoter hypomethylation
RBL LCL
M F M FCCL3L1FCER2SLAMF7 F1Ls
1.0
0 8
1.0
SLAMF7
BLNKIL25IRS2
TRAF1TAP1 B
eta
Valu
e LC
L
Bet
a Va
lue
LCL 0.8
0.6
0.2
0.4
0.8
0.6
0.2
0.4
CD19IL21
COLEC12
MAP3K7IP1 CL
F2
L M
ale 1.0
0.8
1.0
0.8
Beta Value RBLs Beta Value RBL F1
0.00.2 1.00.80.60.0 0.4
0.00.2 1.00.80.60.0 0.4
BLK
CCR7
CD1C
TCL1A
MAP3K7IP1
Bet
a Va
lue
LC
Bet
a Va
lue
LC 0.6
0.2
0.0
0.4
0 2 1 00 80 60 0 0 4
0.6
0.2
0.0
0.4
0 2 1 00 80 60 0 0 4
CD79A
LCK
CD80
CL
Fem
ale
e LC
L F3
1.0
0.8
0.6
1.0
0.8
0.6
Beta Value RBL Male Beta Value RBL F20.2 1.00.80.60.0 0.4 0.2 1.00.80.60.0 0.4
DOK3
B V l RB F l
Bet
a Va
lue
LC
Bet
a Va
lue
B t V l RBL F3
0.2
0.0
0.4
0.2 1.00.80.60.0 0.4
0.2
0.0
0.4
0.2 1.00.80.60.0 0.427K
256 genes hypomethylated in LCLs (FDR ≤ 0.05 & Fold‐change ≥ 2)
0-3 3 Beta Value RBL Female Beta Value RBL F327K
Hernando et al (2013) Genome Biol.
No changes in DNA methylation in repeats in EBV‐mediatedB cell to LCL transformation
Hernando et al (2013) Genome Biol.
Pyrosequencing confirms promoter hypomethylation
Potential pathways to DNA demethylation
• DNA hypomethylation associated with inefficient/defective maintenance of DNAmethylation throughout replication cycles
• Active DNA demethylation
Potential pathways to DNA demethylation
Demethylation occurs as cell start to proliferate
AID not involved in demethylation in RBL to LCL conversionAID not involved in demethylation in RBL to LCL conversion
MERGEMERGEDAPI Anti-HA
-LMB
DAPI Anti-HA
+LMB
OC
KM
OA
ID W
TA
CD19
+87 bp+122 bp
TSS
BLNK
TSS
+149 bp+122 bp
CCL3L1
TSS
+119 bp+122 bp
3 2 2
0
1
2
3
0
0,5
1
1,5
2
0
0,5
1
1,5
2
Demethylation does not occur in CD40L/IL40 stimulated cells
Hypomethylated genes are relevant to B cell function
Hypomethylated genes display binding motifs for NFkBsubunits and other hematopoietic TFs
ChIP‐seq analysis reveals binding of NFkB and Pol II tohypomethylated promoters
Binding of additional TF to hypomethylated promoters
Nucleic Acids Res 39, 874–888 (2011).
Mol Cell Biol 29, 5366‐5376 (2009)
DNMTs are less efficient in maintaining DNA methylation ineucrhomatic sites as proliferation starts
Hernando et al (2013) Genome Biol.
Hypomethylated genes undergo further upregulationHypomethylated genes undergo further upregulation
Demethylating agents promote transformation andDemethylating agents promote transformation andproliferation
Conclusions
• Transformation of resting B cells into proliferating lymphoblasts involveshypomethylation of around 250 genes. No hypermethylation is detected.
• A significant group of those 250 hypomethylated genes are already highly expressed inB cells, are bound by NFkB RELA and REL and other B cell specific transcription factorsand their expression levels do not change during this process.
• Hypomethylation does not appear to occur through an active process and it is likelythat is associated with the inefficient maintenance of DNA methylation at active regions(it does not occur at repetitive heterochromatic regions)(it does not occur at repetitive heterochromatic regions)
• Demethylation may contribute to the efficiency of the process by further enhancinggene upregulation of certain genesgene upregulation of certain genes
Chromatin and Disease Group, IDIBELL, Barcelona SpainLaura CiudadHenar HernandoVirginia RodríguezRoser Vento
Environmental Autoimmunity, NIEHS, NIH, BethesdaTerry O’HanlonLisa G. RiderFred Miller
Lorenzo de la RicaJosé UrquizaLluís PonsJavier Rodríguez‐Ubreva
Computational Medicine Unit, Karolinska Institutet, Stokholm, Sweden
University of OklahomaAmr Sawalha (U Michigan)John Harley (CCHMC)
Computational Medicine Unit, Karolinska Institutet, Stokholm, SwedenDavid Gómez‐CabreroJesper Tegnér
Leiden University Medical CenterRené Toes
University of BirminghamClaire Shannon‐Lowe
INNPACTO, SAF
Claire Shannon‐Lowe
Broad InstituteFatima Al‐Shahrour
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PPEEBCBCPPEEBCBC
FUNDACIÓN RAMÓN ARECES