Integrative Analysis of Epigenomics and miRNA data in Immune System Models

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Integrative Analysis of Epigenomics and miRNA data in Immune System Models

  1. 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 PEBCCOST‐STATEGRA Workshop
  2. 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
  3. 3. Molecular anatomy of CpG sites in chromatin and their roles in gene expressionJones (2012) Nat. Rev. Genet
  4. 4. Histone post‐translational modifications K4 K9 R17 K27 K36H3 K9 K14 K18 K23 R3 K20 H4 Activation K79 K5 K8 K12 K16 Repression R i Acetylation Phosphorylation Methylation Ubiquitylation
  5. 5. Interplay between epigenetic modifications and miRNAs in gene regulation Transcriptional control T i i l lEpigenetics + transcription factors TF p promoter miRNA gene g Post‐transcriptional control miRNAs + RNA binding proteins TF mature miRNA promoter protein gene mature mRNA
  6. 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. 7. DNA methylation changes in different models of immunedisease‐related disease: predominance of DNAhypomethylationh th l ti• 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.
  8. 8. ICF syndrome: mutations in DNMT3b and hypomethylationPNAS 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]
  9. 9. Genetic Elements Hypomethylated in autoimmune diseasesBallestar (2011) Nat. Rev. Rheumatol
  10. 10. MZ twins discordant for autoimmune diseases to investigate the role of DNA methylation in pathogenesis y p gCollection 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, NIHMethylation Arrays807 CpG‐containing gene promoter probesSelected 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. 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 6.0Javierre et al (2010) Genome Res
  12. 12. DNA methylation changes associated with conversion of resting B  cells to proliferating lymphoblasts p g y p Resting B cell EBV LCLsPrimary Infection continuous B cell proliferation (naïve hosts, immunocompromised) type III latency lLatency 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. 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. 14. No changes in DNA methylation in repeats in EBV‐mediated B cell to LCL transformationHernando et al (2013) Genome Biol.
  15. 15. Pyrosequencing confirms promoter hypomethylation
  16. 16. Potential pathways to DNA demethylation• DNA hypomethylation associated with inefficient/defective maintenance of DNAmethylation throughout replication cycles• Active DNA demethylation
  17. 17. Potential pathways to DNA demethylation
  18. 18. Demethylation occurs as cell start to proliferate
  19. 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
  20. 20. Demethylation does not occur in CD40L/IL40 stimulated cells
  21. 21. Hypomethylated genes are relevant to B cell function
  22. 22. Hypomethylated genes display binding motifs for NFkBsubunits and other hematopoietic TFs
  23. 23. ChIP‐seq analysis reveals binding of NFkB and Pol II tohypomethylated promoters
  24. 24. Binding of additional TF to hypomethylated promoters
  25. 25. Nucleic Acids Res 39, 874–888 (2011). Mol Cell Biol 29, 5366‐5376 (2009) 
  26. 26. DNMTs are less efficient in maintaining DNA methylation in eucrhomatic sites as proliferation startsHernando et al (2013) Genome Biol.
  27. 27. Hypomethylated genes undergo further upregulation
  28. 28. Demethylating agents promote transformation andproliferation
  29. 29. 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)• Demethylation may contribute to the efficiency of the process by further enhancinggene upregulation of certain genes
  30. 30. Chromatin and Disease Group, IDIBELL, Barcelona Spain Environmental Autoimmunity, NIEHS, NIH, BethesdaLaura Ciudad Terry O’HanlonHenar Hernando Lisa G. RiderVirginia Rodríguez Fred MillerRoser VentoLorenzo de la Rica University of OklahomaJosé Urquiza Amr Sawalha (U Michigan)Lluís Pons John Harley (CCHMC)Javier Rodríguez‐Ubreva Computational Medicine Unit, Karolinska Institutet, Stokholm, SwedenLeiden University Medical Center David Gómez‐CabreroRené Toes Jesper TegnérUniversity of BirminghamClaire Shannon‐LoweClaire Shannon‐LoweBroad InstituteFatima Al‐Shahrour INNPACTO, SAF FUNDACIÓN RAMÓN ARECESPEBC

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