Deﬁning Epigene*cs Genome DNA § Reversible changes in gene expression/func5on § Without changes in DNA Chroma*n sequence Epigenome § Can be inherited from precursor cells Gene Expression § Allows to integrate intrinsic with environmental signals Phenotype (including diet)
DNA Methylation Differentiates Totipotent EmbryonicStem Cells from Unipotent Adult Stem Cells! Alex Meissner, Henry Stewart Talks
Reprogramming the DNA methylome Paula Vertino, Henry Stewart Talks
Epigene*c Changes are Important in Causing Cancer GENETIC EPIGENETIC Example: Example: Replica*on errors Chroma*n modiﬁca*on errors X X Altered Altered DNA sequence chroma*n structure Oncogenesis Altered Altered levels of DNA/mRNA/proteins mRNA/proteins Tumor
Example of Methyla*on vs Muta*on: Colon & Breast Cancer 120 100 80 60 Dx 40 20 CDx 0 Methylated Mutated Source: Schuebel et al 2007 76-‐100 51-‐75 21-‐50 1-‐20
MGMT Biology O6 Methyl-‐Guanine Methyl Transferase Essen5al DNA Repair Enzyme Removes alkyl groups from damaged guanine bases Healthy individual: -‐ MGMT is an essen5al DNA repair enzyme Loss of MGMT ac5vity makes individuals suscep5ble to DNA damage and prone to tumor development Glioblastoma pa*ent on alkylator chemotherapy: -‐ Pa5ents with MGMT promoter methyla5on show have longer PFS and OS with the use of alkyla5ng agents as chemotherapy
MGMT Promoter Methyla*on Predicts Beneﬁt form DNA-‐Alkyla*ng Chemotherapy Post-‐hoc subgroup analysis of Temozolomide Clinical trial with primary glioblastoma pa5ents show beneﬁt for pa5ents with MGMT promoter methyla5on Median Overall Survival 25 21.7 months 20 plus temozolomide 15 12.7 months radiotherapy 10 radiotherapy 5 Adapted from Hegi et al. NEJM 2005 0 352(10):1036-‐8. Non-‐Methylated Methylated Study with 207 pa5ents MGMT Gene MGMT Gene
Next_next miRNA, (l)ncRNA, CIS/TRANS splicing, SV, fusion loci , bidirec*onal promoters ? RNA_seq: sequence RNA molecules Next Gen Pla`orm Total RNA_seq: all RNA molecules (normalisa*on procedure) Direc*onal Total RNA_seq: before ampliﬁca*on use diﬀerent 5’ and 3’ adaptors Integrated Direc*onal Total RNA_seq: Combine with other datasets eg. enrichment sequencing data, visualise and query in genome browser 32
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT 25% 50% 25% GCATCGTGACTAGCGACTGATCGATGGATGCTAGCATGCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT Dense methylated needed for transcrip5onal silencing Are there alleles with all three posi.ons methylated ?
Deep Sequencing unmethylated alleles methylated alleles less methyla5on more methyla5on GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT!
Conclusion Combina5on of diﬀerent sequencing techniques is emerging as best prac5ce Bioinforma5cs is challenging § Methods for normalisa5on under construc5on § Reference databases are generated Data visualiza5on and integra5on is key 41
Slides availablewww.bioinformatics.be 4th December 2012 Johns Hopkins Bloomberg School of Public Health