Epigenetics and Chromatin Remodeling Bradford Coffee, PhD, FACMG Emory University Atlanta, GA Speaker Disclosure Information Grant/Research Support: none Salary/Consultant Fees: none Board/Committee/Advisory Board: none Membership: none Stocks/Bonds: none Honorarium/Expenses: none Intellectual Property/Royalty Income: none Learning objectives 1. Define epigenetics 2. Describe the relationship between chromatin structure, histone modifications, DNA methylation and gene expression. 3. Explain imprinting and the importance of uniparental disomy on the expression of imprinted regions. 4. Describe the many different molecular mechanisms can lead to loss of imprinted gene expression. 5. Compare the different methodologies available to test for DNA methylation as a marker of epigenetic disease. Definition Epigenetics – stable and heritable (mitotic and/or meiotic) changes in gene expression that do not entail a change in DNA sequence Nucleosomes Felsenfeld and Groudine Controlling the double helix Nature 421: 448-453 Histone Modifications Bhaumik et al. Nat. Struct and Mol. Biol. 14: 1008-1016 DNA Methylation NH2 CH3 N N O occurs at the 5th position in cytosine ~50 million 5-methylcytosines/genome (4-8% of the cytosines) In somatic cells 99.98% of cytosine methlyaion in CpGs In ES cells only ~75% of cytosine methylation in CpGs. The remaining ~25% is in mCHG and mCHH contexts (H=A,C or T) CpG islands encompass the 5’ end of genes 50-60% of all genes in the human genome contain a CpG island Chromatin Structure Modifications of the DNA and histones (and other chromatin proteins) act together to direct specific chromatin structures that control access to regulatory elements of genes. Two general states of chromatin- Euchromatin-open trancriptionally active genes Heterochromatin-closed transcriptionally repressed genes Constitutive-repetitive sequences Facultative-gene repression in specific cell types Imprinting Definition-Exclusive or preferential expression of a gene from one of the two parental alleles ~75-100 known imprinted genes in humans Found in clusters (imprinted regions). Maternally and paternally imprinted genes are clustered Imprinting is an epigenetic process. There is a change in gene expression without a change in the DNA sequence. These changes in gene expression are stable during meiosis. Clusters of Imprinted Genes Prader-Willi and Angelman Syndromes Sahoo et al Nat. Genet. 40: 719 - 721 http://www.angelmanuk.org/ Prader-Willi syndrome (PWS) Angelman syndrome (AS) severe hypotonia in early infancy severe developmental delay excessive eating later in childhood mental retardation morbid obesity severe speech impairment delayed motor milestones and gait ataxia delayed language development tremulousness of the limbs cognitive impairment inappropriate happy demeanor Caused by loss of paternal gene Caused by loss of maternal gene expression. expression. 15q11-q13 Imprinted Gene Cluster Molecular Mechanisms of PWS and AS 1. ~5 Mb deletions (mediated by flanking low copy repeats) ~70% of cases of PWS and AS on the paternal chromosome → PWS on the maternal chromosome → AS 2. Uniparental disomy (UPD) ~25-30% of cases of PWS → maternal UPD ~5% of cases of AS → paternal UPD 3. Single gene mutation N/A for PWS ~10% of AS due to mutation of UBE3A gene 4. Imprinting center mutation ~1% of PWS ~5% of AS 5. Unknown <1% of PWS 10%-15% of AS Uniparental Disomy Trisomy Rescue genetests.org Beckwith-Wiedemann and Russell Silver Syndromes Beckwith-Wiedemann (BWS) Russell Silver (RSS) www.nlm.nih.gov/medlineplus/ http://www.magicfoundation.org macrosomia -prenatal and postnatal growth retardation -prenatal and postnatal macroglossia triangular shaped face abdominal wall defects normal head circumference (omphalocele, umbilical hernia) (pseudohydrocephalus) hemihyperplasia fifth-finger clinodactyly embryonal tumors limb-length asymmetry (hemihypotrophy) Both BWS and RSS caused by defects in imprinted gene expression at 11p15.5 11p15.5 Imprinted Gene Cluster RSS (Lit1) BWS Smith et al. 2007. Pediatr. Res. 61: 43R-47R Beckwith Wiedemann Syndrome Russell Silver Syndrome matUPD7 DMR1 hypomethylation unknown Mechanisms Leading to Epigenetic Diseases 1. conventional sequence changes deletions removing imprinted gene(s) PWS, AS mutations that disrupt resetting imprint PWS, AS, RSS chromosome rearrangements BWS 2. uniparental disomy PWS, AS, UPD6, UPD7 (RSS) and UPD14 3. epimutations BWS, RSS, UPD14 DNA methylation is a marker for detecting if one of these mechanisms has occurred. Methods to Detect Aberrant Methylation Clinical laboratories (locus specific) methylation restriction enzymes Southern blot Methylation Specific-MLPA bisulfite based methods Methylation Sensitive PCR (MSP) COBRA Quantitative-MSP (Q-MSP, Methyl-Light) Research laboratories (whole genome) MeDIP Illumina Infinium methylation assay Methylome sequencing Southern Blot Using Methylation Senstive Enzymes CpG island XbaI KspI XbaI ~1.1 kb ~2.9 kb NEG PWS AS 4kb methylated (maternal) 1.1kb unmethylated (paternal) Methylation Specific MLPA http://www.mlpa.com Sodium Bisulfite Treatment of DNA http://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis Methylation Specific PCR ……………GCCGCGCGGCGGCAG………… methylated unmethylated sodium bisulfite …GUCGCGCGGCGGUAG… …GUUGUGUGGUGGUAG… PCR amplification …GUCGCGCGGCGGUAG… …GUUGUGUGGUGGUAG… GCGCGCCGCCATC… ACACACCACCATC… methylated DNA unmethylated DNA specific primer specific primer Chr15 Methylation Analysis CpG island XbaI KspI XbaI ~1.1 kb ~2.9 kb 174bp methyl 100bp unmethyl H2O 1 2 AS PWS NEG 174bp methyl 100bp unmethyl Askree et al 2011 J. Mol. Diag. 23 Chr15 Methylation Analysis by MSP H2O NEG AS 1 2 3 PWS Original primer set 174bp-methylated 100bp-unmethylated Alternate primer set 152bp-methylated 100bp-unmethylated Askree et al 2011 J. Mol. Diag. 24 UPD7 Methylation Analysis GRB10 (7p11.2-7p12) PEG1/MEST (7q32) Chr7 patient samples Pos Neg H2O methylated (maternal) unmethylated (paternal) GRB10 methylated (maternal) unmethylated (paternal) PEG1/MEST Quantitative Methylation Specific PCR methylated CG CG CG CG CG CG CG CG unmethylated UG UG UG UG UG UG UG UG Amplification Taqman probe unmethylated DNA primers Taqman probe methylated DNA Q-MSP Amplification Plots 1ng (~151 copies) B FAM 8ng (~1208 copies) methylated 64ng (~9664 copies) R2 = 0.987, E = 96.4%, slope = -3.412 1ng (~151 copies) VIC 8ng (~1208 copies) unmethylated 64ng (~9664 copies) Coffee et al. R2 = 0.996, E = 92.6%, slope = -3.514 J. Mol. Diag. in press Qualitative vs. Quantitative Assays If the disease is caused by underlying DNA sequence change (e.g. a deletion in PWS) or caused by UPD, a qualitative methylation assay will detect the disorder. If the disease is caused by epimutation (e.g. loss of methylation DMR2 in BWS), a quantitative assay is needed to detect changes in methylation. Mean methylation Mean methylation of unaffected population of disease population Self-Assessment Questions 1.Define epigenetics? 2.How do epigenetic modifications control gene expression? 3.List the molecular mechanisms of PWS and AS? 4. How does the mechanism of epigenetic disease determine if you use quantitative vs. qualitative methylation analysis in testing? 5. What are some of the molecular techniques used in clinical laboratories to test for aberrant DNA methylation? .