Epigenetics and Chromatin Remodeling

Transcription

1 Epigenetics and Chromatin Remodeling Bradford Coffee, PhD, FACMG Emory University Atlanta, GA

2 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

3 1. Define epigenetics Learning objectives 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.

4 Definition Epigenetics stable and heritable (mitotic and/or meiotic) changes in gene expression that do not entail a change in DNA sequence

5 Nucleosomes Felsenfeld and Groudine Controlling the double helix Nature 421:

6 Histone Modifications Bhaumik et al. Nat. Struct and Mol. Biol. 14:

7 DNA Methylation NH 2 CH 3 N N O occurs at the 5 th 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

8 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 Chromatin Structure Heterochromatin-closed transcriptionally repressed genes Constitutive-repetitive sequences Facultative-gene repression in specific cell types

9 Imprinting Definition-Exclusive or preferential expression of a gene from one of the two parental alleles ~ 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.

10 Clusters of Imprinted Genes

11 Prader-Willi and Angelman Syndromes Sahoo et al Nat. Genet. 40: Prader-Willi syndrome (PWS) severe hypotonia in early infancy excessive eating later in childhood morbid obesity delayed motor milestones and delayed language development cognitive impairment Caused by loss of paternal gene expression. Angelman syndrome (AS) severe developmental delay mental retardation severe speech impairment gait ataxia tremulousness of the limbs inappropriate happy demeanor Caused by loss of maternal gene expression.

12 15q11-q13 Imprinted Gene Cluster

13 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

14 Uniparental Disomy Trisomy Rescue genetests.org

15 Beckwith-Wiedemann and Russell Silver Syndromes Beckwith-Wiedemann (BWS) Russell Silver (RSS) macrosomia -prenatal and postnatal macroglossia abdominal wall defects (omphalocele, umbilical hernia) hemihyperplasia embryonal tumors growth retardation -prenatal and postnatal triangular shaped face normal head circumference (pseudohydrocephalus) fifth-finger clinodactyly limb-length asymmetry (hemihypotrophy) Both BWS and RSS caused by defects in imprinted gene expression at 11p15.5

16 11p15.5 Imprinted Gene Cluster RSS (Lit1) BWS Smith et al Pediatr. Res. 61: 43R-47R Beckwith Wiedemann Syndrome Russell Silver Syndrome matupd7 DMR1 hypomethylation unknown

17 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.

18 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

19 Southern Blot Using Methylation Senstive Enzymes CpG island XbaI ~1.1 kb KspI ~2.9 kb XbaI NEG PWS AS 4kb methylated (maternal) 1.1kb unmethylated (paternal)

20 Methylation Specific MLPA

21 Sodium Bisulfite Treatment of DNA

22 Methylation Specific PCR GCCGCGCGGCGGCAG methylated sodium bisulfite unmethylated GUCGCGCGGCGGUAG GUUGUGUGGUGGUAG PCR amplification GUCGCGCGGCGGUAG GCGCGCCGCCATC methylated DNA specific primer GUUGUGUGGUGGUAG ACACACCACCATC unmethylated DNA specific primer

23 Chr15 Methylation Analysis CpG island XbaI ~1.1 kb KspI ~2.9 kb XbaI 174bp methyl 100bp unmethyl 1 2 NEG PWS AS H2O 174bp methyl 100bp unmethyl Askree et al 2011 J. Mol. Diag. 23

24 Chr15 Methylation Analysis by MSP NEG PWS AS H2O Original primer set 174bp-methylated 100bp-unmethylated Alternate primer set 152bp-methylated 100bp-unmethylated Askree et al 2011 J. Mol. Diag. 24

25 UPD7 Methylation Analysis Chr7 GRB10 (7p11.2-7p12) patient samples Neg Pos H2O PEG1/MEST (7q32) GRB10 methylated (maternal) unmethylated (paternal) PEG1/MEST methylated (maternal) unmethylated (paternal)

26 Quantitative Methylation Specific PCR methylated CG CG CG CG CG CG CG CG unmethylated UG UG UG UG UG UG UG UG Amplification primers Taqman probe unmethylated DNA Taqman probe methylated DNA

27 Q-MSP Amplification Plots 1ng (~151 copies) B 8ng (~1208 copies) FAM methylated 64ng (~9664 copies) R 2 = 0.987, E = 96.4%, slope = ng (~151 copies) 8ng (~1208 copies) VIC unmethylated R 2 = 0.996, E = 92.6%, slope = ng (~9664 copies) Coffee et al. J. Mol. Diag. in press

28 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 of unaffected population Mean methylation of disease population

29 1. Define epigenetics? Self-Assessment Questions 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?