E i p g i e g n e e n t e i t c i s

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1 Epigenetics

2 Introduction to Epigenetics Classical Genetics: The Central Dogma

3 The Human Genome Project (HGP) Key findings: approx. 24,000 genes in human being all human races are 99.99% alike Challenges: about 24,000 genes vs. 100,000 proteins non-coding (98%) vs. coding DNA (2%) genotype vs. phenotype Introduction to Epigenetics

4 Introduction to Epigenetics Epigenetic biology Position-effect variegation They may look the same, but are they? Paramutation at the maize

5 Introduction to Epigenetics Epigenetic differences arise during the lifetime of monozygotic twins 5mC AcH4 AcH3 PNAS, July 26,2005, Vol.102

6 Epigenetics Introduction to Epigenetics Definition: The study of any potentially stable and, ideally, heritable change in gene expression or cellular phenotype that occurs without changes in Watson- Crick base-pairing of DNA. One example: : cellular differentiation/development totipotent stem cells become the various pluripotent cell lines of the embryo which in turn become fully differentiated cells

7 Introduction to Epigenetics Change: Genetics vs. Epigenetics

8 Introduction to Epigenetics Brief History of Epigenetics 1938, Hermann Muller first described the Position- effect variegation 1942, Conrad Waddington defined epigenetics as the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being 1950s, R. Brink discovered Paramutation in maize 1961, Mary Lyon proposed the random inactivation of one female X chromosome to explain the mottled phenotype of female mice heterozygous for coat color genes 1983, discovered the DNA methylation

9 Epigenetics Events paramutation bookmarking imprinting gene silencing X chromosome inactivation position effect reprogramming transvection maternal effects Introduction to Epigenetics

10 Epigenetic Research: techniques ChIP; ChIP-on-chip; ChIP-seq fluorescent in situ hybridization methylation-sensitive restriction enzymes Hpa II (CCGG) and Msp I (CCGG) DNA adenine methyltransferase identification (DamID) Nat Protoc,2007, 2(6): bisulfite sequencing. computational epigenetics Introduction to Epigenetics

11 Introduction to Epigenetics Epigenetic Research: future NoME-Seq: Nucleosome Occupancy and Methylome Sequencing oxbs-seq: Oxidative Bisulfite Sequencing BisChip-seq/ ChIP BS-Seq: Combined Bisulfite Sequencing & Chromatin Immunoprecipitation inform methylation status of histone-modified DNA Nanofluidic Epigenetic Analysis: SCAN (Single Chromatin Analysis at the Nanoscale) & Nanofluidic DNA Methylation Detection

12 Introduction to Epigenetics The Focuses of Epigenetic Reseach Genomic Level Chromatin Remodeling DNA modificaition Histone modification other modifications X-chromosome inactivation: DNA methylation; ncrna genomic imprinting: DNA methylation; ncrna RNA level ncrna RNAi (RNA Interference) & mirna (microrna) lncrna RNA-interacting protein

13 Introduction to Epigenetics The Levels of Epigenetic Reseach Molecular interaction DNA modification Protein modification ncrna regulation RNA modification

14 The nucleosome Chromatin Remodeling Higher-Order Structuring of Chromatin The spacing and positioning of nucleosomes at critical cis-acting control regions (i.e., promoters) are tailored for the proper regulation of the locus.

15 Chromatin Remodeling The Structure of a Nucleosome

16 Chromatin Remodeling Euchromatin vs. Heterochromatin

17 Chromatin Remodeling Chromatin-remodeling: Dynamic structural changes to the chromatin occurring throughout the cell division cycle. These changes range from the local changes necessary for transcriptional regulation to global changes necessary for chromosome segregation

18 Chromatin Remodeling Seven distinct mechanisms of chromatin remodeling

19 Chromatin Remodeler Chromatin Remodeling: mechanisms Non-covalent manner Chromatin remodeling complexes Histone variants Covalent manner Histone modification DNA methylation Non-coding RNA Fact: work coordinately

20 Chromatin Remodeler Chromatin remodeler: Non-covalent manner are ATP-hydrolyzing machines Functions: fully package the genome restructure, mobilize or eject nucleosomes specialize chromatin regions provide regulated DNA accessibility in packaged regions.

21 Chromatin Remodeler Chromatin remodeler Families All contain a SWI2/SNF2-family ATPase subunit characterized by an ATPase domain that is split in two parts: DExx (red ) and HELICc (orange).

22 Chromatin Remodeler Chromatin remodeler: a complex

23 The actions of remodelers on nucleosomes Chromatin Remodeler

24 The actions of remodelers on nucleosomes Chromatin Remodeler

25 Chromatin Remodeling Chromatin Remodeling: covalent manner

26 Chromatin Remodeling DNA methylation: is a type of chemical modification of DNA that can be inherited and subsequently removed without changing the original DNA sequence Histone Modifications: change the chromatin template by cis-effects that alter internucleosomal contacts and spacing, or the trans-effects caused by histone and non- histone protein associations with the template.

27 Methylation ( 甲基化 ) DNA methylation: : typically occurs at CpG sites in vertebrates catalyzed by the enzyme DNA methyltransferase.

28 DNA methylation Hypomethylation Hypermethylation

29 DNA methylation The Roles of DNA Methylation Imprinting X chromosome inactivation Heterochromatin maintenance Developmental controls Tissue specific expression controls

30 DNA methylation DNA methylation and demethylation in a life cycle

31 Regulation of Gene Expression by DNA methylation Interference with transcription factor binding DNA methylation Attraction of Methyl- CpG-binding proteins

32 DNA methylation DNA Methylation and Cancer Nature Reviews Genetics, 6: 597

33 DNA Methylation and Other Human Diseases -- Imprinting Disorder: Beckwith-Wiedemann syndrom (BWS) Prader-Willi syndrome (PWS) Transient neonatal diabetes mellitus (TNDM) -- Repeat-instability diseases Fragile X syndrome (FRAXA) Facioscapulohumeral muscular dystroph DNA methylation -- Defects of the methylation machinery Systemic lupus erythemtosus (SLE) Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome

34 DNA Methylation Analysis Bisulfite Genomic Sequencing (Gold Standard) Bisulfite Genomic Sequencing Pyrosequencing The HELP assay (CCGG CCGGHpaII - CCGG GGMspI) ChIP-on-chip assays Methylated DNA immunoprecipitation (MeDIP) Molecular break light assay for DNA adenine methyltransferase activity For more information, see

35 Bisulfite-mediated conversion of C U

36 Bisulfite Genomic Sequencing Nucleotides in blue are unmethylated cytosines converted to uracils by bisulfite, while red nucleotides are 5-methylcytosines resistant to conversion.

37 DNA methylation analysis methods not based on methylation-specific PCR Following bisulfite conversion, the genomic DNA is amplified with PCR that does not discriminate between methylated and non-methylated sequences. The numerous methods available are then used to make the discrimination based on the changes within the amplicon as a result of bisulfite conversion. For more information, see

38 Be Careful Be aware: : the process of demethylation of 5mC 5mC 5-hydroxymethylcytosine (5hmC) 5-formylcytosine (5fC) 5-carboxylcytosine (5caC) C 5mC and 5hmC, but not 5fC or 5caC, are both resistant to bisulfite conversion

39 Timeline of Sequencing-Based Technologies for Mapping Human Epigenomes Cell, 2013, 155: 39

40 Methylated DNA immunoprecipitation (MeDIP)

41 Chromatin Remodeling Coordinated modification of chromatin The transition of a naïve chromatin template to active euchromatin(left) or the establishment of repressive heterochromatin (right), involving a series of coordinated chromatin modification. In the case of transcriptional activation, this is accompanied by the action of nucleosome-remodeling complexes and replacement of core histones with histone variants (yellow)

42 RNA Epigenetics Nat Chem Biol, 2011, 7(12): N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. fat mass and obesity-associated protein (FTO): oxidative demethylation activity Data demonstrate that methylation of RNA has a critical role in human energy homeostasis

43 RNA Modificaton RNA epigenetics Nature Chemical Biology, 2010, 6:

44 RNA epigenetics RNA Epigenetics? Nature Chemical Biology, 2010, 6:

45 Genetics-epigenetics-environment interations

46 754 Year published: (2013) Further Reading 1. Bird A. Perceptions of epigenetics. Nature,, 2007, 447(7143): Hamilton JP. Epigenetics: principles and practice. Dig Dis, 2011, 29(2): He C. Grand challenge commentary: RNA epigenetics? Nat Chem Biol,, 2010, 6(12): You JS, Jones PA. Cancer genetics and epigenetics: two sides of the same coin? Cancer Cell,, 2012, 22(1): Jang H & Serra C. Nutrition, Epigenetics, and Diseases. Clin Nutr Res , 3(1): Sheppard TL. RNA takes control. Nat Chem Biol , 9: Pan T. N6-methyl-adenosine modification in messenger and long non-coding RNA. Trends Biochem Sci , 38: 204