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Epigenetic regulation of lymphocyte development and function Hodaka Fujii, M.D., Ph.D. Combined Program on Microbiology and Immunology Research Institute for Microbial Diseases April 14, 2015 Today’s topics 1. What is epigenetics? 2. Known mechanisms of epigenetic regulation 3. Methods to analyze epigenetic regulation 4. Future direction 1. What is epigenetics? What is epigenetics? Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- (Greek: επίover, above, outer) -genetics. It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Central issue in the post-genome era: Molecular mechanisms of epigenetic regulation ON Me Me CG CG Ac ON ♀ OFF ♂ Me Me Me CG CG Ac OFF Leads to discovery of potential novel biological phenomena Transcription Lineage commitment Cell differentiation OFF Genome Imprinting Me ON RNA X chromosome inactivation X X Active Inactive 2. Known mechanisms of epigenetic regulation Known mechanisms of epigenetic regulation ・ DNA modification, eg. methylation ・ Modification / replacement of histones ・ DNA-binding proteins ・ Intra / inter-chromosomal interaction ・ Binding of RNA DNA modification: methylation of cytosine 1 DNA modification: methylation of cytosine 2 DNA modification: methylation of cytosine 3 hydroxymethylation Histone modification 1 Histone modification 2 Histone code hypothesis Histone replacement 1 Histone replacement 2 DNA-binding proteins Inter- & intra-chromosome interaction 1 Inter- & intra-chromosome interaction 2 olfactory receptors nuclear receptor target genes CD4 & CD8 Non-coding RNA in epigenetic regulation 3. Methods to analyze epigenetic regulation Conventional methods for epigenetics research I Genetic approaches 1 1. Forward genetics Mutagenize (mutagen, P-element, etc.) ↓ Isolate mutants ↓ Identify causative genes Bacteria Yeasts Plants Nematodes Fruit flies Zebra fish Mouse Human mutant WT Cannot find redundant genes Takes long time to elucidate molecular mechanisms Conventional methods for epigenetics research I Genetic approaches 2 2. Reverse genetics Knockout genes encoding modification enzymes of DNA or histones or other candidate molecules Not non-biased screening Global but not local Conventional methods for epigenetics research II Identification of gene regulatory regions 1 DNase hypersensitivity sites Conventional methods for epigenetics research II Identification of gene regulatory regions 2 Formaldehyde-assisted isolation of regulatory elements (FAIRE) Conventional methods for epigenetics research II Identification of gene regulatory regions 2 Promoter / enhancer analysis Reporter assay Not physiological How to find regulatory regions? Conventional methods for epigenetics research III Analysis of specific genomic regions 1 1. Protein analysis:Identification of chromatin-binding proteins (i) Identification of chromatin-binding proteins DNA affinity purification, Yeast one-hybrid, IP, etc. non-physiological conditions Conventional methods for epigenetics research III Analysis of specific genomic regions 2 1. Protein analysis:Identification of chromatin-binding proteins (i) Identification of chromatin-binding proteins DNA affinity purification, Yeast one-hybrid, IP, etc. non-physiological conditions Conventional methods for epigenetics research III Analysis of specific genomic regions 3 1. Protein analysis:Identification of chromatin-binding proteins (ii) Chromatin Immunoprecipitation (ChIP) Histone modification, detection of DNAbinding proteins not non-bias screening Conventional methods for epigenetics research III Analysis of specific genomic regions 4 1. Protein analysis:Identification of chromatin-binding proteins (iii) Imaging: localization of proteins 2. DNA analysis:Detection of DNA modification and intra/interchromosomal interaction (i) Fluorescence in situ Hybridization (FISH) 3. RNA analysis:Identification of chromatin-bound RNA (i) FISH not non-bias screening low resolution Conventional methods for epigenetics research IV Epigenomics Genome-wide mapping of epigenetic marks 1. DNA modifications 2. Histone modifications 3. ChIP analysis of DNA-binding proteins / Histones 4. 3C (see below) 5. RNA Descriptive Methods to directly identify molecular interaction in the genome Why are methods to directly identify molecular interaction in the genome necessary? 1. Conventional methods are time consuming. eg. Analysis of regulation of gene expression a. Identification of cis-regulatory elements: reporter assay b. Identification of binding proteins: in vitro affinity purification, yeast one hybrid c. Verification of interaction in vivo: ChIP d. Functional analysis: loss-of-function experiments It takes more than ten years to make a story! 2. Frequently, conventional methods produce nonsense results. Chromosome Conformation Capture (3C) and its derivatives Dekker et al., Science (2002) - Incomplete digestion causes amplification of adjacent regions, which inhibits amplification of interacting genomic regions. - Multiple enzymatic reactions in non-optimal conditions - 3C-based methods generate some data, but do they really represent interaction? Another indication of accessibility? de Wit & de Laat, Genes & Dev., 2012 Non-biased analysis of molecules bound to specific genomic regions of interest PURIFY! Biochemical and molecular biological analysis Proteomics of isolated chromatin segments (PICh) Déjardin et al. Cell 2009 LNA: locked nucleic acid - Successful in identification of telomere-binding proteins - Telomere: multiple copies - Applicable to singlecopy or low-copy genes? insertional chromatin immunoprecipitation: iChIP 8 x LexA BE 160 bp Direct detection of molecular interaction in the genome Hoshino & Fujii, J. Biosci. Bioeng., 2009 Fujita & Fujii, PLoS One, 2011 US patent: 8415098; Japan patent 5413924 iChIP by using homologous recombination ZFN or TALEN targeting vector LexA-binding elements Homologous recombination Neor genomic DNA genomic DNA a target region -Neor (Cre-LoxP) iChIP Guidelines: 1. Evolutionarily NON-conserved regions a target region (e.g. promoter) 2. Several hundred base apart from the Random integration Into genomic DNA reporter genomic regions of interest LexA-binding elements iChIP by using random integration genomic DNA Selection marker (e.g. Neor) iChIP Zinc finger nuclease (ZFN), TALEN & Cas9 ZFN Cas9 TAL (trans activator-like) engineered DNA-binding molecule-mediated chromatin immunoprecipitation: enChIP Fujita et al., Sci. Rep. (2013) enChIP using the CRISPR system Fujita & Fujii, BBRC (2013) Advantages of locus-specific ChIP (iChIP & enChIP) 1. Non-biased method: does not need any prior knowledge about targets. 2. Speed: takes much shorter time. eg. p68 in an insulator complex x 70 acceleration!! conventional methods: 17 years iChIP: 3 months 3. Generality: any molecules including DNA, RNA, and proteins can be analyzed. 4. Robustness: calibration is easy. 5. Higher sensitivity: x 103 - 104 more sensitive!! PICh: telomere 103 - 104 copies / genome iChIP: 1 copies / genome Comparison of non-biased methods to analyze specific genomic regions Method iChIP enChIP 3C PICh DNA analysis Protein analysis RNA analysis ○ ○ ○ ○ ○ ○ ○ X X ○ Not reported Not reported Include enzyme reactions (-) (-) (+) (-) Low copy # genes ○ ○ ○ ? Need transgenic Allelespecific analysis LexA BE Insertion ○ Yes X No X No X Yes Contact 藤井穂高 Hodaka Fujii 感染症学免疫学融合プログラム推進室・准教授 (Principal Investigator) Associate Professor Combined Program on Microbiology and Immunology 大阪大学 微生物病研究所 Research Institute for Microbial Diseases [email protected] http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/index.html Tel/Fax: 06-6879-8358