1. Introduction of Dr. Yongfeng Shang Jin-Qiu Zhou jqzhou@sibs.ac.cn Shanghai Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences

2. Greek, epi = above, upon; Epigenetics=above genetics The study of heritable changes in gene function that occur without a change in the DNA sequence. Epigenetics? Genotype Epigenetic regulation Selective gene expression Cell fate Development Disease

3. Epigenetic Signatures Lunyak V & Rosenfeld M, Human Mol Genet, 2008

4. Chromatin  Walther Flemming first used the term Chromatin in 1882. At that time, Flemming assumed that within the nucleus there was some kind of a nuclear-scaffold.  Chromatin is the complex of DNA and protein that makes up chromosomes.  Chromatin structure: DNA wrapping around nucleosomes – a “beads on a string” structure.  In non-dividing cells there are two types of chromatin: euchromatin and heterochromatin.

5. Chromatin Fibers 30 nm chromatin fiber 11 nm (beads) Chromatin as seen in the electron microscope. ( source: Alberts et al., Molecular Biology of The Cell, 3 rd Edition)

6. DNA Methylation 5- methylcytosine S-adenosylmethionine DNA methyltransferase deoxycytosine

7. SAM DNA Methylation

8. CpG Islands  CpG island: a cluster of CpG residues often found near gene promoters (at least 200 bp and with a GC percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 0.6).  ~29,000 CpG islands in human genome (~60% of all genes are associated with CpG islands)  Most CpG islands are unmethylated in normal cells.

10.  The basic repeating unit of chromatin.  It is made up by five histone proteins: H2A, H2B, H3, H4 as core histones and H1 as a linker.  It provides the lowest level of compaction of double-strand DNA into the cell nucleus.  It often associates with transcription. Nucleosome H2A H2B H3 H4 1974: Roger Kornberg discovers nucleosome who won Nobel Prize in 2006.

11. Core Histones are highly conserved proteins - share a structural motif called a histone fold including three α helices connected by two loops and an N-terminal tail

12. Histone Octamer  Each core histone forms pairs as a dimer contains 3 regions of interaction with dsDNA;  H3 and H4 further assemble tetramers.  The histone octamer organizes 146 bp of DNA in 1.65 helical turn of DNA:  48 nm of DNA packaged in a disc of 6 x 11nm

13. The N-terminal tails protrude from the core

14. Histone Modifications Me P Ub Su Ac Me Acetylation Methylation Ubiquitination Sumoylation Phosphorylation ‘Histone Code’

15. Acetylation of Lysines  Acetylation of the lysines at the N terminus of histones removes positive charges, thereby reducing the affinity between histones and DNA.  This makes RNA polymerase and transcription factors easier to access the promoter region.  Histone acetylation enhances transcription while histone deacetylation represses transcription.

16. Methylation of Arginines Arginine can be methylated to form mono-methyl, symmetrical di-methyl and asymmetrical di-methylarginine.

17. Methylation of Lysines Lysine can be methylated to form mono-methyl, di-methyl and tri-methylarginine.

18. LSD1 demethylates H3K4me2/me1 via an amine oxidation reaction using FAD as a cofactor. The imine intermediate is hydrolyzed to an unstable carbinolamine that subsequently degrades to release formaldehyde. Demethylation of Lysines by LSD1

19. The JMJC proteins use KG and iron (Fe) as cofactors to hydroxylate the methylated histone substrate. Fe(II) in the active site activates a molecule of dioxygen to form a highly reactive oxoferryl [Fe(IV) = O] species to react with the methyl group. The resulting carbinolamine intermediate spontaneously degrades to release formaldehyde. Demethylation of Lysines by Jmjc Proteins

20. MarkTranscriptionally relevant sitesBiological Role Methylated cytosine (meC) CpG islandsTranscriptional Repression Acetylated lysine (Kac) H3 (9,14,18,56), H4 (5,8,13,16), H2A, H2B Transcriptional Activation Phosphorylated serine/threonine (S/Tph) H3 (3,10,28), H2A, H2BTranscriptional Activation Methylated argine (Rme) H3 (17,23), H4 (3)Transcriptional Activation Methylated lysine (Kme) H3 (4,36,79) H3 (9,27), H4 (20) Transcriptional Activation Transcriptional Repression Ubiquitylated lysine (Kub) H2B (123/120) H2A (119) Transcriptional Activation Transcriptional Repression Sumoylated lysine (Ksu) H2B (6/7), H2A (126)Transcriptional Repression Chromatin modifications

21. Gene Silencing

22. Genome-wide Distribution Pattern of Histone Modification Associated with Transcription Li et al. Cell (review) 128, 707-719 Source: Li et al. Cell (Review, 2007), 128:707-719

23. DNA Methylation and Gene Silencing in Cancer Cells 1324 1234 X CG M CG Normal Cancer CG M CG C: cytosine m C: methylcytosine CpG island

24. NormalCancer Region-Specific Hypermethylation Accumulation of Epigenetic Abnormalities Global Hypomethylation + Progressive Alterations in DNA Methylation in Cancer

25. CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal Normal Epithelia Dysplasia Carcinoma in situ Carcinoma Metastasis 1 2 3 4 5

26. Normal Epithelia Dysplasia Carcinoma in situ Carcinoma Metastasis 1 2 3 4 5 CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

27. Normal Epithelia Dysplasia Carcinoma in situ Carcinoma Metastasis 1 2 3 4 5 CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

28. Normal Epithelia Dysplasia Carcinoma in situ Carcinoma Metastasis 1 2 3 4 5 CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

29. When bound to its ligands, androgen (A), the AR translocates to the nucleus to interact with histone demethylases on androgen-responsive elements (ARE) on specific genes. Through its interaction with JMJD2C, LSD1 or JMJD1A demethylation is triggered, removing the repressive H3K9 methylation and leading to the transcriptional induction of these androgen-responsive genes. Repressive complexes (RCO), possibly featuring H3K9-methyltransferase (KMT), HDAC, and H3K4 demethylase (JARID1) activities, may potentially act to prevent ligand-independent activation. Histone demethylas in AR-mediated transcription

30. D NA-binding proteins are crosslinked to DNA with formaldehyde in vivo. I solate the chromatin. Shear DNA along with bound proteins into small fragments. B ind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins. U se PCR to amplify specific DNA sequences to see if they were precipitated with the antibody. Chromatin immunoprecipitation (ChIP)

31. ChIP-on-chip  ChIP  Labeling pool of DNA fragments.  Hybridization of DNA onto microarrays featuring 60- mer oligonucleotide probes.

32. Major types of array platforms  NimbleGen Arrays: tiling arrays, promoter arrays, whole genome arrays. (http://www.nimblegen.com/products/chip/index.html)  Agilent Arrays: promoter arrays, whole genome arrays. (http://www.chem.agilent.com/Scripts/Phome.asp)  Affymetrix Arrays: tiling arrays, Chr21,22 arrays, whole genome arrays. (http://www.affymetrix.com/index.affx)

33. Measurement of intensity of probes on the array  The hybridized arrays were scanned on an Axon GenePix 4000B scanner (Axon Instruments Inc.) at wavelengths of 532 nm for control (Cy3), and 635 nm (Cy5) for each experimental sample.  Data points were extracted from the scanned images using the NimbleScan 2.0 program (NimbleGen Systems, Inc.).  Each pair of N probe signals was normalized by converting into a scaled log ratio using the following formula: Si = Log2 (Cy5l(i) /Cy3(i))

34. Promoter 1 Promoter 2 Reproducibility of promoter arrays using biological replicates Top 1000 overlap H3me3K27

35. 500 kb region of chromosome 6 500 kb region of chromosome 1 Reproducibility on tiling arrays

36. 尚永丰 博士 北京大学医学部基础医学院教授 长江学者特聘教授 中国科学院院士 1999 年，美国宾夕法尼亚州立大学 ，博士。 1999 年至 2002 ，美国哈佛大学，博士后。 2000 年 6 月至 2001 年 10 月，美国哈佛大学医学院，讲师 。 2001 年 10 月，美国约翰 霍普金斯大学医学院，助教授 。 2002 年 4 月，北京大学医学部生物化学与分子生物学系，教授， 博导，长江学者。 2009 年 12 月，中国科学院院院士

37. 尚永丰教授主要研究成就 主要从事基因转录调控的表观遗传机制及性激素相关妇科肿 瘤分子机理的研究。提出、验证并从分子机理上诠释了雌激素受 体转录起始复合体在靶基因启动子上循环反复结合的假说以及雌 激素受体所介导的基因转录具有 “ 双相性 ” 和 “ 两维性 ” 的特点，为 基因转录调控的理论增添了新的内容；揭示了雌激素受体拮抗剂 三苯氧胺诱发子宫内膜癌的分子机理，克隆了多个肿瘤相关基因， 为肿瘤分子生物学的理论发展作出了贡献；揭示了组蛋白去乙酰 化和组蛋白去甲基化在染色质重塑中协调作用的机理，对认识表 观遗传调控的分子机制具有创新性的理论意义；在世界上首次建 立了哺乳动物细胞染色质免疫沉淀技术（ ChIP ），为研究 DNA 与蛋白质的相互作用作出了重要贡献。在《 Cell 》、《 Nature 》 和《 Science 》等杂志上发表了一系列的研究论文。

38. 尚永丰教授获奖 2002 年 “ 国家杰出青年基金 ” 获得者，获 2005 年度 “ 中国基础研究十大新闻 ” 、 2006 年度 “ 中国高等学校十大科技进展 ” 等荣誉，并获 2007 年度 “ 中华医学科技奖 ” 一等奖、 2007 年度 “ 教育 部自然科学奖 ” 一等奖和 2008 年度 “ 国家自然科学 奖 ” 二等奖等奖励。尚永丰本人还获得第九届 “ 中 国青年科技奖 ” 、 2006 年度美国 ELI Lilly 公司的 “ 礼来科研成就奖 ” 和 2007 年 “ 何梁何利科学与技 术进步奖 ” ，还是 2007 年度全国百篇优秀博士学 位论文博士生导师。 中国高等学校十大科技进展

39. 尚永丰教授主要学术兼职 2004 年起担任《中国生物化学与分子生物学 学报》副主编。 2007 年被国际著名学术杂志《 Journal of Biological Chemistry 》聘为编委。 美国科学促进会，美国生物化学与分子生物学 学会及美国癌症研究会会员。 中国生物化学与分子生物学学会北京分会常务 理事。

40. THANK YOU FOR YOUR ATTENTION 谢