Download 结构生物学研究组 - 中国细胞生物学学会

Introduction of Dr. Yongfeng Shang
Jin-Qiu Zhou
[email protected]
Shanghai Institute of Biochemistry and Cell Biology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
Epigenetics?
Greek, epi = above, upon; Epigenetics=above genetics
The study of heritable changes in gene function that
occur without a change in the DNA sequence.
Cell fate
Epigenetic
regulation
Genotype
Selective
gene
expression
Development
Disease
Epigenetic Signatures
Lunyak V & Rosenfeld M, Human Mol Genet, 2008
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.
Chromatin Fibers
Chromatin as seen in the electron microscope.
(source: Alberts et al., Molecular Biology of The Cell, 3rd Edition)
30 nm
chromatin fiber
11 nm
(beads)
DNA Methylation
N
N
N
N
O
-O
N
O
OH
CH 3
DNA methyltransferase
S-adenosylmethionine
H
deoxycytosine
O
-O
N
O
OH H
5-methylcytosine
DNA Methylation
SAM
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.
Nucleosome

H2A

H3
H2B
H4


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.
1974: Roger Kornberg discovers nucleosome who won Nobel Prize in 2006.
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
<
11 nm
>
Histone Octamer
<
6 nm
>
 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
The N-terminal tails protrude from the core
Histone Modifications
Acetylation
Me
Ac
Me
Ub
Su
Methylation
Ubiquitination
Sumoylation
P
Phosphorylation
‘Histone
Code’
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.
Methylation of Arginines
Arginine can be methylated to form mono-methyl,
symmetrical di-methyl and asymmetrical di-methylarginine.
Methylation of Lysines
Lysine can be methylated to form mono-methyl,
di-methyl and tri-methylarginine.
Demethylation of Lysines by LSD1
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 Jmjc Proteins
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.
Chromatin modifications
Mark
Transcriptionally relevant sites
Biological Role
Methylated cytosine
(meC)
CpG islands
Transcriptional 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, H2B
Transcriptional 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
Gene Silencing
Genome-wide Distribution Pattern of Histone
Modification Associated with Transcription
Source: Li et al. Cell (Review, 2007), 128:707-719
Li et al. Cell (review) 128, 707-719
DNA Methylation and Gene Silencing
in Cancer Cells
CpG island
CGCG CG
Normal
1
MCGMCG MCG
Cancer
CG
2
3
MCG
1
3
MCG
4
CG
CG
2
X
MCG
CG
4
C: cytosine
mC:
methylcytosine
CG
Progressive Alterations in DNA
Methylation in Cancer
Global
+
Hypomethylation
Normal
Region-Specific
Hypermethylation
Cancer
CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal
Epithelia
Dysplasia
Carcinoma
in situ
Metastasis
CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal
Epithelia
Dysplasia
Carcinoma
in situ
Metastasis
CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal
Epithelia
Dysplasia
Carcinoma
in situ
Metastasis
CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal
Epithelia
Dysplasia
Carcinoma
in situ
Metastasis
Histone demethylas in AR-mediated transcription
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.
Chromatin immunoprecipitation
(ChIP)
DNA-binding proteins are
crosslinked to DNA with
formaldehyde in vivo.
Isolate the chromatin. Shear
DNA along with bound
proteins into small fragments.
Bind 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.
Use PCR to amplify specific DNA
sequences to see if they were
precipitated with the antibody.
ChIP-on-chip
ChIP
Labeling pool of DNA fragments.
Hybridization of DNA onto microarrays featuring
60-mer oligonucleotide probes.
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)
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))
Reproducibility of promoter arrays
using biological replicates
•H3me3K27
•Top
1000
overlap
•Top
1000
overlap
•Promoter 1
•Promoter 2
Reproducibility on tiling arrays
•500 kb region of chromosome 6
•500 kb region of chromosome 1
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
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。
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。
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。
THANK YOU
FOR YOUR ATTENTION
谢 谢