Genomic Imprinting & Epigenetics

基因印迹与表观遗传学

徐国良 研究员
细胞大楼 300
电话 : 6433 5944
E-mail: glxu@sibs.ac.cn
Epigenetics October 17, 2005
1. Introduction to Epigenetics
1.1 Ubiquity of epigenetics
1.2 Epigenetic Inheritance v.s. Mendelian inheritance
1.3 Epigenetic gene regulation v.s. Signal transduction

2. Genomic Imprinting
2.1 Discovery
2.2 Igf2/H19 paradigm
2.3 Imprinting Center
2.4 CTCF
2.5 Evolutional aspects

3. X-chromosome Inactivation
 Epigenetics October 19, 2005
1. DNA methylation
4.1 Basic concepts: methylation patterns, de novo methylation, maintenance methylation
4.2 Techniques to study DNA methylation
4.3 Biological functions of DNA methylation
4.4 Mechanisms of DNA methylation-mediated transcriptional repression
4.5 Methylation dynamics in development
4.6 DNA methyltransferases
4.7 DNA methylation and disease

5. Epigenetic regulation of Phenotypes

6.1 Epigenetic regulation of homeotic genes in the formation of Drosophila body plan
6.2 Epigenetic regulation in the control of flowering
6.3 Implications for cancer etc
 Mammalian DNA Methylation

H2N H2N
H CH3
N N
MTase
SAM
O N O N

dC 5-methyl-dC
CH3
Methyltransferase in action
Detection of genomic methylation by methylation-
sensitive restriction enzymes

HpaII, MspI MspI

CH3

-----CCGG----- -----CCGG-----
-----GGCC----- -----GGCC-----
CH3
Detection of Methylcytosines: Bisulfite Sequencing
 DNA 甲基化的生物学功能

• 遏制基因组内寄生虫（ intragenomic
parasites ）
• 控制基因活性状态 ( 开或关 )
如在基
因印迹 (genomic imprinting) 与 X 染色
体失活 (X-inactivation) 过程中
• 维持染色体结构稳定性
• 维持正常胚胎发育

• 甲基化与癌变和某些遗传病有关
Review

Science 293, 1068. August 10, 2001

Bacterial Restriction/Modification Systems
E. coli Dam methylation
and Mismatch Repair

CH3

---GATC---
---CTAG---
CH3
 How DNA methylation works?

Jones P. & Takai D. Science 293, 1068- (2001).

DNA Methylation and Gene Silencing
 Methylation reprogramming
in the germ line (A) and
Preimplantation embryos (B)

Reik W. et al, Science 293, p1089-

Dynamic changes of DNA Methylation patterns in development
Dnmt1 Protein and mRNA in Germ Cells and Early Embryos
Mammalian DNA Methyltransferases
Facial Features in ICF Syndrome
A
A­Miniou et al. Hum. Mol. Genet. 3(12):2093 (1994)
B­Adapted from Brown et al. Hum. Genet. 96:411 (1995)
 DNMT3B mutations in ICF patients

Ins744STP
A766P
158X G655S V810M
S282P
F/S 53 A585V L656T H814R

I IV VI IX X
∆1aa
6
R54X Q204X V606A V718G D809G
A603T V699G
R832Q

Xu et al., Nature, 1999;

Hansen et al., PNAS, 1999;
Shirohzu et al., Amer. J. Med. Genet., 2002.
 Conclusions

• DNMT3B is the ICF disease gene

• DNMT3B methylates satellite sequences
• Cytosine methylation is essential for
chromosome stability and heterochromatin
formation.
Diseases associated with abnormal
methylation

Diseases Genes involved

ICF DNMT3B
Rett MeCP2
Fragile X Syndrome FMR
α - Thalassaemia ATRX

Cancer Tumor suppressor genes

Azoospermia in Homozygous Males (Dnmt3L-/-)
Abnormal development of embryos from homozygous females
Loss of Maternal Methylation
Imprints in Embryos Derived
Homozygous Mother
Biallelic Expression of Maternally Imprinted Genes in Dnmt3L -/+ Embryos
 Conclusions
• Dnmt3L is essential for the establishment of
maternal imprints (i.e. the de novo methylation
of maternally imprinted genes in oogenesis)
Cell 69, 915-926, 1992

Targeted Mutation of the DNA Methyltransferase

Results in Embryonic Lethality

En Li, Timothy H. Bestor, and Rudolf Jaenisch

Figure 1. Disruption of the 5’ End of the DNA MTase Locus
Figure 2. Southern Blot Analysis of ES Cell Clones Targeted with pMT(N)neo or hyg
Figure 3. Immunoblot Analysis of DNA MTase Figure 4. DNA MTase Enzyme Activity
In Wild-Type and Mutant ES Cell Lines In Wild-Type and Mutant ES Cell Lines
Figure 5. Quantitation of Genomic Figure 6. Southern Blot Analysis of DNA
m5C Content Methylation of Endogenous Retroviral Geno
In Wild-Type and Mutant ES Cell Lines
In Wild-Type and Mutant ES Cell Lines
wt

Figure 7. Gross Morphology of Wild-Type

and Mutant Embryos at 10.5 Days of
Gestation
mutant
Figure 9. Immunoblot Analysis and m5C Quantitation of Wild-Type and Mutant Embryos

---C CGG---
---GGC C---
Epigenetic Regulation of Phenotypes – Example 1

Regulation of Drosophila Body Plans

through Control of Homeotic Genes
Polycomb, Trithorax, and Maintenance of Gene Expression
A key feature of development in metameric animals is the definition of body
segments where groups of cells with a specified fate will give rise to their relative
body structures. Cell fates are specified by particular combinations of homeotic gene
products. During early embryogenesis, maternal and segmentation genes regulate
homeotic genes by binding to specific regulatory sequences located in the promoter
regions.  Later in development, the expression pattern of homeotic genes as well as
other important developmental genes are maintained by a cell memory system
dependent on two groups of genes. The members of these two groups are able to
recognize the active and inactive state of expression and fix it to the cell progeny
through many cell divisions. These components have been classified in two genetic
groups. The trithorax-group (trxG) maintain the active state of expression, while the
Polycomb-group (PcG) counteracts this activation with a stable repressive function.
There is strong evidence that the memory function encoded by these two groups
of genes is achieved through regulation of higher order chromatin structures. PcG
gene products form large multimeric protein complexes in Drosophila, mouse and
human. PcG mediated gene silencing can be directed by DNA elements in cis, defined
as PcG response elements (PRE). On the other hand, several trxG members act at
elements defined as TRE (that overlap with PRE) via chromatin remodeling and
induction of histone modifications that increase chromatin accessibility to
基因表达状态的建立与维持需要
transcription factors. :
顺式调控元件 : specific DNA sequence
反式作用因子 : DNA binding proteins including
transcriptional factors
 Polycomb, Trithorax, and Maintenance of Gene Expression
Early development  OFF ONMaternal, Gap, Pair­rule, Segment polarity
Establishment of patterns Ubx

OFF ON

Polycomb­Group Maintenance phase trithorax­Group

Transmission of 
pattern after 
disappearance of early 
factors
OFF ON

Ubx OFF  Ubx ON

wing  haltere 
Update: December 2004
PcG and trxG Proteins Associate to Multiple Genomic Loci
DAPI Merge

PH

Polytene chromosome staining shows around 100 bands for each PcG protein
 PcG and trxG Proteins Bind to Specific DNA
Elements, Named PRE and TRE

PRE
und by PcG proteins in vivo (in polytene chromosomes and by cross­linking experiments)

inding leads to maintenance of PcG­dependent repression of reporter genes

Repression is enhanced by homologous pairing of the transgenes

TRE
Bound by trxG proteins in vivo

inding leads to maintenance of trxG­dependent activation of reporter genes

PRE and TRE often overlap in the same genomic region
 Members of the PcG and of the trxG
PcG Gene protein motif homologs
Polycomb (Pc) chromo domain M33 (mouse); hPC (human)
(Binding to H3 methyl K9 or K27)
PRC1  polyhomeotic (ph) one zinc finger Mph1/Rae­28 (mouse); hph1; hph2 (human)
comple
x Posterior sex combs (Psc) RING finger bmi­1 (mouse/human); mel­18 (mouse)

Esc/E(z) Enhancer of zeste (E(z)) SET (H3MTase) Ezh1; Ezh2 (human); clf (Arabidopsis)

Complex extra sex combs (esc) WD repeat Eed (mouse); hEED (human)

Pleiohomeotic (pho) Zinc­finger (DNA binding) hYY­1 (human); mYY­1 (mouse)

trxG Gene protein motif homologs

TAC1  trithorax (trx) SET  (H3 HMTase)/ PHD­finger MLL/ALL­1/HRX (human)
complex
Ash­1 SET  (H3/H4HMTase)/ PHD­finger ASH­1 (human); NSD1 (mouse)

bromo domain SWI2/SNF2 (yeast)
Brm complex brahma (brm)
(DNA dependent ATPase/helicase) brg1 (mouse/human); Hbrm (human)

FACT  Trithorax­like (Trl) BTB/POZ (dimerization)

complex zinc finger (DNA binding)
Action of PcG and trxG Complexes on Chromatin
Nucleosome trxG
remodeling
Ac (BRM complex) ON Maintenance of
Histone active states
acetylation and (open
methylation
(TAC1 and Me K4 H3 chromatin)
ASH1
complexes)

Target
PRE gene

OFF PcG
Deacetylation and Me K27 H3
methylation Maintenance of
(ESC-E(Z) repressed states
complex) (compact
- Chromatin chromatin)
compaction Ub H2A
- H2A
Ubiquitination
(PRC1 complex)
 Histone H3 methylation and Polycomb
Pc H3 K9  Merge
triMe

Data from: 
Ringrose et al. 
(2004) Mol. Cell 
16, 641 Pc H3 K27  Merge
triMe
There is a strong but not absolute correlation between trimethylation of K27 (and
K9) trimethylation and Polycomb recruitment at target loci. i.e. there is more to Pc
recruitment
Epigenetic Regulation of Phenotypes – Example 2

Regulation in the Control of Flowering

Vernalization Requirement and Response in Crops and Arabidopsis
“ 春化作用”后才能开花
The Role of FLC in Vernalization Requirement and Response
VRN Genes Are Required to Maintain Suppression of FLC after Vernalization

fca-1

vrn1fca-1
Epigenetic Silencing of FLC