Transcript Epigenetic Alterations in Cancer - Biomedical Informatics

Chromatin Structure
& Dynamics
Victor Jin
Department of Biomedical Informatics
The Ohio State University
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)
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
Nucleosome Assembly In Vitro
4 core histones + 1 naked DNA template at 4C at 2M salt concentration,
from Dyer et al, Methods in Enzymology (2004), 375:23-44.
10,000 nm
DNA compaction in a human cell nucleus
11 nm
30nm
1bp (0.3nm)
DNA
length
compaction
10 m ball
12,000
Mbp
4 m DNA
400,000 x
1200
bp
400 nm
DNA
35 x
nucleus
(human)
2 x 23 = 46
chromosomes
chromatin
fiber
approx. 6 nucleosomes per
‘turn’ of 11 nm
30 nm
diameter
nucleosome
disk 1.65 turn of DNA (146 bp)
+ linker DNA
6 x 11 nm
200 bp
66 nm
DNA
6 - 11 x
0.33 x 1.1
nm
1 bp
0.33 nm
DNA
1x
base pair
92 DNA
molecules
compact
size
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 and Lysines


Arginine can be
methylated to form
mono-methyl,
symmetrical di-methyl
and asymmetrical dimethylarginine.
Lysine can be
methylated to form
mono-methyl,
di-methyl
and tri-methylarginine.
Methylation of Histone H3-K27
SUZ12
EED
DNMT
EZH2
PC
K27
Functional Consequences of
Histone Modification
 Establishing global chromatin environment, such as
Euchromatin, Heterochromatin and Bivalent domains in
embryonic stem cells (ESCs).
 Orchestration of DNA-based process transcription.
Euchromatin




A lightly packed form of chromatin;
Gene-rich;
At chromosome arms;
Associated with active transcription.
Heterochromatin





A tightly packed form of chromatin;
At centromeres and telomeres;
Contains repetitious sequences;
Gene-poor;
Associated with repressed transcription.
Bivalent Domains
Poised state. The chromatin of embryonic stem cells has
“bivalent” domains with marks of both gene activation and
repression. In these domains, the tail of histone protein H3
has a methyl group attached to lysine 4 (K4) that is
activating and a methyl group at lysine 27 (K27) that is
repressive (above). This contradictory state may keep the
genes silenced but poised to activate if needed. When the
cell differentiates (right), only one tag or the other remains,
depending on whether the gene is expressed or not.
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
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.
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
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
Epigenetics
Modifications of DNA (cytosine methylation) and proteins
(histones) define the epigenetic profile.
In 1942, Conrad Waddington first used “epigenetics” to describe
the interactions between genome and environment that give rise to
differences between cells during embryonic development.
Currently, Epigenetics is the study of heritable changes in gene
function that occur without a change in DNA sequence.
Summarizes mechanisms and phenomena that affect the
phenotype of a cell or an organism without affecting the genotype.
Epigenomics is the study of these epigenetic changes on a
genome-wide scale.
Normal Cellular Functions Regulated
by Epigenetic Mechanisms
Correct organization of chromatin
Genomic imprinting
Silencing of repetitive elements
X chromosome inactivation
X-chromosome Inactivation
5
me


3’..pGpCp..5’
5
me
5’..pCpGp..3’
transcriptional repressor MeCP2
co-repressor
 X-inactivation (also called lyonization) is a
process by which one of the two copies of the X
chromosome present in female mammals is
inactivated.
 The inactive X chromosome is silenced by
packaging in repressive heterochromatin.
 The choice of which X chromosome will be
inactivated is random in higher mammals such
as mice and humans. Once an X chromosome is
inactivated it will remain inactive throughout the
lifetime of the cell.
 Silencing initiated at Xic/XIC and spreads along
chromososme.
 5meC CpG DNA modification is observed in
inactivated X chromosomes.
 5meC binds transcriptional repressor MeCP2
(MethylC-binding Protein-2).
 MeCP2 binds Sin3 with RPD3 histone
deacetylase.
Sin3
Histone Deacetylase
RPD3
Source: Jones et al. Nat.Genet. 19, 187 (1998)
Epigenetic Diseases
 Some human disorders such as Angelman syndrome
and Prader-Willi syndrom are associated with
genomic imprinting.
 Involvement in cancer and development
abnormalities.
 The emerging hypothesis of cancer stem cells (CSC).
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
Epigenetic Mediation of Gene Silencing
DNMT
Polycomb Repressors
Histone-modifying Proteins
Methyl-Binding Domain
Proteins
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
Epigenetic Alterations in Cancer
Stem Cells
Cancer Stem Cells: Stem cells arising through the
malignant transformation of adult stem cells.
Cancer Stem Cells Hypothesis: Cancer stem cells are the
main driving force behind tumor proliferation and
progression.
Hallmarks of Cancer Stem Cells
A cell residing in a tumor that –
1. has a capacity to remain in an undifferentiated state
2. has properties of asymmetric divisions and self-renewal
3. has metastatic and repopulation capacities at specific niches
(microenvironment) in the body
4. gives rise to a tumor that is histologically identical to the one
from which the CSC is derived
The Evidence of Cancer Stem Cells
 First isolated from the patients of acute myeloid
leukemia in 1997 by John Dick and colleagues at
the University of Toronto.
 Isolated from two solid tumors, breast and brain
cancers.
 ~1% cancer cells may be really cancer stem cells.
More ChIP-chip
Step 1: Rapid fixation of cells chemically cross-links DNA binding proteins to their
genomic targets in vivo.
 Step 2: Cell lysis releases the DNA-protein complexes, and sonication fragments the
DNA.
 Step 3: Immunoprecipitation (IP) purifies the protein-DNA fragments, with specificity
dictated by antibody choice.
 Step 4: Hydrolysis reverses the cross-links within the released DNA fragments.
 Step 5: PCR amplification of ChIP DNA
 Step 6: PCR amplification on a known binding-site region for that protein will need to be
performed using either conventional PCR methods followed by agarose gel
electrophoresis or by quantitative PCR.
 Step 7: Labeling pool of protein-DNA fragments.
 Step 8: 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))
Antibody Validation
 Confirming on a known target
 Different antibodies to same factor
 Antibodies to different family members
 siRNA-ChIP
 Antibodies to two components of a complex
 Antibodies to an enzyme/modification pair
Confirming on a known target
Comparison of biological replicates and
antibodies to different E2Fs
Loss of E2F6 ChIP signal after
knockdown of E2F6 siRNA
Reproducibility of promoter arrays
using biological replicates
•H3me3K27; Ntera2 cells
•Top
1000
overlap
•Top
1000
overlap
•Promoter 1
•Promoter 2
Biological reproducibility
on tiling arrays
•500 kb region of chromosome 6
•500 kb region of chromosome 1
Amount of Sample Per ChIP
Number of cells
Chromatin
input
ChIP output
1x107
200 µg
150 ng
1x106
20 µg
10 ng
5x105
10 µg
1.3 ng
1x105
2 µg
300 pg
1x104
200 ng
30 pg
Amount of Sample Per ChIP
Number of cells
Chromatin
input
ChIP output
1x107
200 µg
150 ng
1x106
20 µg
10 ng
5x105
10 µg
1.3 ng
1x105
2 µg
300 pg
1x104
200 ng
30 pg
Miniaturization
•Standard ChIP Protocol (1x107 cells; WGA2)
•
Promoter Arrays
•
Genome Tiling Arrays
•MicroChIP Protocol (10,000-100,000 cells; WGA4)
•
Promoter Arrays
•
Genome Tiling Arrays
Reproducibility of MicroChIP Protocol