ChIP’ing (non-)histone modifications
Efrén Riu Pastor
Center of Animal Biotechnology and Gene Therapy
(CBATEG)
Table of contents
1. Epigenetics
1.1.- DNA Methylation
1.2.- Chromatin/Histone modifications
2. Methods to unveil target genes
3. Introduction to Chromatin Immunoprecipitation
4. ChIP detection
4.1.- Array hybridization (ChIPonCHIP)
4.2.- High-throughput sequencing (ChIPseq)
5. Other high-throughput NGS applications
Epigenetic modifications
• ‘Epi’genetics - ‘On’ or ‘over’ the genetic information
encoded in the DNA
•Epigenetics is the study of gene regulation that does
not involve making changes to the SEQUENCE of the
DNA, but rather to the actual BASES within the
nucleotides and to the HISTONES
Epigenetic mechanisms
DNA methylation
Covalent modifications of
histones
Protein complexes
RNA-mediated gene regulation
The DNA methylation reaction
NH 2 NHN
N
N
CH3 C CH
CH 2SCH 2CH 2CH(NH 2)
N N +
COOH + O C
N
CH
HO HO
S-Adenosylmethionine (SAM)
DNA methyltransferase
(DNMT)
NH2
N NHN
N
C C CH3
N N CH 2SCH 2CH2CH(NH 2)
+
+ O C CH
COOH N
HO HO
S-Adenosylhomocystein (SAH)
DNAmethylation silences gene expression by
two independent mechanisms
Transcription
factor
CH3
X CH3
Ac
Transcription
factor Ac Ac
X
Sin3A
Ac
MECP2 HDAC
CH3 CH3
CH3 CH3
SUV39
X
HP1
DNA Methylation Analysis: Bisulfite treatment
Chromatin Structure and Organization
Chromosomes are Composed of Chromatin:
A. DNA – long, acidic polymer (2 meters,
3 billion (3 x 109) base pairs per cell)
B. Protein
1. Histones – small basic (positively
charged) proteins whose mass equals
that of the DNA
2. Non-histone proteins and RNAs
(replication, transcription etc.)
Nucleosome/Histone modifications
Acetyl
Ubiquityl
Methyl
Phosphoryl
Luger et al.
Nature, (1997)
Histone tails can be
covalently modified in
multiple ways at multiple Felsenfeld and Groudine,
sites Nature, (2003)
Histone code hypothesis
“… multiple histone modifications, acting in a
combinatorial or sequential fashion on one or multiple
histone tails, specify unique downstream functions …”
― Strahl and Allis,
Nature, (2000)
Histone modification cross-talk
HISTONE H3 MODIFICATIONS
Histone methylation, histone deacetylation and DNA methylation interactions:
Euchromatin and heterochromatin plasticity
(Inactive)
(Active)
2. Analysis of Target Genes: Informatics
Bioinformatics prediction
- Consensus Binding Sites
2. Analysis of Target Genes: Immuno-FISH
Bioinformatics prediction
- Consensus Binding Sites
Immuno-Fluorescent in situ Hybridization
- In vivo assay
- Low resolution
2. Analysis of Target Genes: Immuno-FISH
TBP TBP
TATAAA TATAAA
ATATTT ATATTT
AUAUUU
F F FF
Lavrov, S., Déjardin, J., and Cavalli, G. (2004). Combined immunostaining and
FISH analysis of polytene chromosomes. Methods Mol Biol, 247: 289-303.
2. Analysis of Target Genes: EMSA
Bioinformatics prediction
- Consensus Binding Sites
Immuno-Fluorescent in situ Hybridization
- In vivo assay
- Low resolution
Electrophoretic Mobility Shift Assay
- In vitro assay
- High resolution
2. Analysis of Target Genes: EMSA
TBP P* TATGAA P*
TBP
TATAAA
P* TATAAA
2. Analysis of Target Genes: DNA footprinting
Bioinformatics prediction
- Consensus Binding Sites
Immuno-Fluorescent in situ Hybridization
- In vivo assay
- Low resolution
Electrophoretic Mobility Shift Assay
- In vitro assay
- High resolution
DNA Footprinting
- In vivo or in vitro assay
- High resolution
2. Analysis of Target Genes: DNA footprinting
DNAseI DNAseI naked DNA chromatin
DNAseI TBP DNAseI
TATAAA
T
A
T
A
A
A
TATAAA
TATAAA
TATAAA
2. Analysis of Target Genes: ChIP
Bioinformatics prediction
- Consensus Binding Sites
Immuno-Fluorescent in situ Hybridization
- In vivo assay
- Low resolution
Electrophoretic Mobility Shift Assay
- In vitro assay
- High resolution
DNA Footprinting
- In vivo or in vitro assay
- High resolution
Chromatin Immunoprecipitation
- In vivo assay
- High resolution
3. Introduction to Chromatin Immunoprecipitation
1. Chromatin crosslinking double crosslinking with disuccinimidyl
glutarate and formaldehyde
crosslinking with
formaldehyde amino and imino groups of proteins
react with formaldehyde, and the
intermediate reacts with another amino
group (DNA or protein) and condenses
2. Sonication
sonication
IMPORTANT: REACH A BALANCE BETWEEN
CROSSLINKING AND SONICATION
3. Introduction to Chromatin Immunoprecipitation
3. Immunoprecipitation
addition of the
specific antibody
As a negative control, an IP without Ab or
with a non-related Ab must be performed
3. Introduction to Chromatin Immunoprecipitation
4. Purification of the immunocomplexes
addition of
conjugated beads protA/G
Conjugated
beads
(agarose, sepharose or
magnetic beads)
3. Introduction to Chromatin Immunoprecipitation
5. Decrosslinking and purification
protA/G reversal of crosslinks
conjugated by heating
Seph/Magnet.
As input, a 10% of the IP extract must be
also decrosslinked
DNA PURIFICATION
Chromatin ImmunoPrecipitation detection
e3
4m
b
tA
3K
t
pu
Conventional detection
ou
H
x
in
Tr
ith
w
- Southern Blot / PCR rp49
- qPCR
High-throughput detection
ChIPonCHIP
ChIP-Seq
4. ChIPonChip: Preprocessing
1. End repair
Klenow
+ dNTPs
2. Linker ligation
Ligase
+ ATP Ligase
3. Linker-mediated PCR amplification
4. ChIPonChip: Hybridization
Detection (comparison of two samples: different tissues or treatments)
control or hybridization control or
sample 1 sample 1 hybridization
NimbleGene / Agilent
sample 2 Arrays sample 2
Affymetrix Arrays
4. ChIPonChip: Normalization
1. Filter the images
2. Assign a value to each spot
3. Convert the spots into sequences
4. Align to genome in genome browser
4. High-throughput detection: ChIPonChip
-High resolution - Availability of the array
-Genome wide analysis - Array cross-hybridization
- Faster and cheaper than conventional - Spacing between oligos
detection
4. High-throughput detection: ChIPseq
Cluster amplification of purified chromatin
Sequencing by incorporation of fluorescence
labeled nucleotides
Data processing
1. Filter the images
2. Convert spots into sequences
3. Map sequences to the genome (not allowing more than 2 missmatches)
4. Discard all sequences that may align to more than one region
5. Align to genome in genome browser
4. ChIPseq: Library preparation
Cluster amplification of purified chromatin
1. End repair
Klenow
+ dNTPs
2. Addition of “A” base to 3’ ends
A
+ dATP A
Klenow Exo minus
4. ChIPseq: Library preparation
3. Adapter ligation
T A
A ligase T
4. Size Selection and PCR Amplification
e3
b
tA
4m
2
ou
3K
sh
t
pu
ith
αA
αH
in
400 bp
300 bp
200 bp
100 bp
4. ChIPseq: Cluster amplification
5. Denature the DNA and load the flowcell
8 lines per flowcell
300 tiles per line 7,2x107 reads per flowcell
30000 reads per tile
4. ChIPseq: Amplification
6. Amplification
- Hybridization
- Amplification
- Denaturation
4. ChIPseq: Amplification
4. High-throughput detection: ChIPseq
Cluster amplification of purified chromatin
Sequencing by incorporation of fluorescence
labeled nucleotides
Data processing
1. Filter the images
2. Convert spots into sequences
3. Map sequences to the genome (not allowing more than 2 missmatches)
4. Discard all sequences that may align to more than one region
5. Align to genome in genome browser
4. ChIPseq: Sequencing
Sequencing in the Genome Analyser
[Link] of fluorescent nucleotides
[Link] the image of emitted fluorescence
[Link] the fluorophore
4. High-throughput detection: ChIPseq
Cluster amplification of purified chromatin
Sequencing by incorporation of fluorescence
labeled nucleotides
Data processing
1. Filter the images
2. Convert spots into sequences
3. Map sequences to the genome (not allowing more than 2-3 missmatches)
4. Discard all sequences that may align to more than one region
5. Align to genome in genome browser
Analysis of ChIP-Seq data (quality control)
· fastQC is a free tool to analyse sequence quality after
sequencing
- Good quality sequence
- Poor quality sequence
Visualization of ChIP-Seq data
· At genome browsers data sets are upload in different formats
signals (peaks) are often visualized as
wiggle (wig) files
targets (enriched regions) are often
visualized as Bed Extensible Data (bed)
files
GENOME DISTRIBUTIONS
GENOME BROWSERS
HISTONE PROFILES
4. High-throughput detection: ChIPseq
- It works with a very little amount of DNA (10 ng - Sequencer facilities
or less)
- Standard normalization has
- It allows to perform ChIPs of all previously not been set up
sequenced species
- It does not depend on the annealing temperature
of the products and avoids problems of cross-
hybridization or secondary structures
Comparison of ChIP-chip and ChIPSeq
5. RIP/RNASeq
quantitative
PCR
high-throughput
detection (RNA Seq)
5. MeDIPSeq
5. MNase-Seq
5. FAIRE-Seq
Efrén Riu Pastor
[Link]@[Link]
