Transcript ChIP-seq - FaPGenT

Lecture-5
ChIP-chip and ChIP-seq
Huseyin Tombuloglu, Phd
GBE423 Genomics & Proteomics
• A PROTEIN INTERACTS WITH DNA.
• Q. HOW YOU CAN FIND ITS INTERACTION
REGION ON DNA?
• ChIP-sequencing, also known as ChIP-seq, is a
method used to analyze protein interactions
with DNA
• Also ChIP-chip is the array-based method for
the same purpose
Figure 15.2
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15-7
The recognition helix recognizes and makes contact
with a base sequence along the major groove of DNA
Hydrogen bonding between an a-helix and nucleotide
bases is one way a transcription factor can bind to DNA
Figure 15.3
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Composed of one a-helix and
two b-sheets held together by
a zinc (Zn++) metal ion
Two a-helices intertwined
due to leucine motifs
Note: Helix-loop-helix motifs can
also mediate protein dimerization
Figure 15.3
Alternating leucine residues in
both proteins interact (“zip up”),
resulting in protein dimerization
Homodimers are formed by two
identical transcription factors;
Heterodimers are formed by two
different transcription factors
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Positioned at regular intervals from -3,000 to + 1,500
Disruption in nucleosome positioning
from -500 to + 200
Figure 15.11
Changes in nucleosome position during the activation of the b-globin gene
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Chromatin Remodeling

there are two common ways in which chromatin
structure is altered

1. Covalent modification of histones

2. ATP-dependent chromatin remodeling
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
1. Covalent modification of histones

Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation
Adds acetyl groups, thereby
loosening the interaction
between histones and DNA
Figure 12.15
Removes acetyl groups,
thereby restoring a
tighter interaction
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
2. ATP-dependent chromatin remodeling

The energy of ATP is used to alter the structure of
nucleosomes and thus make the DNA more accessible
Figure 12.15
These effects may significantly alter
gene expression
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DNA Methylation
Only one strand is
methylated
Both strands are
methylated
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Transcriptional
activator binds to
unmethylated DNA
This would inhibit the
initiation of transcription
Figure 15.14 Transcriptional silencing via methylation
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Chromatin Immunoprecipitation
DNA Sequencing (ChIP-seq)
Mapping protein-DNA interactions by ChIP-seq
Why ChIP-seq?
• Protein-DNA interactions
• Chromatin States
• Transciptional regulation
ChIP experiment
In Nutshell
•Protein cross-linked to DNA in vivo
by treating cells with formaldehyde
•Shear chromatin (sonication)
•IP with specific antibody
•Reverse cross-links, purify DNA
•PCR amplification*
•Identify sequences
•Genome-wide association map
*-unless using a single molecule sequencer
History: From ChIP-chip to ChIP-seq
ChIP-chip (c.2000)
• Resolution (30-100bp)
• Coverage limited by
sequences on the array
• Cross-hybridization between
probes and non-specific targets
creates background noise
ChIP-seq experiment (2007-present)
Chromatin immunoprecipitation (ChIP)
An approach to detect specific DNA regions/sequences
associated with a protein of interest, in vivo.
Became a powerful tool to analyze protein/DNA interactions,
furthermore to detect any signal/modification associated with
DNA/Chromatin.
Made a huge impact on
chromatin biology, epigenetics
transcription research, etc.
Chromatin immunoprecipitation (ChIP)
1. Cross linking with FA
Optimization is crucial.
2. Cell lysis and sonication
Chromatin immunoprecipitation (ChIP)
3. Immunoprecipitation (IP)
The protein of interest is immunoprecipitated together with
the crosslinked DNA
Epitope tagging of protein of
interest (HA, myc)
-no need for raising antibody,
-utilize commercial antibodies
better sensitivity
decreased noise
Anything associated with
chromatin can be ChIPed, if an
antibody can be raised.
Chromatin immunoprecipitation (ChIP)
4.Decrosslinking and purification of the DNA
Reverse the FA
crosslinking
Chromatin immunoprecipitation (ChIP)
5. Analysis of ChIP DNA
Identification of DNA
regions associated with
the protein/modification of
interest
- real-time PCR
- DNA microarray
(ChIP-chip)
- Sequencing
(ChIP-seq)
Controls for ChIP (ChIP-seq)
- Input DNA
the Chromatin sample processed parallel to the other
samples but lacks the immunoprecipitation step.
- No antibody control (IgG)
the Chromatin sample processed parallel to the other
samples and immunoprecipitated without specific
antibody
- No tag control
chromatin processed in the same way as samples
but from a strain not having a tag on the protein to be
analysed.
Input DNA
Chromatin immunoprecipitation (ChIP)
Analysis of ChIP DNA
-
Problems with real-time PCR - Not genome wide, making the
identification of unknown, potentially interesting binding sites unlikely.
-
By the development of microarray technologies
ChIP on chip
ChIP DNA is amplified, labeled and hybridized to microarray
Genome wide landscape of binding, good but a little laborious.
-
By the development of high throughput sequencing methods
ChIP-seq
Workflow overview of the wet-lab portion of a ChIP-on-chip experiment.
Workflow overview of the dry-lab portion of a ChIP-on-chip experiment.
ChIP-seq technology
novel high-throughput sequencing methods
Chip-seq technology
Instead of hybridizing the ChIP DNA to a microarray, each sample is
processed directly into a DNA library for sequencing and subsequent
bioinformatics analysis.
ChIP-Seq has improved sensitivity and reduced background
over ChIP-chip.
2-4 times more TFBS are determined, compared with ChiP-chip
DNA library preparation (Illumina)
1. Oligonucleotid adapters are introduced to the ends of small DNA fragments
DNA library preparation and sequencing (Illumina)
2. Adapter ligation, PCR amplification (16-17 cycles)
Barcode sequencing – multiplex illumina sequencing
DNA library preparation and sequencing (Illumina)
4. Library sequencing.
~260000 reads for yeast
genome
13 M reads for human
genome
One flowcell generates
~8 M reads.
Sequence data analysis
- Determination of binding sites from the sequence data is a challenge.
Conceptually, genomic regions with an increased number of sequencing
reads (tags) compared to control is considered to be a TFBS
- Statistical filtering criteria is used to determine if these putative sites
represent true binding sites.
- After statistical analysis of binding sites a further analysis of data is
required. These may include analysis of location of binding, relative to
transcription factor binding sites or potential nearby target genes.
2nd and 3rd Generation DNA Sequencers and
Applications
Roche 454 (2nd)
Sequencing Platforms
Applications
Illumina Solexa(2nd)
De novo sequencing
ABI SoLid (2nd)
Targeted resequencing
Helicos (3rd)
Digital Gene Expression
(DGE)
RNA-seq
ChIP-seq
Chromatin Immunoprecipitation Visual Protocol
https://www.youtube.com/watch?v=bHsfSteiy4E