1. Lecture-5 ChIP-chip and ChIP-seq
Huseyin Tombuloglu, Phd
GBE423 Genomics & Proteomics

2. A PROTEIN INTERACTS WITH DNA.
Q. HOW YOU CAN FIND ITS INTERACTION REGION ON DNA?

3. 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

4. Figure 15.2
15-7
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5. 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
15-9
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6. Composed of one a-helix and two b-sheets held together by a zinc (Zn++) metal ion
Alternating leucine residues in both proteins interact (“zip up”), resulting in protein dimerization
Two a-helices intertwined due to leucine motifs
Homodimers are formed by two identical transcription factors;
Heterodimers are formed by two different transcription factors
Note: Helix-loop-helix motifs can also mediate protein dimerization
Figure 15.3
15-10
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7. Positioned at regular intervals from -3,000 to + 1,500
Disruption in nucleosome positioning from -500 to + 200
Changes in nucleosome position during the activation of the b-globin gene
Figure 15.11
15-37
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8. 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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9. Removes acetyl groups, thereby restoring a tighter interaction
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
Removes acetyl groups, thereby restoring a tighter interaction
Figure 12.15
15-39
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10. These effects may significantly alter gene expression
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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11. Only one strand is methylated Both strands are methylated
DNA Methylation
Only one strand is methylated
Both strands are methylated
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12. 15-47 Figure 15.14 Transcriptional silencing via methylation
Transcriptional activator binds to unmethylated DNA
This would inhibit the initiation of transcription
Transcriptional silencing via methylation
Figure 15.14
15-47
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13. Chromatin Immunoprecipitation DNA Sequencing (ChIP-seq)
Mapping protein-DNA interactions by ChIP-seq

14. Why ChIP-seq? Protein-DNA interactions Chromatin States
Transciptional regulation
Histone modifications (methylation at K and R) play role in gene regulation; both expression and repression. Enzymes that catalyze methylation reaction have been implicated in playing a critical roles in development and pathological processes. Promotor regions of active genes have reduced nucleosome occupancy and elevated histone acetylation.

15. ChIP experiment In Nutshell Shear chromatin (sonication)
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

16. 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
Tiled arrays cover most of the non-repetitive genome. Cost increases with size of genome. Ex. Yeast has been very well characterized, but human not so much due to genome size

17. ChIP-seq experiment (2007-present)

18. 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.

19. Chromatin immunoprecipitation (ChIP)
1. Cross linking with FA
Optimization is crucial.

22. 2. Cell lysis and sonication

23. 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.

24. Chromatin immunoprecipitation (ChIP)
4.Decrosslinking and purification of the DNA
Reverse the FA
crosslinking

25. 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)

26. Controls for ChIP (ChIP-seq)
Chromatin immunoprecipitation (ChIP)
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.
IgG
Input DNA
no tag

27. 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

28. Workflow overview of the wet-lab portion of a ChIP-on-chip experiment.

29. Workflow overview of the dry-lab portion of a ChIP-on-chip experiment.

30. ChIP-seq technology novel high-throughput sequencing methods
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

31. DNA library preparation (Illumina)
Oligonucleotid adapters are introduced to the ends of small DNA fragments

32. DNA library preparation and sequencing (Illumina)
2. Adapter ligation, PCR amplification (16-17 cycles)
Barcode sequencing – multiplex illumina sequencing

33. DNA library preparation and sequencing (Illumina)
4. Library sequencing.
~ reads for yeast genome
13 M reads for human genome
One flowcell generates ~8 M reads.

39. 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.

40. 2nd and 3rd Generation DNA Sequencers and Applications
Roche 454 (2nd)
Illumina Solexa(2nd)
ABI SoLid (2nd)
Helicos (3rd)
Applications
De novo sequencing
Targeted resequencing
Digital Gene Expression (DGE)
RNA-seq
ChIP-seq
Sequencing Platforms

41. Chromatin Immunoprecipitation Visual Protocol