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 15-7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Chromatin Remodeling there are two common ways in which chromatin structure is altered 1. Covalent modification of histones 2. ATP-dependent chromatin remodeling 15-38 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 15-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Only one strand is methylated Both strands are methylated DNA Methylation Only one strand is methylated Both strands are methylated 15-45

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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Chromatin Immunoprecipitation DNA Sequencing (ChIP-seq) Mapping protein-DNA interactions by ChIP-seq

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.

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

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

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) 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

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 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) 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) Illumina Solexa(2nd) ABI SoLid (2nd) Helicos (3rd) Applications De novo sequencing Targeted resequencing Digital Gene Expression (DGE) RNA-seq ChIP-seq Sequencing Platforms

Chromatin Immunoprecipitation Visual Protocol https://www.youtube.com/watch?v=bHsfSteiy4E