AM Patil III Sem

8/3/2019 AM Patil III Sem

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Genome Wide Detection of

Protein-DNA Interactions

A. M. Patil


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Contents

Introduction

Detection techniques DNA-Protein

ChIPChIP-on-chip

ChIP-SAGE

ChIP-PETApplication

Conclusion


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Genome

Predominantly composed ofnon-coding sequences (>98%)

Significant portion of non-CDS serve as transcriptional

regulatory element

Previous investigations: molecular, genetical and biochemicalapproach identified several DNA binding proteins.

Identification and characterization of gene regulatorysequences, reveal gene regulatory networks in cells. Whichserves as a fundamental in understanding developmentalbiology and in determining the molecular basis of manybiological processes.


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Continued

The interaction b/n DNA & protein has an important role in

Repairing damaged DNA.

Recombination & replication.

Genomic integrity.

Controls of epigenetic changes.Differentiation & Development.


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These proteins interact with DNA by means ofvarious structural Motifs


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Detecting: Protein-DNA interaction

Gel retardation assays

DNase foot printing

DNA modification assay

Nitrocellulose filter binding assay

Crystallography


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Gel retardation assay:

Principle: DNA Protein complexes move slowly through an electrophoretic

gel than DNA.

Retarded band

DNA Protein


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DNase foot printing

5 10 15 20

Principle: DNA with bound protein is protected from nuclease digestion

20

15

10

5

Footprint

5 10 15 20

P


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Modification Protection assay: Protein bound to DNA can protect specific bases from

chemical modification

G G

Modification G G

G G G Gmm m

P

Residue

protected

Missing cleavage product

because protected residue

notmodified

DNA


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Incubate labeled DNA with protein

Filter the mixture through a filter disk

made of nitrocellulose Proteins bind to nitrocellulose, but

DNA does not. Any DNA that is retainedon the filter is there because it isinteracting with the protein.

Nitrocellulose filter binding assay


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DNA sequences interacting with proteins(TF)..contd.

Conventionally, studied in test tubes using purifiedproteins and naked DNA fragments.

Demerit: In vitro methods do not replicate the in vivoconditions faithfully physiological condition.

DNA inside cell exist in a compact chromatin state withdistinct properties from that of naked DNA

Besides chromatin organization and distributionof histone variants histone modification: acetylation,methylation.


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ChIP

Allows study of protein-DNA interactions directly inside cell

under in-vivo physiological conditions.

Protocol:

Cross linking of proteins and DNA (Formaldehyde)

Extraction and fragmentation of chrom DNA (mechanical

shearing / enzym

atic digestion)Immuno-affinity purification (using specific antibody

against a protein)

Assay purified DNA (southern blotting/ PCRs)


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Preparation of cross linked chromatin-cell line, tissue or whole organism.

Formaldehyde-common ,Dimethyapitimidate (DMA) & disuccinimedyl glutrate

(DSG)

O

II

H-C-H

Readily permeable & generates zero length cross link.

Formaldehyde reactive dipolar in which protein or DNA carbon acts as nucleophiliccentre.Amino & Imino gp of A.A & of DNA readily react with formaldehyde give Schiff

base. Which further react with 2nd amino group & condense to give the DNA-Protein

complex.

Continued


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Cells or tissues incubated with 1%-10-15min RT or4.c > time.

Increase cross link time make fragmentation of chromatin difficult.

Cell lysed isolate nuclei.

Chromatin released from nuclei by ultrasonicsonication , solubilizes & fragments chromatin 500-2000bp.

Sonication important step to optimize-Longfragment not immunoprecipitated efficiently

/detected on microarray againstshort fragment

Empirical test :time of cross linking no. of

sonication session

Continued ..


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Chromatin immuno precipitationcontd.

Immunoprecipitation : affinity & specificity crucial.

Not all available antibody efficiently immunoprecipites the protein DNA

complexes.Masking of epitome by formaldehyde cross link.

Incubation with chromatin (2ng) with magnetic / agarose beads coupled

with antibodies, followed by many wash to remove unbound fraction.

Assaying purified DNA

Southern blottingPCR


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Chromatin immuno-precipitationcontd.


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Chromatin immunoprecipitationcontd.

Demerits

Allows to study interaction between proteins & limitedno. of suspected DNA sequences.

Genome wide interaction cannot be studied.


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ChIP-on-chip

Genomic binding location of regulatory factors can be

determined using Chromatin immunoprecipitation

(ChIP) followed by determination of enriched fragments

by DNA microarray (chip) hybridisation.


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G

eneral features of DNAm

icroarray


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Microarray features

Density of the arrayNumber of features in one array.

ResolutionDistance between each feature on the contiguous

genomic region.

Level of genome representation

Total amount of genomic sequence represented by

the array.


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(ChIP)-enriched targets. Probes Cy5/Cy3 ratios significantly high confidence (P < 0.001) level.


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Contd

Approach to detect ChiP enriched DNA 2 color

competitive hybridisation .ChiP DNA one color

Control DNA other fluorescent color hybridized to

array. Ratio b/w ChiP & control DNA correlates with

enrichment of specific DNA sequences.

Alternately, to detect ChiP enriched DNA is detected

through 1 color hybridization. Test & control to

different arrays.


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Microarray data analysis

Single array error model

Rank based

Chip & control DNA signal Ratio.

To test specific enrichment is significant

Single error model assumes ofnormal distribution & significance is than

calculated by one sided probability.

Rank based model no assumption but require the multiple replicates.

ChIPOTle (Chromatin Immuno- Precipitation On Tiled arrays), Implemented as

a Microsoft Excel macro written in Visual Basic, ChIPOTle uses a sliding

window approach that yields improvements in the identification of bona fide

sites of protein-DNA interaction.

Michael et.al. 2005.


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Data interpretation

Microarray & ChIP-on-chipExpression experiments each element on microarraymeasures the abundance of RNA molecules of fixedlength.

ChIP-on-chip each element measures the abundance ofpopulation of fragments of various lengths due toeffect of chromatin shearing.Expression experiment the data are two tailed &

roughly symetric ,there is biological significanceassociated with both low & high ratio measurement.ChIP-on-chip measurement derived from experimentsarise as a mixture of two distribution .first correspondsto population of genomic fragments specifically enrichedby Chip, & second corresponds to remaining population ofgenomic DNA that is not Chip enriched representingbackground or noise.


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Validating the Identified Protein-Binding Sites

Microarray- and SAGE-coupled ChIP assaysMultiple steps to identify the protein-bindingsites in the genome, errors introduced at

various stepsValidation :Testing the relative enrichment ofthe identified sequences in the ChIP DNA incomparison to the total genomic DNA.

Quantitative PCR


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Merits of CHIP on chip

Availability of high density oligo-nucleotide arrays representing entiregenomes allows comprehensive mapping of

protein-DNA interactions

Technique doesnt rely upon previousknowledge of transcriptional regulatory

elementsIs a unbiased and comprehensive approach


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Contd..

Major drawback

Requirement of highly specific antibodies for the

proteins

Specific antibodies for all proteins are unavailable

There is immunoprecipitation oflarge no. of non-

specific of DNA sequences


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Alternate ways to purify DNA binding proteins nospecific antibodies

Using a Engineered cell systemsBy introducing epitope tag to endogenous locus (TF)

DNA adenine methyl transferase (DamID) identification

Method: expression of protein of interest fused with E.coli DamID,

binding of fusion protein cause methylation of local DNA sequences,

extract and digest DNA with methylation sensitive RE

Demerit: Dam fusion proteins may have distinct properties fromthat of endogenous proteins


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Chip-SAGE


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Principles of SAGE

A short sequence tag (10-14bp) contains scientificinformation to uniquely identify a transcript provided thatthe tag is obtained from a unique position within eachtranscript.

Sequence tags can be linked together to form long serialmolecules that can be cloned and sequenced .

Quantification of the number of times a particular tag isobserved , provides the expression level of thecorresponding transcript.


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Advantages over ChiP-chip. It does not depend on preselectedsequences & it can scan the whole genome

Disadvantages

Majority of ChIP DNA actually corresponds to non-specifically

immunoprecipitated sequences and requires sequencing a large

number of clones to distinguish the truly enriched sequences

from background.

Cost of sequencing is high.


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Chip-SAGE

Does not require priorknowledge of sequence to be

analyzed

Discovery of new sequencesis possible

Quantification of sequences is

possible.

ChiP-Chip

Require prior knowledge ofsequence to be analyzed

Comparison is only possiblewith existing sequences.

Quantification is not easy.


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Applications

Decoding the Transcriptional Regulatory Function ofGenome

Sequences.

Dissecting the Transcriptional Regulatory Circuits.

Unravelling Epigenetic Mechanisms.


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ChiP-SAGE

High resolution genome wide mapping of histone modification in

Yeast

Distribution of hyperacetylated histones H3 & H4

AcH3, AcH4that recognize hyperacetylated histones in yeast.

Compared directly the acetylation levels b/w promoter & codingregion among 6040 genes.

Tae-young Roh, 2004


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NlaIII site appears once every 316 bp

class II enzyme, MmeI


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Generally believed thatpromoter region are highly acetylated.

The analysis unexpectedly revealed that Highest histone H3acetylation observed afterATG start codon & within the first500 bp of ORFs.

The acetylation of H4 followed similar pattern.

Deletion of gene encoding GCN5 ,GCN5 is critical catalytic subunit

ofADA & SAGA complexes & plays a global role in regulatinghistone H3 acetylation.

Deletion of GCN 5 specifically eliminated the peak acetylation inpromoter , increased acetylation in 3 ORF.

Tae-young Roh,2004


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These suggest that GCN 5 functions to maintain higher levels of H3

acetylation in promoter & 5 end of coding region of transcription unit


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Transcription regulator networks in yeastTonglee et al., 2002

Epitome Tagging


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Genome wide analysis for yeast transcription regulatory bind topromoter sequences across genome.

Epitome tagging.

All 141 transcription factorsYeast Proteome Database for study.

Yeast strains were constructed so that each of transcription factor containedcMyc epitome tag.

Epitope tag might be expected to affect the function of some

transcriptional regulators; for 17 of the 141 factors, were not able toobtain viable tagged cells, despite three attempts to tag each regulator.

106 TFonly showed immunoblot.Each strain one TF .

Continued .


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Calculated a confidence value (P value) for each spotfrom each array by using an error model.

Generally describe results obtained at a P value

threshold of 0.001 because this threshold maximizesinclusion of legitimate regulator-DNA interactions andminimizes false positives.

Various experimental and analytical methodsindicate that the frequency of false positives

in the genome-wide location data at the 0.001

threshold is 6% to 10%

Continued .


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Nearly 4000 interactions between regulators and promoter

regions at a P value threshold of 0.001.

The promoter regions of2343 of 6270 yeast genes (37%) were

bound by one or more of the 106 transcriptional regulators.

Many yeast promoters were bound by multiple transcriptional

regulators a feature previously associated with gene regulation in

higher eukaryotes.

Suggesting that yeast genes are also frequently regulated

through combinations of regulators.

Continued .


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Effect ofPvalue threshold. The sum ofalregulator-promoter region

interactions is displayed as a function of varying Pvalue thresholds applied

to the entire location data set for the 106 regulators.More stringent P

values reduce the numberofinteractions reported but decrease the

likelihood of false-positive results .

Continued .


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Chromosome wide analysis for differential methylation innormal and transformed human cells.

Michael et al 2005

Methylation of cytosine at CpG dinucleotide is a major epigenitic

modification observed in mammalian cells.

CpG islands are exceptions and less methylated (are associated

with promoter regions of several house keeping genes).

Abberant promoter hypermethylation is believed to contribute to

sporadic cancer.


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Relevance of DNA methylation in normaldevelopment and disease

Immunocapturing technique combined

with DNA microarray for enrichment of

methylated DNA and detection

Use of methylation sensitive RE

(HpaII) 3.9% of all CpGs

Antibodies specific to -5 mcytosine

MeDIP (Methylated DNA

immunoprecipitation)


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Relevance of MeDIP technique


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Assessing the abberant CpG island methylation intransformed cell

CpG island methylation profile for both SW48 colon cancer

and normal colon mucosa was generated

Microarray representing ~ 12,000CpG island probes (derived

from CpG island library, 75% unique sequences, remaining

repeatative elements (SINES,LINES,satellites) as well as

ribosomal and mitochondrial genes.


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Observation

Normal cells

Most of CpG islands including ribos and mito. DNA showed basal/low level

methylation

Sequences with highest methylation included repetitive DNA, promoters of

imprinted genes or genes residing on X-chromosomes

Transformed cells

Methylation profile was almost same.

Hypermethylation was observed in 108 clones of which 82 clones

represented ribos DNA (reported in cells at the time of aging/neoplasia but

physiologic al role unknown), remaining 26 clones were unique


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Ne

wtargetsforhypermethylationi

ncolo

Cell matrix interactionapoptosisCell cycle progression


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Contd

Combining MedIP with hybridization on CpG island microarray

could be used to detect epigenetically silenced genes in cancer

cellsPattern ofCpG island methylation is conserved and no. of

genes that are hypermethylated in transformed cell are

unexpectedly low.


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Transcriptional regulatory circuitry in human ESCLaurie et al 2005

Mammalian development requires over 200 specialized cell types

All derived from single totipotent zygotic cell

Embryonic StemC

ellsDerived from inner cell mass ofblastocyst.

Are capable of generating any cell type of body (Pluriotent)

Realizing their therapeutic potential, underastanding the

transcriptional regulatory circuitry is fundamental in knowing

molecular basis ofpluripotency and self renewal of ESC


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Three major TF OCT4, SOX2 and Nanog

Key stem cell regulators in self renewal ability of stem

cells

They regulate genes encoding other transcriptional

regulators involved in determining the developmental

potential of cells

Known to interact with one another in a complex

synergistic way

Contd..


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Genome wide location analysis (CHIP on chip)

Objective:

To identify the target sites for three key stem cellregulators

DNA microarray design:Oligo-nucleotide covering (-8 to +2 Kb relative to trans.

start site) for 17,917 annoatated genes


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Examples of target sites for OCT4


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OCT4 showed binding to 623 (3%) of protein coding genes

SOX2 to 1271 (7%)

Nanog to 1681 (9%)

OCT4 and SOX2 shared half of bound proteins, while > 90% of Nanog

bound genes were of both OCT4 and SOX2

Regulators bind to both active and inactive/ repressed genes (that

encode for TF involved in regulation of gene required for meso, endo

and ectodermal diffrn)

RESULTS


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Examples of protein coding genes co-occupied by OCT4, SOX2and Nanog in close proximity


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Myogenic differentiationAlexandre et.al 2005

Genome wide TF binding & expression profiling to assemble

regulatory network controlling Myogenic differentiation in

mammalian cells.

Myoblast Myotubes (multinucleate)

Governed by TF ,MRFs(MyoD & Myogenin) MEF2

Myod bHLH bind to E boxes of genes.

First step of regulatory cascade involves expression ofMyoD

which subsequently leads to myogenin & MEF2 promoting

myogenesis.


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Contd..

Beyond these first steps knowledge is somewhat fragmentary:

Relatively few physiological targets of MRFs and MEF2 have

been identified.

The number of genes known to be regulated by these factors is

considerably smaller than the number of genes induced upon

myogenic differentiation


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Contd..

Recent attempts to identify MyoD-binding sites exploitedgene expression profiling of cells that ectopically expressMyoD.

complications

Ectopic expression of a bHLH transcription factor canlead to promiscuous binding to E boxes throughout thegenome. The fact that another E-box-binding factor,Myc, binds to an extensive portion of the genome


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To know contribution of MRF family members

on gene expression patterns.

ChiP chip using promoter Chip from C2ci2

myoblast .


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Confirmation for binding by MyoD, myogenin, and MEF2 to asubset of targets


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Growing myoblasts, MyoD bound a

Set of genes involved in synapse specification andutilization (NMj) and neuromuscular function

Bound by MyoD in MT play a role in muscle

development & contraction.


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MRFs ,MEF2-Triangle

Square TF

Circle Non TF


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ChiP-PETChiP-PET

Ruan and colleagues :Pair of signature tags,paired-end tags (PET), both ends of a DNAconcatenate.

Like SAGE, the PET method can be used to obtainlarge numbers ofsequence tags with moderatecost.

PET additional information of start and end ofeach ChIP DNA fragment, used precisely locate the

binding sites. To achieve this, one identifies theoverlapping PET, and the overlap between differentPET fragments would correspond to the binding sites.


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