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ZOOLOGY Molecular Cell Biology

Promoter analysis, DNA foot printing

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Paper : 15 Molecular Cell Biology

Module : 34 Methods for analysis of gene expression:


Co-Principal Investigator : Prof. D.K. Singh

Department of Zoology, University of Delhi

Paper Coordinator : Prof. Kuldeep K. Sharma

Department of Zoology, University of Jammu

Content Writer : Dr. Jasvinder Kaur, Dr. Poonam Sharma

Gargi College, University of Delhi

Ms Poornima Vishwakarma, Research Scholar, DU

Content Reviewer : Prof. Rup Lal


Principal Investigator : Prof. Neeta Sehgal


Development Team



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Description of Module

Subject Name ZOOLOGY

Paper Name Zool 015: Molecular Cell Biology

Module Name/Title Methods for analysis of gene expression

Module ID M34: Promoter analysis, DNA foot printing

Keywords DNA Footprint Assay, DNase I, DNA foot print, Promoter characterization,

Cleavage agent, Autoradiography

Contents

1. Learning Outcomes

2. Introduction

3. Procedure

4. Cleaving Agents

4.1. DNase I

4.2. Hydroxyl radicals

4.3. Ultraviolent radiation

5. Applications

5.1. Advanced applications

5.1.1. In vivo footprinting

5.1.2. Quantitative footprinting

5.1.3. Detection using capillary electrophoresis

5.2. Limitations

6. Summary



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1. Learning Outcomes

After studying this module, you shall be able to learn

The basic principle and procedure of DNA footprinting.

A brief account of the labelling and cleavage agents that should be used is also

presented.

You will also be able to learn the various applications for which this technique can be

used and recount it with promoter analysis.

2. Introduction

DNA Footprint Assay is used to determine the binding site of a protein regulator to DNA

which is typically at a promoter for a gene. Promoters play a fundamental role in regulating

gene expression. It is at this site that the RNA polymerase binds for transcription. Information

on promoter strength and regulation can help understand gene expression. Binding of the

polymerase to DNA involves multiple functional sites. Binding sites for transcription factors

include elements like the TATA box, GC box, and CAAT box. By studying these distinct

elements within the promoter sites, as well as their combinatorial effects, our understanding

of promoter strength and regulation will be enhanced, subsequently expanding our perception

of gene expression.

Examples of some online Promoter- prediction programs for eukaryotes are:

1. PROMOSER - Human, Mouse and Rat promoter extraction service (Boston University,

U.S.A.) - maps promoter sequences and transcription start sites in mammalian genomes

2. Promoter and gene expression regulatory motifs search (Softberry, U.S.A.) - offers a

variety of promoter-scanning programs

3. GP Miner - identifies promoter regions and annotates regulatory features in user-input

sequences. After identifying the promoter regions, the regulatory features such as

transcription factor binding sites, CpG islands, tandem repeats, the TATA box, the CCAAT

box, the GC box, over-represented oligonucleotides, DNA stability and GC-content are

graphically visualized to enable the observation of gene promoters.



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For promoter characterization, DNA footprinting is often used. DNA foot printing was

developed by Galas and Schmitz in 1978. It is an in-vitro system for tracing protein binding

regions on a DNA molecule. The technique is also known as DNase I foot printing. A large

number of proteins interact with the DNA to control the cell exercises like replication,

transcription and translation. The technique to find out these DNA-binding regions is called

DNA foot printing. For instance, for locating regions in a gene where transcription factors

(proteins) bind (control elements) and initiates transcription.

ProfessorDavid J. Galas (in picture) and Albert Schmit developed DNA foot printing technique in 1978.

Source:https://www.researchgate.net/publication/22923884_DNAase_footprinting_Simple_method_for_detecti

on_of_protein-DNA_binding_specificity

Principle

Footprinting is a technique to determine the exact DNA sequence to which a particular DNA-

binding protein binds. In this method, nucleases like DNase I is used to degrade the DNA

molecule. However, nucleases cannot degrade DNA if it is bound by proteins. Consequently

that region is shielded from degradation by nucleases. The protected DNA region (bound by

proteins) is called a “foot print” (Fig. 1).

The method enables the recognition of the nucleotide(s) that binds a DNA binding protein.

DNA foot printing is primarily used to identify the transcription factors of a gene that bind (s)

to regulatory regions like promoters, enhancer and silencer (Fig.1). This technique is based

on the principle that the DNA sequences that are involved in the direct binding with DNA

binding proteins, are protected by nucleases (DNase I) or chemical reagents

(hydroxyl radicals and copper-phenanthroline ions) that cleave the phosphodiester backbone.

By employing DNA foot printing technique, the segregation of an unknown DNA binding



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protein can be carried out by means of the crude cell extract or purified proteins. It is very

crucial that the conditions created during cleavage are such it leads to reproducible alterations

when the DNA is with or without DNA binding protein. The true potential of this technique

is the simultaneous visual assessment of cleavage products with and without protein on

sequencing gels.

Fig. 1: Sample of a DNase I footprinting gel (for a DNA-binding protein). Lanes 2-4 had increasing amounts of

the DNA-binding protein (lambda protein cII); lane 1 had none.

Source: http://image.slidesharecdn.com/footprint-150907080647-lva1-app6891/95/footprint-3-

638.jpg?cb=1441613474

3. Procedure

1. A DNA fragment thought to contain sequences for a potential protein-binding site is

isolated and amplified by Polymerase chain reaction (PCR).

First the DNA region of interest is digested into fragments using restriction enzymes.

Amplicon length between 50 to 200 base pairs is preferred. The fragments are radio-labelled

for easy detection. For this, the 5' phosphate groups of the fragments are removed using an

alkaline phosphatase enzyme. The dephosphorylated DNA is then incubated with radio-

labelled 32P-ATP and polynucleotide kinase so that the 5' phosphate groups are reintroduced

to the fragments.

Footprint



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2. The DNA is treated with a nuclease such as DNase I that digests only unprotected

DNA.

The radio-labelled DNA must then be incubated with a mixture of nuclear protein extract that

includes the protein of interest, the protein will bind to their complementary regions on the

DNA. The labelled fragments are then mixed with a cleavage agent such as DNase. The

cleavage agent is a chemical or enzyme that will cut at random locations in a sequence

independent manner. The reaction should occur just long enough to ensure that each fragment

is broken approximately once, DNase (a DNA-hydrolysing enzyme) is added to the mixture

in low concentration for this. A protein that specifically binds a region within the DNA

template will protect the DNA it is bound to from the cleavage agent.

DNase I results in a complete base ladder (one base difference) when electrophoresed in 6-8

% polyacrylamide gel. As a control for comparison; in a separate test tube, labelled DNA is

mixed with DNase without the suspected protein.

3. X-ray film exposure and autoradiography.

The digested fragments are then run on high-resolution polyacrymalide gel to separate them

by size. X-ray autoradiography of the radio-labelled fragments is done thereafter. Blank

regions on the gel correspond to regions where there was no digestion of the fragment due to

the bound protein. This will allow inference of the specific DNA sequence which binds the

protein.

Comparison of both samples reveals foot prints or protein binding sites. Maxam-Gilbert

chemical DNA sequencing can be run along with the samples on the polyacrylamide gel to

allow the prediction of the exact location of ligand binding site. The result will be a mixture

of radioactive fragments of varying lengths, with the smallest increment in length represented

by a single nucleotide. In DNA sample with protein, protein binding regions are protected

from degradation by DNase I.



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Fig. 2: a) DNA sample without protein; b) DNA sample with protein

Source: http://www.biology-pages.info/F/footprinting.gif

In DNA sample A without protein, consistent degradation by DNase I, results in a continuous

ladder. In DNA sample B with protein (lac repressor), interrupted degradation by DNase I.

Protein or lac repressor bound regions are protected from cleavage by DNase I. When the

fragments are separated by electrophoresis, those representing the lengths covered by the

repressor will be missing from the autoradiogram. This protected DNA region is seen as a

gap called the “DNA foot print”. The same sample of DNA (unprotected by the repressor) is

subjected to normal DNA sequencing and the resulting ladder aligned with the footprint

autoradiogram. Missing bands (footprint) indicate where protein was bound to DNA.

Labelling

Depending on the location of the binding site, the DNA template can be labelled at the 3' or 5'

end. Radioactivity or fluorescence can be used as labels. Traditionally, radioactivity has been

used to label DNA fragments for footprinting analysis, as the method was originally

developed from the Maxam-Gilbert chemical sequencing technique. Radioactive labelling is

very sensitive and is ideal for visualizing small quantities of DNA. On the other hand,

fluorescence is a desirable improvement due to the hazards of using radio-chemicals.

However, it has been more challenging to optimize because it is not always sensitive enough

to detect the low concentrations of the target DNA strands used in footprinting experiments.

For analyzing footprinting of fluorescent tagged fragments, electrophoretic sequencing gels

or capillary electrophoresis have proved successful.



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4. Cleaving Agents

A number of cleavage agents can be selected. An ideal desirable agent should be sequence

neutral, simple to utilize, and is effortless to manage. But unfortunately no existing cleavage

agents satisfy these all standards; therefore, a suitable agent can be selected, considering the

DNA sequence and ligand of interest.

4.1. DNase I:

DNase I is a double-strand endonuclease that binds the DNA in the minor groove o cleaves

the phosphodiester backbone. Since, the action of DNase I can be controlled by EDTA, it is

an excellent cleavage agent for footprinting. As DNase I action depends on the structure and

sequence of DNA it results in an uneven ladder. Additionally, the prediction accuracy of

protein specific DNA sequence is also as affected.

4.2. Hydroxyl radicals:

When the iron salts are reacted with hydrogen peroxide, they are reduced to form free

hydroxyl radicals. The DNA fragment is cleaved when the free hydroxyl molecule react with

the DNA backbone. Owning to the smaller size of these free hydroxyl radicals, very high

resolution DNA footprining occurs. These free hydroxyl radicals are not dependent on

sequence and result in more equally distributed ladder. Because of slower reaction and

digestion time, the downside of using the hydroxyl radicals is that they are more time

consuming. DNA footprinting using hydroxyl radical has been extensively employed for

studying the DNA structure and DNA-protein complexes. Given that the hydroxyl radical

have high reactivity and no base specificity, they are first rate probe for high-resolution

footprinting and assist in revealing minute structural details can be accomplished which is not

possible by using DNase I footprinting. Furthermore, the hydroxyl radical footprinting can be

done using easily accessible and low-cost reagents and lab apparatus. In this technique, the

hydroxyl radicals cleave the nucleic acid molecule attached to protein, subsequently the

cleavage products are separated on a denaturing electrophoresis gel to ascertain the protein-

binding sites on the nucleic acid molecule.



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4.3. Ultraviolent radiation:

The excited nucleic acid using ultraviolet radiations can lead to DNA fragments damage.

Once both protected and unprotected DNA has been treated, this process involves another

extra step which is the primer extension of the cleaved products. This primer extension gets

terminated when it reaches a damaged base and consequently as the PCR products are run

alongside on a gel; the protected DNA sample (the DNA is attached to protein) will display

an extra band. There are many advantages of using ultraviolet radiations in DNA footprinting

as they react very quickly and as a result are capable of capturing very brief interactions also.

Furthermore, as UV can pierce the cell membranes, this technique can even be applied to in

vivo experiments. But the major drawback is that the gel is very intricate to infer and

understand as the attached protein does not shield the DNA, it only modifies the

photoreactions in its surroundings.

Since the DNase I molecule is comparatively larger than all other footprinting reagents, its

attack on the DNA is considerably quite sterically hindered. Therefore, DNase I footprinting

is the most certain of all the footprinting techniques used to identify a specific DNA-protein

interface. The electrophoresis gel (Fig. 3) results show the bands corresponding to DNase I

cleavage sites. As the quantity of protein present on DNA increases, the footprint area is

gradually more protected from cleavage and hence, a bigger footprint will be observed on the

gel.

Other agents that break or damage DNA can be used in addition to DNase I.

G residues can be methylated with the help of of Dimethyl sulfate (DMS), creating a DNA

damage site that can be chemically broken. The position of the breaks is then determined by

primer extension. It can be carried out under conditions (e.g., absence of magnesium) in

which DNase I is inactive. Also, it can be done on covalently closed, supercoiled DNA,

which often binds proteins differently than linear DNA.

Additionally, Potassium permanganate (KMnO4) can be employed which is specific for

thymine residues in single-stranded regions of DNA. Again, it is usually used in conjunction

with primer extension.



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Fig. 3: Electrophoresis gel picture showing the bands corresponding to DNase I cleavage sites. As the quantity

of protein present on DNA increases, a bigger footprint area is observed on the gel.

5. Applications

DNA footprinting is a method used to study the DNA-binding proteins based on

sequence specificity. This technique can study both the exterior and interior protein-

DNA interactions of cells.

Various transcription factors that bind with operators, enhancers and silencers can be

known and studied using DNA footprinting technique.

The transcription regulation has been investigated to a large extent but even now so

much is still to be acknowledged. These factors and their related proteins which bind

the promoters, enhancers, or silencers to drive or suppress transcription are essential

to understand and examine the extraordinary regulation of individual genes of the

entire genome. Such techniques similar to DNA footprinting will assist in clarifying

which all proteins bind to different regions of DNA and solve the complexities of

regulation of transcription.

Scientists can use DNA footprinting to ascertain the various functional genes present

in human genome



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Diverse hormone-receptor and their complexes that bind to their hormone response

constituents can be recognized.

DNA footprinting facilitates the identification of the promoter region and by this

means also the expression levels of the gene. These bound proteins on DNA

sequences both close to and far away from promoter also influence gene expression

levels.

Example: binding of the lac repressor (which shuts down the lac operon in E. coli) to

a 24 base pair sequence in the operator region stops DNase I from attacking this

region of the operon.

5.1. Advanced applications:

5.1.1. In vivo footprinting:

To analyze the protein-DNA interactions taking place in a cell at a certain time point, the in

vivo footprinting technique utilized. If the cell membrane can be permeabilized, DNase I can

be employed as a cleavage agent. However, UV irradiation is one of the most widespread

cleavage agent used as it is capable of penetrating the cell membrane without disrupting the

cell’s normal state and consequently catch the interactions that are receptive to cellular

changes. To investigate the region of interest, the purified DNA sample can be isolated from

lysed cells after the UV has cleaved the DNA. Another alternative method to carry out in vivo

footprinting technique is the ligation-mediated PCR. A linker is added onto the break points

produced by cleavage agent on the genomic DNA. Using a gene-specific primer, the area of

interest is amplified and after the sample is run on a polyacrylamide gel, the footprint where a

protein was bound can be obtained. In vivo footprinting in amalgamation with

immunoprecipitation can be employed to study and evaluate the protein specificity at several

positions spread all over the entire genome. DNA attached to a protein of interest can be

immunoprecipitated with an antibody and next the specific region binding can be anylazed

using the DNA footprinting method.

5.1.2. Quantitative footprinting:



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The DNA footprinting procedure can be customized to evaluate the binding strength of a

protein to an area of DNA. By using different concentrations of the protein for the

footprinting test, the emergence of the footprint can be detected because as the concentrations

increase and the proteins binding affinity can then be predicted.

5.1.3. Detection using capillary electrophoresis:

To modify the footprinting procedure to modernized detection means, the labelled DNA

fragments are identified using capillary electrophoresis mechanism as a substitute of

polyacrylamide gel. When the DNA fragments to be investigated are produced by PCR, the

fluorescent molecules like carboxyfluorescein (FAM) are simply coupled to the primers. In

this way, the DNase I digestion produce fragments containing FAM and consequently can be

visualized by the capillary electrophoresis machine. In general, standards labeled with

carboxytetramethyl-rhodamine (ROX) are mixed to the combination of fragments to be

examined. Transcription factors binding sites on DNA have been effectively acknowledged

using this method.

5.2. Limitations:

1. The methodology is tedious and technically demanding.

2. DNase I can only be used under conditions that support its activity (presence of Mg++ and

Ca++ ions).

3. DNase I has a small degree of specificity, so that it does not cut uniformly at all sites.

4. Binding is not instantaneous. The protein needs to be incubated with the DNA for a period

of time to allow binding.

6. Summary

DNA footprinting is often used for promoter characterization. Developed by Galas and

Schmitz in 1978, it is an in-vitro system for tracing protein binding regions on a DNA

molecule. A large number of proteins interact with the DNA to control the cell exercises like

replication, transcription and translation. The technique to find out these DNA-binding

regions is called DNA foot printing. For instance, for locating regions in a gene where

transcription factors (proteins) bind (control elements) and initiates transcription.



13

Footprinting is a technique to determine the exact DNA sequence to which a particular DNA-

binding protein binds. In this method, nucleases like DNase I is used to degrade the DNA

molecule. However, nucleases cannot degrade DNA if it is bound by proteins. Consequently

that region is shielded from degradation by nucleases. The protected DNA region (bound by

proteins) is called a “foot print”.

It is a modest procedure involving four steps (Fig. 4).

A DNA fragment thought to contain sequences for a potential protein-binding site is

isolated and amplified by Polymerase chain reaction (PCR).

The DNA is treated with a nuclease such as DNase I that digests only unprotected

DNA.

The digested fragments are then run on high-resolution polyacrymalide gel to separate

them by size.

X-ray autoradiography of the radio-labelled fragments is done.

The result will be a mixture of radioactive fragments of varying lengths, with the smallest

increment in length represented by a single nucleotide. Depending on the location of the

binding site, the DNA template can be labelled at the 3' or 5' end. Radioactivity or

fluorescence can be used as labels. In DNA sample with protein, protein binding regions are

protected from degradation by DNase I. A number of cleavage agents can be selected. An

ideal desirable agent should be sequence neutral, simple to utilize, and is effortless to

manage. Various transcription factors that bind with operators, enhancers and silencers can be

known and studied using DNA footprinting technique.

The technique has varying applications ranging from- recognition of diverse hormone-

receptor and their complexes that bind to their hormone response constituents to

identification of the promoter region and by this means also the expression levels of the gene.



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Fig. 4: DNA footprinting procedure. The DNA is amplified and labelled. The protein of interest is then bound to

the amplified fragments. The DNA is now cleaved using a preferable cleaving agent. The fragments are now run

on a polyacrylamide gel and “footprint” identified by autoradiography or fluorescence tracking.

https://en.wikipedia.org/wiki/DNA_footprinting#/media/File:Courtney_2008.jpg

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