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:
Promoter analysis, DNA foot printing
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
Department of Zoology, University of Delhi
Principal Investigator : Prof. Neeta Sehgal
Department of Zoology, University of Delhi
Development Team
ZOOLOGY Molecular Cell Biology
Promoter analysis, DNA foot printing
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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.
ZOOLOGY Molecular Cell Biology
Promoter analysis, DNA foot printing
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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