Determination of Protein-DNA Interactions

Question 1

How are protein interactions studied?

Answer 1

DNA footprinting

1. foot printing is a method for determining the exact DNA sequence to which a particular DNA binding protein binds
2. label the DNA fragments to be tested
3. Digest the DNA with a nuclease (DNase) microccal nuclease) or cleave with specifice chemicals
4. separate fragments by size (electrophoresis) using denaturing conditions (ssDNA)
5. Dry gel and expose to X-ray film to locat bands

Is most often done in vitro but can be done in vivo. The DNA sequence needs to be known.
 Multiple enzymes (DNAse 1, Micrococcal Nuclease, Exo nuclease III) can be used. Certain chemicals generating hydroxyl radical that will cleave the DNA between every bp can also be used.

Question 2

Electrophoretic-mobility shift assay

Answer 2

1. EMSA used to study nucleic acids (DNA or RNA) interactions with proteins
2. Binding or proteins to the nucleic acid fragments will alter their electrophoretic mobility in a gel (agrose or acrylamid)

EMSA: migration is a function of mass, conformation and charge. Most commonly the addition of the DNA-binding protein will result in a lower mobility in the electrical field.
Gross approximation of the principles of electrophoretic analysis, where the charge and conformation are equally important.

1. DNA fragment
2. divide sample

• 1st sample

1. protein-incubate with protein
2. protein bound to DNA

• 2nd sample

1. incubate without protein
2. Electrophoresis
3. Autoradiography

• DNA-protein complex
• Free DNA

Question 3

Purification of Sp1 by DNA affinity Chromatography

Answer 3

Affinity chromatography.
If a specific transcrip[tion factor can recognize a specific sequence, that sequence can be attached to beads and use to trap the cognate TF or DNA-binding protein. The TF will bind the DNA under low ionic strength conditions (low salt).At medium salt concentration, the proteins that do bind in a non-specific manner will be eluted (referred to as wash conditions). The protein will be released by increasing the ionic strength (high salt) Elution of purified TF or DNA-binding protein.

Question 4

Chromatin immunoprecipitation

Answer 4

1. 1. treat cells with formaldehyde
2. sonicate to produce fragments of chromatin
3. fragments of chromatin with transcription factors cross-linked to DNA
4. immunoprecipitate with antibody against transcription factor of interest

• Chromatin-Immunoprecipitation is a laboratory procedure to analyze target proteins bound to DNA with antibodies.

Procedure:

• Most important feature is the use of an antibody recognizing and binding to a protein of interest.
• Targets a protein associated with DNA, and allows immunoprecipitation of DNA through protein-antibody complex.

Questions of interest are:

• Whether a given protein binds to a specific DNA sequence
• To investigate protein binding under different experimental conditions
• To scan for specific target genes by finding responsive elements in upstream regions of the gene.

Question 5

Chromatin immunoprecipitation

Answer 5

1. Antibody binds specific transcription factor
2. Collect chromatin-antobody complex
3. Reverse cross-links
4. purify DNA
5. DNA fragment containing specific transcription factor binding site

Antibodies are the most important factor for a successful ChIP experiment. It is crucial to choose an antibody carefully, especially when antibodies
are raised against non-plant proteins. Papers reporting the use of a specific antibody for ChIP are a good indication of the suitability of that
antibody, but one should realize that quality can differ between antibody batches. The successful use of a specific antibody in experiments other than
ChIP (i.e. Western blotting, immunocytochemistry) does not automatically mean the antibody is suitable for ChIP; that has to be tested.
To ensure the reliability of ChIP data, two control samples specific for the ChIP experiment should be included: the input sample and the ‘no-antibody’ (NoAb) control sample.

Both the input sample and the NoAb control sample provide essential information about the ChIP experiment, and should be analyzed with
every primer set used.
ChIP-QPCR
Accurate, trusted, quantitative
Not high throughput or global
Good for testing and validation

ChIP-chip
Agilent 244,000 custom 50mer chips
Affymetrix 25mer Whole Genome Tiling chips Nimblegen tiling arrays
ChIP-Seq
Sequence census, or counting, method New ultrahigh throughput sequencing Solexa [Illumina]
Cheap, quantitative, precise, comprehensive (but requires unique infrastructure)

Question 6

Enhancers and Insulators

Answer 6

1. Cis-acting regulatory elements
2. orginially discovered in mammalian but habe been described in E coli
3. multiple functional sequence elements
4. proteins can bind to enhancer regions (activators)
5. Enhancers are positioned and orientation-independent
6. enhancers involved in transcriptional regulation (protein-protein interactions activators and co-activators)
7. Insulators: Cos-acting elements that divide chromosomes into independent domains. They also are known to prevent heterochromatin from spreading to neighboring domains

Enhancers and insulators are cis elements (sequences to which specific proteins will bind and promote a conformation that will be conducive to the formation of a particular structure. Such structure will have the role of bringing far apart regions of DNA in cleos proximity or to prevent heterochromatin from invading euchromatin

Question 7

Actions of enhancers

Answer 7

Enhancers are position (upstream or downstream of +1 position) and orientation-independent (forward or backward)

How does and enhancer work? It is needed to increase the transcription efficiency (simulated transcription)

Question 8

Enhancers and DNA looping

Answer 8

enhancers can generate loops that will promote the long distance interactions