Analyze the Data 9-2: DNA Binding with EMSA An electrophoretic mobility shift as

Question

Analyze the Data 9-2: DNA Binding with EMSA

An electrophoretic mobility shift assay (EMSA) was performed using a radiolabeled DNA fragment from the sequence upstream of gene X. This DNA probe was incubated with (+) or without (-) nuclear extract isolated from tissues A (bone); B (lung); C (brain); and D (skin). The DNA;protein complexes were then fractionated on nondenaturing polyacrylamide gels. The gels were exposed to autoradiographic film; the results are presented in the figure.

a. Which tissues contain a binding activity that recognizes the sequence upstream of gene X? Is the transcription factor the same in each tissue?

b. If the binding activity was purified, what test could be done to verify that this factor is in fact a transcription factor?

c. What type of assay would be performed to determine the specific DNA sequence(s) within this probe that the transcription factor binds?

Explanation / Answer

From the given gel, it is clear that gel shift is seen only in nuclear extracts from bone and skin tissues. This shows that a transcription factor is present specifically in these tissues. It is given that these tissues do not express gene X. So, the transcription factor in bone and skin tissues must be a negative regulator. i.e. repressor of gene X transcription. Since this transcription factor is absent in lung and brain tissues, gene X is expressed in these tissues.

The speculated transcription factor might recruit transcription silencing complex (could be epigenetic) to the promoter region of gene X. This leads to the repression of gene X expression.

a. Bone and skin
b. We can perform yeast-1-hybrid assay or ChiP to confirm that it is indeed a transcription factor.
c. We can perform SELEX with the given protein. We can also perform EMSA with mutant oligos to determine which nucleotide positions are important for binding.

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