Question

OPEN QUESTIONS: You are researching mechanisms involved in wdf1 gene expression regulation in yeast

1. Please shortly describe how to identify enhancers regulating expression of the wdf1 gene.

2. You have discovered that Wou1 protein is the transcriptional factor that is involved in wdf1 activation. You next goal is to identify other factors, that are interacting with Wou1 protein and are required for wdf1 activation. Please shortly describe experimental approaches to achieve this goal.

3. You have discovered that Wou1 protein is the transcriptional factor that is involved in wdf1 activation. You next goal is to identify other factors, that are interacting with Wou1 protein and are required for wdf1 activation. Please shortly describe alternative experimental approaches to achieve this goal.

There is no other information provided.

Answer 1

1. Identification of enhancers
Enhancers can be identified by a method called the Enhancer trap.

• In enhancer trap, a reporter gene, say, GFP is fused to a minimal promoter - that contains the core promoter elements like TATA box and transcription start site.
• Using a P-element transposon, the reporter gene- promoter complex is transformed into a host cell, in this case, yeast.
• During random insertion by transposons, if the gene complex inserts near the Enhancer of the wdf1 gene, then the GFP expression will increase.
• The upstream region of the insertion can be restriction digested, PCR analyzed and sequenced to identify the Enhancer.
• Additional NGS techniques can be used to identify the protein binding motifs and other CRMs supporting the evidence.

2.

• The Wou1 is the transcription factor involved in wdf1 activation.
• To identify other factors interacting with Wou1 and activating gene expression, we can go for the EMSA technique.
• Here, the protein complex of Wou1 and associated protein binds to DNA and is isolated in gel electrophoresis.
• Upon isolation, the protein complex can be sequenced to identify other proteins complexed with wou1.

3.

• To identify the other proteins in the complex Co-immunoprecipitation(co-IP) is commonly used.
• Here, we can pull down the entire protein using bait(antibodies or antigens) and immobilized to a support.
• The binding partners can then be detected by SDS-PAGE and western blot analysis.