What Are Effective Methods to Investigate Gene Downregulation in Cancer?

Thread starter Ygggdrasil
Start date Jul 17, 2016
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Biology Cell biology Gene Regulation

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SUMMARY

This discussion focuses on investigating gene downregulation in cancer, specifically targeting transcription factors and signaling pathways involved in the expression decrease of a particular gene. The participant proposes using computational tools to analyze promoter DNA for transcription factor binding sites and exploring distal enhancer sequences. Additionally, generating a reporter cell line with GFP under the target gene's promoter and conducting an siRNA screen is suggested as a method to identify perturbations affecting gene expression. The conversation seeks further insights into effective computational and experimental approaches for unbiased investigation.

PREREQUISITES

Understanding of transcription factor binding sites and their role in gene regulation.
Familiarity with enhancer sequences and their identification methods.
Knowledge of reporter assays, specifically using GFP for gene expression studies.
Experience with siRNA screening techniques for gene expression modulation.

NEXT STEPS

Research computational tools for identifying transcription factor binding sites, such as JASPAR or TRANSFAC.
Explore methods for enhancer identification, including Hi-C and ChIP-seq techniques.
Learn about designing and implementing reporter assays using GFP and other fluorescent markers.
Investigate best practices for conducting siRNA screens, including controls and analysis of results.

USEFUL FOR

Researchers in molecular biology, cancer biologists, and geneticists interested in gene regulation mechanisms and experimental approaches to study transcriptional changes in cancer.

Jul 17, 2016

Ygggdrasil

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I am studying a particular gene whose expression decreases in certain types of cancer. I'm interested in finding out the mechanisms for this downregulation, specifically, which transcription factors and signalling pathways are involved. I have a hypothesis for the pathway involved, but in case that hypothesis turns out to be incorrect, I want to look into some ways of approaching the problem in an unbiased manner. Here are my thoughts for some starting approaches:

1. Feed the promoter DNA for this gene into a computer program to look for transcription factor binding sites. (https://abc.med.cornell.edu/education/introtobio/t-promoter.html seems to list a number of resources. Does anyone have experience with any of these?) However, a lot of gene regulation in mammals occurs though distal enhancer sequences. Are there computational or experimental ways to identify enhancers for a target gene?

2. Generate a reporter cell line by placing GFP under the control of my target gene's promoter and perform an siRNA screen to see what perturbations increase/decrease expression of the reporter.

Are there better ways of computationally and/or experimentally approaching this problem? Any suggestions or pointers to helpful references would be appreciated.

Last edited: Jul 17, 2016