What Are Effective Methods to Investigate Gene Downregulation in Cancer?

[Ygggdrasil]

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.

 

[Zefram]

Dear fellow researcher,

Thank you for sharing your research topic and your proposed approaches for investigating the downregulation of a gene in certain types of cancer. I have some experience in this area and would like to offer some suggestions and references that may be helpful to you.

1. Computational approaches for identifying transcription factor binding sites and enhancers:
- As you mentioned, there are several resources available for identifying transcription factor binding sites, such as TRANSFAC, JASPAR, and PROMO. These databases provide information on predicted binding sites based on known transcription factor binding motifs. However, it is important to note that these predictions may not always accurately reflect the actual binding sites in vivo.
- To complement the computational approach, you could also consider using experimental methods such as chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) or ChIP coupled with microarray analysis (ChIP-chip). These techniques allow for the identification of transcription factor binding sites in a more direct and unbiased manner.
- As for identifying enhancers, there are also computational tools available, such as EnhancerFinder and EnhancerAtlas, which use histone modification patterns to predict enhancer locations. However, similar to transcription factor binding site predictions, these may not always be accurate and should be validated experimentally.
- Another experimental approach for identifying enhancers is through the use of CRISPR/Cas9 genome editing technology. This allows for the targeted deletion of potential enhancer regions, followed by analysis of the effect on the target gene's expression.

2. Reporter cell line and siRNA screen:
- Generating a reporter cell line and performing an siRNA screen is a good approach for identifying potential regulatory factors involved in the downregulation of your target gene. However, it may be more effective to use a genome-wide CRISPR/Cas9 screen, which allows for the simultaneous knockdown of all genes in the genome. This can provide a more comprehensive understanding of the regulatory network involved in the downregulation of your gene of interest.
- Additionally, you could also consider using RNA sequencing (RNA-seq) on samples where your gene is downregulated, followed by pathway analysis to identify potential signaling pathways involved in the downregulation.

Here are some references that may be helpful for your research:
- ENCODE Project Consortium et al. (2012). An integrated encyclopedia of DNA elements in the human genome. Nature, 489(7414), 57-74.