I have transcription factors and want to know their target genes. Can anyone please give suggestions for how I can do so, and what kind of tools I should use?
This is a "big" question, and the answer depends on several factors, i.e. on where you are in the process, and what resources you have available (time, money, reagents, model system, wet-lab vs bioinformatics?). It also depends on your biological question: Are you looking for new targets? (==> genome-wide search, RNA-Seq), or are you looking at targets within a particular pathway, and already can come up with putative target lists? (==> targeted RT-PCR studies). First, of course, literature search. Second, experimental loss-of-function and gain-of-function experiments: Ideally: knock-out or knock-down model (depending on your model system) with genome-wide analysis of genes that changes upon perturbation/loss of your TF. This will give both direct target genes, as well as all indirect target genes. Similar for gain-of-function studies (over-expression of your factor). This can then be refined with time-course studies, traditional CHX treatment to find only direct targets (ask if this is not clear), and merged with ChIP-Seq to add another layer to deduce putative direct targets. Alternatives include, but are not limited to: bioinformatic studies of already published datasets (if available), DNA binding site motif analysis/search (if known), using information from various websites to supplement your literature search (pathways, "interactomes", etc). Confirming/further solidify target genes by reporter assays (i.e in vitro luciferase assay, or in vivo assays) to confirm regulatory function at promoter or enhancer, etc,
@Hilde Schjerven Yes you are right but actually I am wondering about bioinformatics tools. If I can use those and know the putative genes. Based on that, I can go for further analysis.
Since I don't know your TF's of interest, I don't know how much is already known about them (or if they are non-characterized), but you can for instance search the NCBI GEO database for what is known with either ChIP-Seq (of ChIP on microarray) or expression analysis of cells with your TF of interest knocked down/knocked-out or over-expressed (RNA-Seq or microarray). Question is if any datasets are available , and if they are in a relevant cell type/organism. Indirect methods might also be possible if you know the consensus DNA binding motif(s) of your TF (if available, websites/tools like Transfac database and others have motif info), then you can do motif search combined with expression data/gene annotation/chromatin data for your cell of interest (but you would need well-defined and longer-stretch motifs to get usable information). I am not a bioinformatic specialist, and I only know of a handful of the tools that are available out there, so perhaps you can edit your question and specify that you are asking about bioinformatics tools (and add any other info that might help people understand your starting point).
Hi, if you are working on plant transcription factors, you may use the efficient CRES-T strategy, followed by RNAseq analysis of the transformed lines
@Hilde Schjerven Thanks for nice suggestions @David Lecourieux Yes that might be possible.... thanks so much
This is a big question. If you know the transcription factors, you look for the new m-RNAs produced and their proteins. Blotting methods and sequence analysis one can follow, C-DNA also one can prepare for verification
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