Question

How will you determine the transcription factor binding sites in the whole genome? Give at least one example from literature.

Answer 1

ChiP-ChiP or ChiP Sequencing are methods that can be used to detect transcription factor binding sites on the genome. They are based on Chromatin immunoprecipitation, that can detect the DNA protein interactions. This technique captures proteins at the site of its interaction with DNA. The DNA is extracted and then fixed with formaldehyde. Formaldehyde cross links the DNA with protein, so that the transcription factor remains bound to the nucleus. The DNA protein complexes are sonicated to fragments that are 200-1000 bp in length. These fragments are immunoprecipitated using specific antibodies to the transcription factor. The DNA protein crosslinks are reversed and then the DNA sequence is obtained. The expression of genes regulated by this transcription factor can then be identified by PCR analysis.

Whole genome ChiP sequencing will sequence numerous DNA fragment simultaneously. It is more specific than ChiP-ChiP tiling arrays as it involves direct sequencing. ChiP sequencing has been used to detect transcription factor binding sites of many transcription factor. It has been used to detect transcription factor binding sites for genes: ZIC2, OTX2, SOX2, POU5F1 and POU3F1 (all stem cell related genes) in epiblast stem cells. Microfluidic platforms can be used to identify transcription factors binding sites of several transcription factors simultaneously for a genome. Genomic libraries are prepared from the DNA fragments identified by the Chip, which are then used for sequencing.

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