I am new to these concepts in biology and need some help understanding. My main concern is how to find the enhancer of my gene of interest, specifically the sequence of the enhancer. I am working on a project where I will be attempting to use CRISPR- mediated deletion of the promoter region and enhancer region in my gene of interest FOXN2, in MCF7 Tamoxifen resistant breast cancer cells. I already have the sequence for the promoter region from UCSC's GenomeBrowser, but I'm not sure if the enhancer region sequence can just be looked up on there or anywhere else. Do I need to do a protocol to find the enhancer region myself? I have seen many things about ChIP-Seq and that I may need to do that to get data to find my enhancer but I am just not sure. I plan to use the sequences of the enhancer and promoter region of my gene of interest to create gRNA for CRISPR. Thank you to anyone who can help.
There are several ways to identify enhancers:
(1) Chip seq / chip-chip recognize transcription factor binding sites,
(2) Enhancer specific factors can also be used to identify enhancers, such as EP300, the binding sites of EP300 are often used to predict enhancers;
(3) RNA polymerase II can bind thousands of enhancers, so POLR2A2a, the largest subunit of RNA polymerase II, can also be used to search for enhancer sites;
(4) The high sensitive site of deoxyribonuclease I (DHS) represents the open chromatin region, many of which are covered with enhancers;
(5) A large number of active regulatory elements, including enhancers, can be identified by formaldehyde assisted separation regulatory elements (face) combined with sequencing;
(6) Some histone modification patterns reflect different chromatin states, such as the binding of h3k4me1 and h3k27ac, which are widely used for enhancer labeling;
(7) Enhancer sequence can be transcribed, and the transcriptional enhancer RNA (enrna) is also a marker of enhancer activation;
(8) The method of chromosome 3D conformation (e.g. 5C and Chia pet, capture-c) can also provide the information of enhancer promoter interaction.