How to use H3K27ac, H3K4me1, and RNA-seq to identify enhancers and their target genes?

1. Identify the overlapping peaks of H3K27ac and H3K4me1 using tools like BEDTools intersect or HOMER mergePeaks. H3K4me1 (histone H3 lysine 4 monomethylation) is commonly found in both active and poised enhancer regions. On the other hand, H3K27ac (histone H3 lysine 27 acetylation) is more specific to active enhancers. By combining both H3K27ac and H3K4me1 histone marks, we can more confidently identify active enhancer regions, as the overlapping regions marked by both histone modifications are more likely to represent true enhancers with regulatory roles in gene expression. This approach helps to reduce the number of false positives.

2. Exclude promoter regions (TSS +/- 2 kb) from the overlapping peaks using BEDTools subtract or HOMER mergePeaks with the -exclude option. This is because the histone marks H3K27ac and H3K4me1 can be present in both promoters and enhancers. By excluding the promoter regions, we can reduce the likelihood of identifying promoter-driven regulatory elements and focus on the distal enhancers that are more likely to have long-range regulatory effects.

3. For each remaining distal enhancer region (overlapping peak), find the nearest gene(s) using tools like BEDTools closest or HOMER annotatePeaks.

4. Analyze the expression patterns of these nearest genes in the RNA-seq data to determine if they are differentially expressed or show any interesting expression patterns that could be related to the putative enhancer activity.

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