Epigenomics
Epigenetics Track • Chapter 7
Estimated Study Time: 22 minutes
Learning Objectives
- Understand 5-methylcytosine (5mC) CpG methylation and compare WGBS vs Enzymatic Methyl-seq vs long-read native calling.
- Decode the histone code: active promoters (H3K4me3), active enhancers (H3K27ac), and Polycomb repression (H3K27me3).
- Differentiate ChIP-seq vs CUT&Tag tagmentation protocols for low-input chromatin profiling.
- Execute peak calling and signal visualization using MACS3 and deepTools.
1. The Epigenetic Layers Regulating Genome Activity
Epigenomics studies stable, heritable, and reversible chemical modifications of chromatin that alter gene expression profiles without changing the primary DNA sequence:
- DNA Methylation: Addition of a methyl group to the carbon-5 position of cytosine (5-methylcytosine, 5mC), predominantly in CpG dinucleotide islands. Promoters with dense hypermethylated CpG islands recruit methyl-CpG-binding domain (MBD) proteins, repressing transcription.
- Post-Translational Histone Modifications: Enzymatic methylation, acetylation, phosphorylation, and ubiquitination on N-terminal histone tails:
H3K4me3:Trimethylation of histone H3 lysine 4 marks actively transcribing promoters.H3K27ac:Acetylation of histone H3 lysine 27 distinguishes active enhancers from primed enhancers.H3K27me3:Trimethylation of H3 lysine 27 catalyzes Polycomb Repressive Complex (PRC2) mediated facultative heterochromatin.H3K9me3:Trimethylation of H3 lysine 9 marks dense, constitutive heterochromatin and centromeric regions.
- Chromatin Accessibility (ATAC-seq): Uses hyperactive Tn5 transposase loaded with sequencing adapters to probe accessible open chromatin regulatory regions (nucleosome-free regions at active promoters and enhancers).
2. Peak Calling & Matrix Heatmap Workflow
# 1. Call narrow peaks for transcription factors or ATAC-seq
macs3 callpeak -t atac.bam -f BAMPE -g hs -n sample_atac \
--nomodel --shift -100 --extsize 200 -q 0.01 --outdir macs3_out/
# 2. Call broad peaks for histone marks (H3K27me3 / H3K9me3)
macs3 callpeak -t h3k27me3.bam -c input.bam -f BAMPE -g hs \
--broad --broad-cutoff 0.05 -n sample_h3k27me3 --outdir macs3_broad/
# 3. Generate BigWig coverage normalized to 1x genome coverage
bamCoverage -b atac.bam -o atac_normalized.bw \
--normalizeUsing RPGC --effectiveGenomeSize 2913022398 -p 16
# 4. Generate matrix and profile heatmap across transcription start sites (TSS)
computeMatrix reference-point --referencePoint TSS \
-b 2000 -a 2000 -R gencode.v44.genes.bed \
-S atac_normalized.bw -o atac_matrix.gz -p 16
plotHeatmap -m atac_matrix.gz -out atac_heatmap.pdf --colorMap viridis