Questions: Epigenomics: ChIP-seq and ATAC-seq

Question 1

What is the fundamental difference in what ChIP-seq and ATAC-seq measure?

• A. ChIP-seq measures gene expression while ATAC-seq measures chromatin structure
• B. ChIP-seq maps the locations of specific proteins or histone modifications while ATAC-seq maps regions of open chromatin regardless of which factors are bound
• C. ChIP-seq works on DNA while ATAC-seq works on RNA
• D. ChIP-seq requires more input material but ATAC-seq requires more computational analysis

Answer: (B)

ChIP-seq is targeted: you choose an antibody against a specific protein (e.g., CTCF) or histone modification (e.g., H3K27ac), and the experiment tells you where in the genome that factor or mark is located. ATAC-seq is untargeted: it identifies all regions of open, accessible chromatin using a transposase that inserts into accessible DNA. ATAC-seq tells you which regions are open to regulatory activity without specifying which factors are responsible. They are complementary: ATAC-seq provides a map of potentially active regulatory regions, and ChIP-seq identifies which specific factors or marks are present at those regions.

Question 2

An ATAC-seq peak at a genomic region proves that the region is actively driving transcription of a nearby gene.

• T. True
• F. False

Answer: False

ATAC-seq identifies regions of open chromatin — DNA that is physically accessible to proteins. While active regulatory elements (promoters, enhancers) are typically in open chromatin, accessibility alone does not prove active transcription. A region may be accessible but unoccupied, poised but not active, or accessible due to recent transcription factor departure. Functional validation requires additional evidence: H3K27ac ChIP-seq for active enhancer status, RNA-seq for gene expression, reporter assays for regulatory activity, or CRISPR perturbation experiments.

Question 3

Explain why ChIP-seq experiments require an input control or IgG control, and what artifacts can arise without one.

Model answer: The input control (sequencing of DNA before immunoprecipitation) or IgG control (using a non-specific antibody) captures background signal — regions that are enriched in the sequencing library for reasons unrelated to the protein of interest. These include open chromatin regions (which are more easily fragmented and sequenced), repetitive regions, PCR amplification biases, and mappability artifacts. Without a control, peak callers cannot distinguish true binding sites from these systematic background enrichments, producing false-positive peaks at highly accessible or highly amplified regions.

MACS2 and other peak callers work by comparing the ChIP signal to the control signal at each genomic position. Regions where the ChIP signal significantly exceeds the control are called as peaks. The control effectively normalizes out the biases inherent in chromatin fragmentation, library preparation, and sequencing, making the experiment interpretable.