ChIP-seq — chromatin immunoprecipitation followed by sequencing — maps, at genome scale, the sites where a protein binds DNA: a transcription factor or a histone bearing a modification. With its variants (CUT&RUN, CUT&Tag, ATAC-seq), it reveals the state of the chromatin and the epigenetic regulatory programmes that govern the identity and fate of cells.
Principle and workflow
In ChIP-seq, the chromatin is cross-linked then fragmented; a specific antibody immunoprecipitates the DNA associated with the target protein. The fragments are sequenced, aligned to a reference genome, and enriched regions are identified by peak calling (MACS2). Peak annotation, motif discovery (HOMER) and functional enrichment follow. Reliability rests on a specific antibody, appropriate controls (IgG, input) and sufficient sequencing depth.
Variants and options
CUT&RUN and CUT&Tag replace cross-linking and sonication with targeted enzymatic cleavage (protein A-MNase or -Tn5) in intact nuclei: less background, up to roughly 200-fold fewer cells and 10-fold less sequencing, which makes them valuable on rare or clinical samples (peak calling by SEACR). ATAC-seq maps open chromatin without an antibody (Tn5 transposase); hMeDIP targets 5-hydroxymethylcytosine. ChIP-seq and CUT&Tag combine with RNA-seq to link chromatin state and expression.
When and why these techniques
ChIP-seq answers a precise question: where, in the genome, does a protein bind, and how does the chromatin state vary between conditions? It is used to locate transcription-factor binding sites, map histone marks (H3K27ac of active enhancers, repressive H3K9me2 or H3K27me3), or track an epigenetic reprogramming under treatment.
Everything rests on the quality of the antibody and the controls. Classical ChIP-seq suffers from a low signal-to-noise ratio, possible epitope masking by fixation, and a heterochromatin bias linked to sonication; it requires roughly one to ten million cells and adapts poorly to single-cell. CUT&Tag attenuates these pitfalls but may bias towards open chromatin and capture indirect events. Poorly controlled fixation or washing silently distort the enrichment.
Inovarion’s expertise
Inovarion profiles chromatin to decode epigenetic regulation in oncology and immunology. Three published studies show this: profiling of H3K27ac and methylation linked, in chronically activated mast cells, an epigenetic signature to a TET2-dependent immune tolerance; CUT&Tag (H3K9me2) and ATAC-seq revealed a disorganisation of heterochromatin at retroelements in the leukaemic stem cells of chronic myelomonocytic leukaemia; and ChIP-seq / RNA-seq coupling showed how hypermethylation favours the immune escape of adrenocortical carcinoma. The choice of technique and of the targeted marks — ChIP-seq, CUT&Tag or ATAC-seq — is decided according to the available material and the resolution sought.
See also: “Bulk” RNA-seq & differential transcriptomics ; Single-cell RNA-seq ; Bioinformatics.
Key publications
- Rigo et al. TET2 regulates immune tolerance in chronically activated mast cells. JCI Insight, 2022. PubMed
- Hidaoui et al. Targeting heterochromatin eliminates chronic myelomonocytic leukemia malignant stem cells through reactivation of retroelements and immune pathways. Communications Biology, 2024. Record → · PubMed
- Kerdivel et al. DNA hypermethylation driven by DNMT1 and DNMT3A favors tumor immune escape contributing to the aggressiveness of adrenocortical carcinoma. Clinical Epigenetics, 2023. PubMed