MB06 Histone Modifications and ChIP-Seq

Histone modification & ChIP-seq

Epigenetics studies changes in gene expression not encoded in DNA. Mechanisms: DNA methylation, histone modification, non-coding RNAs (e.g., microRNAs), regulatory repeating regions. Histone modification = major epigenetic regulator.

Histones and nucleosomes

DNA wraps around histone proteins forming nucleosomes (basic repeating unit of chromatin). Nucleosome = 146 bp of DNA wrapped around histone octamer (H2A, H2B, H3, H4), with H1 holding structure together. N-terminal tails protrude and are modified post-translationally.

Histone packing and gene expression

Histone modifications affect how densely DNA is packed. Compact nucleosomes → less accessible DNA (gene silencing). Relaxed chromatin → more accessible DNA (transcriptional activation). Modifications can attract histones closer or force them apart.

Major histone modifications

Types: acetylation, phosphorylation, ubiquitination, methylation (lysine/arginine). Effects:

Acetylation: first linked to active transcription.

Phosphorylation (H3): cooperates with acetylation in transcriptional activation.

Methylation: can activate or silence transcription, depending on site.

Functional outcomes

Histone modifications govern transcription, replication, DNA repair, apoptosis. Modifications act individually or combinatorially, creating complex regulatory patterns (“histone code”).

Examples of histone modifications

H3K4me3: trimethylation at lysine 4 of histone H3 → activation.

H3K27me3: trimethylation at lysine 27 of H3 → repression.
Notation = histone type + position + modification.

Histone modification effects

Methyl groups: increase packing (gene silencing).

Acetyl groups: decrease packing (activation).

Phosphoryl groups: decrease packing.
These changes alter DNA accessibility for transcription machinery.

ChIP (Chromatin Immunoprecipitation)

Experimental method to study histone modifications, transcription factor binding, and nucleosome positioning. Uses antibodies against specific proteins (histones, TFs).

ChIP procedure (overview)

Cross-linking: fixes proteins (histones/TFs) to DNA.

DNA fragmentation: chromatin sheared into pieces.

Immunoprecipitation: antibodies (attached to magnetic beads) isolate DNA–protein complexes.

Purification: separate DNA from protein.

Sequencing: prepare libraries, sequence DNA fragments.

ChIP-seq readout

Sequencing produces short reads (tags) mapping to genome. Binding sites = regions with peaks of read accumulation compared to control. Peaks represent protein-DNA binding positions.

Controls in ChIP-seq

Input DNA (no IP) sequenced in parallel as control. Comparing ChIP vs input identifies true signal vs background. Strand-specific analysis helps refine exact binding site location.

Importance of antibodies

Antibody specificity critical for high-quality ChIP data. Must be designed for target protein (e.g., H3, H3K27, H3K4). Key factors: monoclonal vs polyclonal, species specificity, and ability to recognize modification state.

ChIP-seq applications

Genome-wide mapping of:

Histone modifications (active vs repressive marks).

Transcription factor binding sites.

Nucleosome positioning.
Results = peak profiles aligned to reference genome, analyzed for functional interpretation.