High-resolution digital profiling of the epigenome 815-817

Chromatin Footprinting

A set of techniques used to map the accessibility of chromatin and the precise locations where proteins (like transcription factors and nucleosomes) are bound to DNA, often with base-pair resolution.

Chromatin Accessibility

A property of the genome defining how "open" or "closed" a region is, which correlates with its potential for being transcribed. It is measured by sensitivity to nucleases, transposases, or sonication.

Micrococcal Nuclease (MNase)

A nuclease used in chromatin profiling. It cleaves DNA with a preference for AT-rich regions and "nibbles" away DNA until it hits an obstruction like a nucleosome or a bound protein. It can map both nucleosomes and non-histone proteins.

MNase-seq

A chromatin footprinting method: Isolate nuclei, digest with MNase, purify DNA, and sequence. It maps nucleosomes and non-histone protein binding sites in a single, cost-effective experiment without needing antibodies. Data is often visualized using V-plots.

Limitations of MNase-seq

1. Cannot conclusively identify the specific non-nucleosomal particles it footprints. 2. Has a cleavage bias towards AT-rich regions. 3. Does not work well for identifying specific transcription factors without additional experiments.

DNase I

A nonspecific endonuclease that cleaves single and double-stranded DNA. It is used to identify regions of chromatin accessibility (DNase Hypersensitive Sites).

DNase-seq (DGF)

A chromatin footprinting method: Digest chromatin with DNase I, purify DNA, and sequence. Digital Genomic Footprinting (DGF) uses this data to map transcription factor binding sites at single-base resolution by identifying protected "footprints".

DNase-FLASH

A modified DNase-seq method that involves sequencing different size ranges of digested chromatin fragments. It allows for the creation of V-plots to footprint both nucleosomes and transcription factors from a single experiment.

Limitations of DNase-seq

1. Requires very high sequencing depth for large genomes. 2. DNase I is inhibited by high concentrations of actin. 3. Suffers from cleavage bias that can create artefactual footprints.

ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing)

A chromatin footprinting method: Treat isolated nuclei with the Tn5 transposase loaded with sequencing adapters. Tn5 simultaneously fragments ("cuts") and tags ("pastes" adapters into) accessible DNA in a process called tagmentation. The DNA is then purified and sequenced.

Advantages of ATAC-seq

1. Requires very low cell input (as few as 500 cells). 2. Fast and simple protocol (can be done in hours). 3. Maps both chromatin accessibility and nucleosome positioning. 4. Avoids dense, inactive chromatin, reducing required sequencing depth.

Limitation of ATAC-seq

Steric hindrance between Tn5 transposomes creates a minimum spacing (~38 bp) between integration events, limiting its resolution and biasing it towards nucleosomes near open regulatory regions.

Tagmentation

The process, catalyzed by Tn5 transposase, of simultaneous DNA fragmentation and adapter ligation. It is the core step in the ATAC-seq protocol.

Targeted Chemical Cleavage

A high-precision footprinting method that uses a engineered histone H4 with a S47C mutation. The cysteine is labeled with a phenanthroline compound that, upon addition of Cu²⁺ and H₂O₂, generates hydroxyl radicals to cleave DNA at specific positions relative to the nucleosome dyad.

Application of Targeted Chemical Cleavage

Primarily used to map nucleosome centers and occupancy with very high precision. It can also be adapted to map non-histone proteins by engineering cysteine mutations into their DNA-binding domains, though this risks altering their function.

DNA Methylation Footprinting

A gentle footprinting method that uses bacterial methyltransferases (e.g., M.CviPI) to methylate accessible DNA (e.g., at GC dinucleotides). The methylation pattern is then read via bisulfite sequencing, revealing regions protected from methylation by bound proteins.

NOMe-seq (Nucleosome Occupancy and Methylation sequencing)

A specific DNA methylation footprinting method. Isolated nuclei are treated with M.CviPI methyltransferase, which methylates GC dinucleotides in accessible DNA. DNA is then purified, subjected to bisulfite conversion, and sequenced to simultaneously read nucleosome occupancy (via protection) and endogenous CG methylation.

Advantages of Methylation Footprinting

1. Does not damage DNA. 2. Causes minimal disruption to chromatin structure. 3. Can be performed in vivo (e.g., with DamID). 4. Can interrogate two epigenomic features at once (e.g., occupancy and endogenous methylation in NOMe-seq).

Limitations of Methylation Footprinting

1. Cannot conclusively identify the specific protein causing the protection. 2. Relies on the presence of the specific dinucleotide target for the methyltransferase (e.g., GC for M.CviPI).

V-plot (Fragment midpoint-versus-length plot)

A two-dimensional representation of paired-end sequencing data. The x-axis is the distance of a read's midpoint from a genomic feature, and the y-axis is the length of the fragment. It is used to visualize patterns of cleavage protection indicative of specific nucleosome positions or transcription factor binding.

Digital Epigenomic Analysis

The use of high-throughput sequencing-based methods to study the epigenome, including DNA methylation, histone modifications, and chromatin accessibility.

Sequencing Library

A collection of DNA fragments that have been prepared for sequencing by having specific adapter sequences ligated to their ends.

Affinity Reagents

Antibodies or other molecules (e.g., aptamers) used to immunoprecipitate a specific protein or modification from a complex mixture. A key component of ChIP experiments.

Sonication-based Methods (FAIRE, Sono-seq)

Methods that rely on the differential sensitivity of open vs. closed chromatin to breakage by sonication. They measure general accessibility but lack the base-pair resolution for footprinting provided by enzymatic cleavage methods.

Key Difference: Cleavage vs. Sonication Methods

Enzymatic cleavage methods (MNase, DNase) can provide base-pair resolution footprints of protein binding. Sonication methods (FAIRE, Sono-seq) only measure general chromatin accessibility with lower resolution.

Primary Use of MNase-seq

Mapping nucleosome positions and occupancy genome-wide. It can also provide information on other bound proteins.

Primary Use of DNase-seq

Identifying DNase Hypersensitive Sites (DHS) as markers of open chromatin and mapping transcription factor binding footprints at base-pair resolution.

Primary Use of ATAC-seq

A rapid, sensitive method for profiling genome-wide chromatin accessibility and nucleosome positioning, especially useful for low-input samples.

Primary Use of Targeted Chemical Cleavage

Precisely mapping the center (dyad) of nucleosomes and characterizing non-canonical nucleosome structures.

Primary Use of NOMe-seq

Simultaneously mapping nucleosome occupancy (via GC methylation protection) and endogenous DNA methylation patterns (via CG methylation) on the same DNA molecule.