Like a fine wine, chromatin analyses are only getting better with age. Through continued innovation these techniques remain at the forefront of the quest to understand development and disease.
Chromatin analyses utilize a range of techniques to ascertain the 3D structure and chemical modifications of chromatin. These techniques examine the locations of either histone modifications or transcription factors. Histone modifications alter how open or closed chromatin is, and transcription factors bind to open chromatin and promote gene expression. These processes are vital for the control of diverse cellular processes. Chromatin analysis is utilized by researchers examining many questions, from basic development and differentiation to cognitive disorders and complex behaviors.
Chromatin Analysis Methods
Chromatin Immunoprecipitation (ChIP) Techniques
ChIP is the tried and true workhorse of chromatin analysis; new and improved variations arise, but the basic idea of ChIP is at the heart of all chromatin analyses.
ChIP-chip: The first, and most simple of the ChIP technologies (it also has the most fun to say name). ChIP-chip couples chromatin IP to microarray analysis allowing genome-wide analysis of protein or modifications of interest distribution.
ChIP-Seq: The star of chromatin analysis until ChIP-seq came along and stole the limelight. ChIP-seq uses the same chromatin IP procedures as ChIP-chip; however, it couples it with quantitative next-generation sequencing technology to detect enrichment peaks
ChIP-exo: A specialized version of ChIP used to very specifically map protein of interest (POI) binding sites in the genome via the addition of a DNA digestion step to ChIP-seq.
ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing): Combines ChIP with chromatin conformation capture (3C) technology to detect when distant DNA regions interact with each other via a protein of interest.
Chromosome Conformation Capture (3C) Techniques
3C is an important technique used to study chromatin structure, as well as the basis for several other derivative techniques. 3C provides information on 3D chromatin structures that occur in living cells.
Circularized Chromosome Conformation Capture (4C): The sequel to 3C offers several innovations to the basic protocol, and reveals how unknown DNA regions interact with a region of interest.
Carbon Copy Chromosome Conformation Capture (5C): Useful for examining interactions with particular loci of interest in detail.
ChIP-Loop: If 3C is chocolate and ChIP is peanut butter then ChIP-Loop is a peanut butter cup: the combination is better than either on its own. The major advantage of ChIP-Loop is it reduces the background noise in 3C experiments and increases the specificity by selecting for a known protein mediating the DNA-DNA interaction.
Hi-C: Tthe first of the 3C derivate technologies to be truly genome-wide. Biotin pull-down isolates specific fragments and the resulting library is then sequenced and mapped to the genome allowing fragment identity and abundance to be detected.
Capture-C: The newest, and in many ways most advanced 3C technology to be developed, is both high resolution and high throughput. It combines 3C and next-gen sequencing with oligonucleotide capture technology (OCT).
Chromatin Analysis Additional Reading
This review gets into the basics of histone methylation, how it is regulated and read and how it controls cellular functions.
This review examines the primary, secondary, and tertiary structure of chromatin. It goes into the biochemical details how each level is formed and their relevance to cellular function.