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Find out more about the structure of chromatin and the methods used to investigate chromatin accessibility and interactions.
The genome is efficiently packaged into the nucleus. DNA is wrapped around histones to form a nucleosome, comprised of 147 base pairs of DNA and eight-core histone proteins. Nucleosomes are strung together like beads on a string and packaged into higher-order chromatin architecture (Figure 1).
Figure 1: Chromatin structure. DNA winds around nucleosomes to form chromatin fiber and then chromosomes
DNA that is tightly bound in nucleosomes or compacted into higher-order heterochromatin is inaccessible, preventing the binding of transcription factors, transcriptional machinery, and other DNA-binding proteins, resulting in gene silencing. Meanwhile “linker” DNA and open euchromatin architecture are accessible to binding, allowing for active gene transcription.
Chromatin is actively and dynamically remodeled to alter gene expression and cellular programming, for example, during different developmental stages or in response to particular stimuli. Large genomic regions may be silenced or activated, or nucleosomes may be unraveled to access specific genes and DNA sequences.
Examining the chromatin structure and nucleosome positioning reveals epigenetic programs and mechanisms involved in specific cellular processes and disease states.
Surveying the genome for exposed regions accessible for active transcription vs those bound tightly into heterochromatin can be an essential first step to understanding the relationship between chromatin structure and function in different contexts. To take a snapshot of genomic architecture, researchers may use one of three methods: DNase-seq, MNase-seq, or ATAC-seq.
DNase-seq and ATAC-seq map exposed regions of DNA, whereas MNase-seq maps regions protected by nucleosomes. It is important to keep in mind that these methods provide snapshots of a dynamic process, often averaged across thousands of cells. If a particular region is dynamically changing, or different between cells within the population, the data may seem conflicting between methods. Some single-cell analysis methods are evolving to resolve these challenges.
DNAse-seq uses DNase to digest exposed regions of the genome, whereas nucleosome-bound DNA is protected from DNase digestion. The small fragments generated by DNase digestion are then sequenced and mapped to the genome to identify regions of active transcription.
Figure 2: ATAC-seq protocol. Our step-by-step guide to ATAC seq can be found here
We can assess the three-dimensional chromatin architecture with chromatin contact mapping to reveal physical interactions between distant genomic regions. This type of mapping is made possible by the advent of chromatin conformation capture (3C) and subsequent methods developed based on this approach. Each of these approaches has particular strengths for particular applications, but selecting a method for a specific purpose can be challenging due to the sheer variety of methodologies.
Figure 3: Chromosome conformation techniques. Various steps of 3C, 4C, 5C, ChIA-PET, and Hi-C.
Chromatin conformation capture (3C)
3C uses formaldehyde cross-linking to lock the three-dimensional chromatin structure in place, followed by restriction enzyme digestion. Excised DNA fragments are then analyzed by qPCR and sequencing to identify where distant DNA regions are connected. This approach for analyzing 3D chromatin structure and interactions in vivo was first developed in 2002 (Dekker et al., 2002), and has since become the foundation for a host of related techniques that have been developed to achieve greater scale, throughput, or specificity.
Circularized chromosome conformation capture (4C)
4C enables identification of previously unknown DNA regions that interact with a locus of interest, which makes 4C ideal for discovering novel interactions within a specific region (Dekker et al., 2006).
4C helpful hints:
Carbon copy chromosome conformation capture (5C)
5C generates a library of any ligation products from DNA regions that associate with the target loci, which are then analyzed by NGS. 5C is ideal when great detail about all the interactions in a given region is needed, for example when diagramming a detailed interaction matrix of a particular chromosome. However, 5C is not truly genome-wide, since each 5C primer must be designed individually, so it is best suited to a specific regions (Dotsie and Dekker, 2007).
5C helpful hints:
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET)
ChIA-PET takes aspects of ChIP and 3C to analyze the interplay of distant DNA regions through a particular protein.
ChIA-PET is best used for discovery experiments involving a protein of interest and unknown DNA binding targets. Transcription factor binding sites, for example, are best studied with ChIA-PET since this technique requires the DNA to be bound by the transcription factor in vivo for the interaction to be called (Fullwood et al., 2009).
ChIA-PET helpful hints:
ChIP-loop is a mix of ChIP and 3C that employs antibodies targeted to proteins suspected to bind a DNA region of interest. ChIP-loop is ideal to find out if two known DNA regions interact via a protein of interest. ChIP-loop is also well suited to confirmation of suspected interactions, but not the discovery of novel ones (Horike et al., 2005).
ChIA-loop helpful hints:
Hi-C amplifies ligation products from the entire genome and assesses their frequencies by high-throughput sequencing. Hi-C is a great choice when broad coverage of the entire genome is required, and the resolution is not of great concern, mapping the genome-wide changes in chromosome structure in tumor cells (Lieberman-Aiden et al., 2009), for example.
Hi-C helpful hints:
Capture-C uses a combination of 3C and oligonucleotide capture technology (OCT), together with high-throughput sequencing to study hundreds of loci at once. Capture-C is ideal when both high resolution and genomic-wide scale are required. For example, analyzing the functional effect of every disease-associated SNP in the genome on local chromatin structure (Hughes et al., 2014).
Capture-C helpful hints:
Belton JM, McCord RP, Gibcus JH, Naumova N, Zhan Y and Dekker J (2012). Hi-C: a comprehensive technique to capture the conformation of genomes. Methods, 58, 268-76.
Dekker J, Rippe K, Dekker M and Kleckner N (2002). Capturing chromosome conformation. Science, 295, 1306-1311.
Dekker J. (2006). The three ‘C’ s of chromosome conformation capture: controls, controls, controls. Nat Methods, 3, 17-21.
Dostie J and Dekker J (2007). Mapping networks of physical interactions between genomic elements using 5C technology. Nat Protoc, 2, 988-1002.
Dostie J, Zhan Y and Dekker J (2007). Chromosome conformation capture carbon copy technology. Curr Protoc Mol Biol, Chapter 21, Unit 21.14.
Horike S, Cai S, Miyano M, Cheng JF and Kohwi-Shigematsu T (2005). Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat Genet, 37, 31-40.
Fullwood MJ, et al. (2009). An oestrogen-receptor-alpha-bound human chromatin interactome. Nature, 462, 58-64.
Lieberman-Aiden E, et al. (2009). Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science, 326, 289-293.
Hughes JR (August 2014). Email interview.
Hughes JR, et al. (2014). Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat Genet, 46, 205-212.
Simonis M, Kooren J and de Laat W (2007). An evaluation of 3C-based methods to capture DNA interactions. Nat Methods, 11, 895-901.
van de Werken H, de Vree PJ, Splinter E, Holwerda SJ, Klous P, de Wit E and de Laat W (2012). 4C technology: protocols and data analysis. Methods Enzymol, 513, 89-112