Whole-genome methods to define DNA and histone accessibility and long-range interactions in chromatin

Abstract

Defining the genome-wide chromatin landscape has been a goal of experimentalists for decades. Here we review highlights of these efforts, from seminal experiments showing discontinuities in chromatin structure related to gene activation to extensions of these methods elucidating general features of chromatin related to gene states by exploiting deep sequencing methods. We also review chromatin conformational capture methods to identify patterns in long-range interactions between genomic loci.

Keywords: ATAC-seq; CHIA-PET; CHIP-seq; FAIRE; HiC; whole-genome techniques.

Figure 1:. Common methods for mapping chromatin accessibility.

The first step in each technique is cell lysis and nuclear isolation. A. DNase-seq: Utilizes cleavage by DNaseI endonuclease to identify the open chromatin regions. Cleavage is attenuated where chromatin proteins (Green and yellow ovals) are bound to DNA, including nucleosomes. Short DNA fragments resulting from the DNaseI cleavage are sequenced. B. FAIRE-seq: Chromatin proteins are crosslinked to the DNA using formaldehyde and chromatin is fragmented by sonication. Protein-free DNA fragments remain in the aqueous phase during phenol:chloroform extraction and are sequenced. C. MNase-seq: MNase is used to digest away linker DNA regions and nucleosomal DNA is sequenced. D. ATAC-seq: A hyperactive Tn5 transposase simultaneously cleaves and inserts DNA adapters at accessible regions of the genome, which are then used for sequencing.

Figure 2:. Methods to assess genomic occupancy of chromatin proteins.

A. ChIP-seq: Proteins of interest are crosslinked to DNA, complexes pulled down via a specific antibody, and associated DNA is sequenced and mapped to the reference genome. B. DamID-seq: A chromatin protein of interest is expressed in cells in a DNA adenine methyltransferase (Dam) fusion. The fusion protein localizes to the appropriate genomic regions resulting in methylation of nearby adenines in Dam cognate sites. The DNA is digested with the methylation-dependent restriction enzyme DpnI. Part of the DpnI digested product is further digested by isoschizomer DpnII enzyme which cleaves the unmethylated DNA. DNA from both digestions is subjected to sequencing. C. ChEC-seq: The chromatin protein of interest is expressed in a fusion with MNase, direting MNase to digest nearby DNA. Released DNA fragments are sequenced and mapped back to the reference genome. D. CUT&RUN: Protein A-MNase fusion is used to bind specific antibody against protein of interest. MNase is activated by the addition of Ca²⁺, cleaving the DNA nearby, releasing the fragments out of the nucleus, which are sequenced. E. CUT&Tag: Protein A-Tn5 fusion is used to bind specific antibody against a protein of interest. Addition of Mg²⁺ activates Tn5 and integrates adapters at cleavage sites. The released DNA is purified and subjected to sequencing.

Figure 3:. Overview of chromosome conformation capture techniques:

All methods share the common step of formaldehyde crosslinking in situ, capturing interaction between different chromatin segments. For 3C, 4C and 5C, the DNA is fragmented by restriction enzyme digestion then the free ends ligated, capturing ends brought in close proximity by chromatin structure, crosslinking is reversed, and the DNA purified. The DNA is either used for PCR with specific primers (3C), or is further digested by a second restriction enzyme, followed by religating shorter fragments and inverse PCR (4C). The PCR product is subjected to microarray hybridization or deep sequencing. In 5C, all the steps are same as 3C except the specific PCR primers tagged with universal T7 and T3 primer sequences are annealed to sequences abutting the ligation site and ligated, and PCR products are sequenced. In Hi-C, the free DNA ends in the crosslinked, digested chromatin is filled with biotinylated nucleotides before ligation, crosslinking reversed, and ligated DNA is isolated and subjected to deep-sequencing.