Emerging Approaches to Profile Accessible Chromatin from Formalin-Fixed Paraffin-Embedded Sections

Abstract

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

Keywords: FFPE; chromatin; nucleosome; nucleosome-depleted region; nucleosome-free region.

Figure 1

Local features that define ‘open’ and ‘closed’ chromatin. Chromatin states (e.g., active transcriptional enhancers or repressed heterochromatin) can be functionally defined by integrating a range of data elements, including nucleosome-depleted regions (NDRs; mapped by one of the methods discussed in this review), DNA methylation (DNAme (primarily 5-methylcytosine); mapped by bisulfite sequencing or EM-seq [18,19]), transcription factors (TFs) and histone post-translational modifications (PTMs, such as H3K27ac, H3K4me3, H3K36me3, H3K27me3, and H3K9me3) mapped by ChIP-seq, or newer approaches like CUT&RUN or CUT&Tag [20,21]. The figure was adapted from [22]. The extended stretch of nucleosome-free DNA at an active promoter represents an NDR.

Figure 2

Publication trends. (A,B) The publication frequency of chromatin accessibility approaches using PubMed search terms “DNase-seq”, “MNase-seq”, “FAIRE-seq”, “NicE-seq”, and “ATAC-seq” (last is the focus of (B) to accommodate its overwhelming field adoption rate). (C) The publication frequency of PubMed search term “FFPE”. (D) Accumulated publications/the first incidence of each search term on PubMed.

Figure 3

Schematic of techniques used for chromatin accessibility profiling. (A) DNase-seq. (B) MNase-seq. (C) FAIRE-seq. (D) ATAC-seq. (E) NicE-seq.

Figure 4

Bioinformatic pipeline for NGS data processing and analysis.