Defining Regulatory Elements in the Human Genome Using Nucleosome Occupancy and Methylome Sequencing (NOMe-Seq)

Abstract

NOMe-seq (nucleosome occupancy and methylome sequencing) identifies nucleosome-depleted regions that correspond to promoters, enhancers, and insulators. The NOMe-seq method is based on the treatment of chromatin with the M.CviPI methyltransferase, which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin (GpC^m does not occur in the human genome and therefore there is no endogenous background of GpC^m). Following bisulfite treatment of the M.CviPI-methylated chromatin (which converts unmethylated Cs to Ts and thus allows the distinction of GpC from GpC^m) and subsequent genomic sequencing, nucleosome-depleted regions can be ascertained on a genome-wide scale. The bisulfite treatment also allows the distinction of CpG from C^mpG (most endogenous methylation occurs at CpG dinucleotides) and thus the endogenous methylation status of the genome can also be obtained in the same sequencing reaction. Importantly, open chromatin is expected to have high levels of GpC^m but low levels of C^mpG; thus, each of the two separate methylation analyses serve as independent (but opposite) measures which provide matching chromatin designations for each regulatory element.NOMe-seq has advantages over ChIP-seq for identification of regulatory elements because it is not reliant upon knowing the exact modifications on the surrounding nucleosomes. Also, NOMe-seq has advantages over DHS (DNase hypersensitive site)-seq, FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)-seq, and ATAC (Assay for Transposase-Accessible Chromatin)-seq because it also gives positioning information for several nucleosomes on either side of each open regulatory element. Here, we provide a detailed protocol for NOMe-seq that begins with the isolation of chromatin, followed by methylation of GpCs with M.CviPI and treatment with bisulfite, and ending with the creation of next generation sequencing libraries. We also include sequencing QC analysis metrics and bioinformatics steps that can be used to identify nucleosome-depleted regions throughout the genome.

Keywords: DNA methylation; Enhancers; Insulators; NOMe-seq; Nucleosome-depleted regions; Open chromatin; Promoters.

Figure 1. Schematic overview of NOMe-seq

Left: Experimental workflow, representing Sections 3.1–3.7 in the Methods. Right: Analytical workflow, representing Sections 3.8.1–3.8.5 in the Methods.

Figure 2. Refinement of a regulatory element using NOMe-seq

Shown is a nucleosome- depleted region (NDR) flanked by nucleosomes harboring the histone modification H3K27ac; the centers of the NDR and the region covered by H3K27ac are also indicated.

Figure 3. Size distribution analysis of a NOMe-seq sample and library

Shown is a Bioanalyzer trace, obtained using an Agilent 2100 Bioanalyzer instrument and an Agilent High Sensitivity DNA chip, of the DNA after M.CviPI treatment and fragmentation using a Covaris S220 sonicator (a) and of the resultant NOMe-seq library (b). The leftmost and rightmost peaks (labeled 43 and 113) are size markers of 35 bp and 10380 bp, respectively. The average length of the fragmented DNA is calculated to be 150 bp whereas the average length of the library fragments is calculated to be 280 bp. (c) For comparison to the Bioanalyzer traces, the gel images of the fragmented DNA and the NOMe-seq library are also shown.

Figure 4. Quality analysis of NOMe-seq data using CTCF peaks

A set of 3,216 CTCF peaks that were commonly identified in 58 different human cell types and that have a CTCF motif were used to compare endogenous DNA methylation (HCG) and accessibility (GCH). In each panel, data is shown for a 2 kb region, centered on the CTCF motifs within the CTCF peaks; the genomic locations of the set of 3,216 CTCF sites are provided in Supplementary Table S1. (a) The density (Z scores) of HCG methylation and GCH methylation, centered on the CTCF motif, is shown for all common CTCF peaks. (b) The average methylation levels of HCG (endogenous DNA methylation) and the average methylation of GCH (accessibility) are shown for all common CTCF peaks. (c) A heatmap representing the percentage of GCH methylation (left) and endogenous HCG methylation (right) is shown for all common CTCF peaks. The heatmap was made by first clustering the GCH values at the CTCF peaks, then plotting both the GCH and HCG values in the same order.

Figure 5. Quality analysis of called NDRs

Data is shown for a 2 kb region centered on 92,482 called NDRs (P-value cut-off = 10⁻¹²) for a NOMe-seq dataset. (a) The density (Z scores) of HCG methylation (endogenous DNA methylation) and GCH methylation (accessibility), centered on the NDRs, is shown. (b) The average methylation levels of HCG (endogenous DNA methylation) and the average methylation of GCH (accessibility) are shown for all NDRs. (c) A heatmap representing the percentage of GCH methylation (left) and endogenous HCG methylation (right) is shown for all NDRs. The heatmap was made by first clustering the GCH values at the NDRs, then plotting both the GCH and HCG values in the same order.

Figure 6. Comparison of DNA methylation levels at NDRs identified using different P- value cut-offs

Shown are heatmaps indicating the percentage of endogenous methylation at HCG sites for a 2 kb region centered on NDRs selected using different P-value cut-offs. The heatmaps were made by first clustering the GCH values at each NDR, then plotting the HCG values in the same order.

Figure 7. Examples of NDRs at regulatory elements

Shown is a genome browser screen shot (hg19) of a region from chr21q22.2 with tracks representing accessibility (GCH), endogenous DNA methylation (HCG), called NDRs, and H3K4me3, H3K27ac and CTCF ChIP- seq data; all data is from CNON cells. The purple box highlights an NDR classified as an insulator (a genomic region bound by CTCF that does not have the histone modifications found at promoters or enhancers), the red box highlights an NDR representing an enhancer (a distal genomic region marked by H3K27ac), the orange box highlights an NDR in the promoter of the EIF1 gene, the green box highlights an NDR that lacks promoter, enhancer, and insulator marks, and the black box highlights an NDR at the promoter of the HAP1 gene, which is not actively transcribed in these cells (as shown by the small H3K4me3 signal).