Methods

Subjects
The FAIRE-chip data, covering human chromosomes 8, 11, and 12, derived from MCF-7 cell lines, were downloaded from the NCBI Gene Expression Omnibus (GEO) database (GSE11579) [23]. The ChIP-Seq data (SRA045635) of H3K4me1, H3K4me3, H3K9/14ac, and input DNA (mononucleosome digested with micrococcal nuclease) in MCF-7 were obtained from the Sequence Read Archive (SRA) at the National Center for Biotechnical Information (NCBI) [26]. The H3K27me3 ChIP-Seq data were generated with Genome Analyzer IIx (Illumina Inc., San Diego, CA, USA) according to the method described by Choe et al. [26]. The gene expression data using the Human Genome U133Plus 2.0 array platform (Affymetrix Inc., Santa Clara, CA, USA) were collected from the GEO database (GSM276046-GSM276048 for MCF-7) [27].

Peak identification and statistical analysis
The FAIRE-chip data were processed by using CisGenome [28] for normalization, signal detection, and identification of significant FAIRE regions. The 26-bp ChIP-Seq reads for H3K4me1, H3K4me3, H3K9/14ac, H3K27me3, and input DNA were aligned to a human reference sequence (hg18) using the CASABA 1.6 program (Illumina Inc.), and the resulting mapped tag counts were normalized for the comparative analysis. To identify peaks enriched with a specific histone modification, the Hypergeometric Optimization of Motif EnRichment (HOMER) package version 3.2 [29] was used with the following options: approximate fragment length, 150 bp; peak size, 150 bp; minimum distance between peaks, 370 bp (equivalent to peak size × 2.5); Poisson p-value threshold relative to local tag count, 0.0001; default false discovery rate threshold, 0.001; and center switch for centering peaks on maximum ChIP fragment overlap and calculating focus ratios. The FAIRE sites coinciding with histone-modified peaks were defined when the distance between the center positions of FAIRE sites and histone-modified peaks was shorter than 100 base pairs. The co-existing sites of FAIRE and histone modifications were plotted, centered at the genes' TSSs using seqMINER with k-mean clustering method [30]. Expression levels of genes associated with the FAIRE-histone-modified regions were examined, and gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by using the DAVID Functional Annotation Tool [31]. In addition, functional, enriched motifs in FAIRE-histone-modified regions were also found by using MEME suite [32] and the TOMTOM motif database [33].