Results

Chromatin structure defined by FAIRE and histone modifications
The chromatin structure was analyzed by comparing genomic regions defined by FAIRE with histone modification sites detected by ChIP-Seq in a breast cancer cell line, MCF-7. Due to the limited information of FAIRE-chip data, we analyzed regions enriched in human chromosomes 8, 11, and 12. The overall positions of regulatory elements and histone modifications relative to TSS are depicted in Fig. 1A. The k-clustered pattern, depending on their enrichment level, showed that most H3K4me3 and H3K9/14ac modifications, known as active chromatin marks, were enriched near TSS, whereas H3K27me3, a repressive mark, was not. Many regulatory elements detected by FAIRE-chip were also located near TSS, reflecting that promoters are one of the major nucleosome-free sites. The relative positions of 3 histone modification enrichments (H3K4me1, H3K4me3, and H3K9/14ac) were aligned to the center of FAIRE signals and overlapped within ± 1-kb regions from TSSs (Fig. 1B). The H3K4me1 had a broader spectrum than the other 2 modifications in ± 0.5-1.5-kb regions. These results implied that regulatory elements were highly correlated with active histone modifications and associated with open chromatin structure.
A total of 2,804 regulatory elements from FAIRE-chip data were identified by CisGenome analysis, and the number of ChIP-Seq peaks was calculated using HOMER program: 18,938 for HK4me1, 4,516 for H3K4me3, 5,763 for H3K9/14ac, and 3,324 for H3K27me3 (Table 1). The FAIRE sites were located in especially functional element-related regions: promoters (32.2% of FAIRE sites analyzed), 4 kb upstream of promoters (3.2%), gene bodies (39.1%), and intergenic regions (25.4%). In particular, the highest enrichment of FAIREs in promoters could be identified by the normalization in quantitative comparison of FAIRE profiles (i.e., the total number of peaks/total length of each of genomic feature). We selected the overlapping regulatory elements of FAIRE signals with ChIP-Seq peaks and looked at their co-occurrence; the regulatory elements with H3K4me1 (FAIRE-H3K4me1) were 1,006; FAIRE-H3K4me3, 1,000; and FAIRE-H3K9/14ac, 1,264. Among them, 334 elements showed enrichment of both H3K4me3 and H3K9/14ac (Fig. 1C). This relationship was further confirmed by the distribution of FAIRE-histone modifications, shown in Fig. 1D. The highest population of FAIRE-H3K4me3 and FAIRE-H3K9/14ac was observed immediately downstream of TSS (Fig. 1D). A weak enrichment of FAIRE-H3K4me1 elements was detected upstream of TSS, where a shoulder peak of FAIRE-H3K4me1 and H3K4me3 was positioned. For example, 4 FAIRE regulatory elements of cyclin D1 (CCND1), involved in tumorigenesis as a cell cycle regulator, were located at the promoter and overlapped with peaks of H3K4me3 and H3K9/14ac but not with H3K4me1 or H3K27me3 (Fig. 1E). Instead, the H3K4me1 peaks in the CCND1 gene locus were expanded along the gene body and far upstream of TSS.

Gene expression and FAIRE-histone modifications
The gene expression program is tightly controlled by a dynamic chromatin environment, which epigenetic factors, like histone modifications and DNA methylation, play a crucial role in determining. As shown in Fig. 1, we found the linkage of FAIRE regulatory elements with histone modification profiles. To further assess the FAIRE-histone modifications, the gene expression profiles for MCF-7 cells were integrated (Fig. 2). From the overlapped regulatory elements determined by comparison of FARE-H3K4me1 (1,006), FAIRE-H3K4me3 (1,000), and FAIRE-H3K9/14ac (1,264), we selected 229 genes associated with at least 2 of 3 FAIRE-histone modification combinations. Scatter plots were produced to see how the expression level agreed with degree of histone modification (Fig. 2A-2C). The Pearson correlation coefficients between histone modifications and the expression level of genes with FAIRE elements were generally low. The highest coefficient was r = 0.50 for the pair of gene expression and FAIRE-H3K9/14ac (Fig. 2C), and the next was r = 0.4 for gene expression and FAIRE-H3K4me3 (Fig. 2B). However, FAIRE-H3K4me1 showed almost no correlation with gene expression level (r = -0.03) (Fig. 2A). To examine whether breast cancer-related genes were up-regulated in MCF-7 and appeared to have a high level of H3K9/14ac, as found in our previous study [26], we selected 68 genes, the expression levels of which ranked in the top 30% among FAIRE-H3K9/14ac-associated genes, and performed DAVID Functional Annotation analysis. We could isolate 29 functionally significant genes with p < 0.05: ATM, BTG1, CCND1, CDK4, CDKN1B, CRADD, CSDA, CTR9, DDB2, DUSP6, ERBB3, ESPL1, FADD, H2AFX, KRT18, KRT8, MADD, MDM2, MYC, NR4A1, POLA2, RIPK2, RRM2B, SART3, SMARCC2, TSG101, UBE2N, XPOT, and YWHAZ. These genes were associated with the following GO categories: regulation of apoptosis, programmed cell death, nuclear lumen, protein ubiquitination, DNA damage checkpoint, mitotic cell cycle, and small conjugating protein ligase activity (Fig. 2D). Moreover, the KEGG pathway analysis displayed their involvement in the cell cycle, p53 signaling pathway, mitogen-activated protein kinase signaling pathway, and pathways in cancer (Fig. 2E).

Sequence motif analysis for FAIREs marked by active histone modifications
As many FAIRE regulatory elements were linked with active histone modifications, we explored the possible existence of functional sequence motifs for known transcription factors in FAIRE-H3K4me3 and FAIRE-H3K9/14ac sites. The binding motifs, such as CTCF, MYB, GFY-staf, ETS, and NRF1, were common in both FAIRE-H3K4me3 and FAIRE-H3K9/14ac sites (Fig. 3A). However, NFY and RUNX motifs existed only in FAIRE-H3K4me3 sites, and the GATA3 motif was specifically detected in FAIRE-H3K9/14ac sites. For FAIRE-H3K4me1 sites, AP-1/2, NF1, CTCF, AP-2, FOXA1, USF1, and MAFA motifs were identified (Fig. 3B). Interestingly, the CTCF motif was commonly found in FAIRE-H3K4me1, FAIRE-H3K4me3, and FAIRE-H3K9/14ac. The genomewide positioning of regulatory elements marked by histone modifications is illustrated in a Venn diagram (Fig. 3C-3E). More than 60% of the FAIRE-H3K4me3 and FAIRE-H3K9/14ac regions carrying binding motifs were distributed at gene promoters; over 20% in the gene body; and at small portions far upstream of promoters (Fig. 3D and 3E). In contrast, the population of FAIRE-H3K4me1 was highly enriched in gene body regions (66.4%) as well as upstream of promoters (31.3%) but almost negligible at promoters (2.3%) (Fig. 3C).