Discussion
FAIRE has been known to enrich functional DNAs located in DNase I hypersensitive sites, active promoters, and transcriptional start sites [22]. The enrichment of such regulatory regions in the aqueous phase might result in easy identification of genomic function without bias. Eeckhoute et al. [23] demonstrated that FOXA1-bound enhancers defined by FAIRE in human chromosomes 8, 11, and 12 were closely related with cell-type specific chromatin remodeling. In addition, it was reported that FAIREs are highly associated with DNase I hypersensitivity sites, RNA polymerase II, and TAF1 binding sites [22]. In combination with the information on these FAIRE regulatory elements in MCF-7 cells, we analyzed our genomewide histone modification data (H3K4me1, H3K4me3, and H3K9/14ac) generated by ChIP-Seq [26]. It was shown that the H3K4me1 distribution, covering the entire human genome, which was different from the pattern of H3K4me3 and H3K9/14ac, was 53% of the gene body, 42% of the intergenic region, and 5% of the promoter. The promoter regions covered 54% and 52% of total sequence reads in H3K4me3 and H3K9/14ac, respectively. The pair-wise colocalization analysis between 2 histone modifications gave poor correlation coefficients in the H3K4me1-H3K4me3 pair (r = 0.14) and H3K4me1-H3K9/14ac pair (r = 0.19) but good coincidence in the H3K4me3-H3K9/14ac pair (r = 0.86) when the Pearson's correlation coefficients were calculated with normalized tag counts detected within 1 kb upstream and downstream of TSSs [26]. The co-occupancy of FAIRE elements with histone modifications is examined in Fig. 1, where it is clear that the promoters were the most abundant regulatory elements. Two active histone modifications, H3K4me3 and H3K9/14ac, were highly enriched within ± 1 kb from TSSs (Fig. 1A), and a comparative analysis showed that the FAIRE regulatory elements associated with histone modifications were positioned at promoters (Fig. 1D). Such epigenetic relationship between FAIRE and active histone modification marks has also been demonstrated in various cell types [22, 23, 34-36]. As exemplified in Fig. 1E, H3K4me1 was differentially positioned, and the distribution of FAIRE-H3K4me1 sites was away from promoter regions, meaning that FAIRE-H3K4me1 sites might be distal regulatory elements, such as enhancers [37].
The level of gene expression is also modulated, depending on the degree and position of various kinds of histone modifications. As shown in Fig. 2A-2C, genes with FAIRE sites carrying H3K4me3 and H3K9/14ac had relatively high expression levels compared to those with FAIRE-H3K4me1. Some genes related with breast cancer were up-regulated and showed high levels of H3K9/14ac in our previous study [26]. We therefore examined 68 genes with FAIRE-H3K9/14ac sites with the DAVID GO analysis tool. These genes were significantly related with cell cycle, apoptosis, DNA damage, and signaling pathways, reflecting that their in vivo functions are essential for cell survival and proliferation. Many of the regulatory sites associated with histone modifications contained transcription factor binding motifs (Fig. 3A and 3B). CTCF binding sites, commonly found in FAIRE-H3K4me1, FAIRE-H3K4me3, and FAIRE-H3K9/14ac, are related to a function of insulators and involved in high-order chromatin structure [38], and DNA demethylation is also known to be coincident with FAIRE-related open chromatin [36, 39]. Using computational motif analysis coupled with ChIP assay, Waki et al. [36] demonstrated that enrichment of a binding motif for nuclear family I (NFI) transcription factors was highly associated with adipocyte-specific FAIRE signals as well as active histone modifications, like H3K4me3 and H3K27ac, providing a global view of cell type-specific regulatory elements in the genome and an identification of transcriptional regulators of adipocyte differentiation [35]. Such selective activities of regulatory elements were also reported by monitoring the chromatin structure at FOXA1-bound enhancers defined by FAIRE [26]. This evidence supports the possibility that open chromatin structures are subject to be bound by many transcription factors and that histone modifications function as markers for these factors to be recruited.
In conclusion, our results suggest that genomic regions defined by FAIRE in breast cancer cells should be highly associated with active histone modifications, such as H3K4me1, H3K4me3, and H3K9/14ac, and play a crucial role in controlling gene expression programs. This analysis will provide an understanding of epigenetic regulatory mechanisms with open chromatin in breast cancer cells.