The epigenetic regulation by DNA methylation and post-translational modifications of histones without altering DNA sequences is tightly linked to the gene expression program in eukaryotic genomes. The N-terminal tails of histones are subject to various types of modifications, such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, and sumoylation [1]. Dynamic changes by histone-modifying enzymes affect the chromatin accessibility to transcriptional machinery and thus modulate gene activation or silencing in diverse biological processes, such as transcription, DNA repair, development and differentiation, and genome stability [2-4]. Moreover, nucleosome positioning can also be controlled by specific histone modifications, leading to alteration of cross-talk among chromatin structure, exon-intron architecture, and RNA polymerase II binding [5, 6]. Genomewide profiles of histone modifications with chromatin immunoprecipitation sequencing (ChIP-Seq) have revealed the characteristic genomic distribution and the association of gene functions and activities in eukaryotic genomes [7-12]. ChIP-Seq is a popularly used method to find genomewide protein binding sites with a high resolution by chromatin-immunoprecipitation by massive DNA sequencing using high-throughput sequencing technology [11]. For example, trimethylated histone H3 lysine 4 (H3K4me3), which is catalyzed by trithorax complex, as well as histone acetylation (e.g., H3K9/16ac) are usually enriched at promoter regions or transcription start sites (TSSs) with open chromatin structure and positively correlated with gene transcriptional activation level. H3K4me1 is also found to be enriched at enhancer-associated regions. These modifications recruit the transcriptional machinery at target sites. Trimethylation of H3K27, catalyzed by polycomb group protein complex 2 (PRC2), contributes to gene silencing by promoting chromatin condensation and chromatin stabilization and is likely to spread over larger regions around TSSs of silent genes. The signals of H3K9me3 are high in silent genes or repressed chromatin domains, such as heterochromatin. The comprehensive and comparative analysis of many histone modifications in the human genome demonstrates that the combination of several histone modifications shows a modular pattern and regulates transcriptional activation in a cooperative manner [13]. Interestingly, many loci of H3K27me3 are often colocalized with those of H3K4me3 around TSSs of lowly expressed genes in stem-like cells, many of which are positioned at developmental regulators, including transcription factors and signaling proteins [14]. This suggests that the formation of a bivalent chromatin domain can be a signature of epigenetic memory and that programmed gene expression during differentiation is dependent on chromatin modifications [14].

The interactions of variations in the genome, epigenome, and transcriptome with sensing of environmental stimuli can determine phenotypic plasticity. Traditionally, phenotypic variation has been explained primarily through genetic variation with sequence changes during evolution. However, epigenetic variations that are potentially sensitive to environmental inputs can alter transcriptional activity, which in turn contributes to diversity of complex traits [15]. Such observations have been demonstrated in human cancers and plant development, such as floral symmetry and vernalization response (reviewed in [15, 16]). For example, silencing of tumor suppressor genes, including p16, VHL, MLH1, APC, and E-cadherin, is associated with DNA hypermethylation in the gene promoters [17]. In relation to histone modifications, overexpression of EZH2, a histone H3K27 methyltransferase, has been observed and positively correlated with the progression of multiple malignancies, including prostate cancer, breast cancer, lymphoma, myeloma, colorectal cancer, endometrial cancer, bladder cancer, and melanoma [18]. In addition, generalized loss of H4K16ac and H4K20me3 is found in lymphoma and colorectal cancer, leading to transcriptional silencing [16].

Organization of genomic DNA into higher-order chromatin structures is involved in transcriptional regulation in eukaryotes [19, 20]. In particular, cis-regulatory elements complexes located in open chromatin regions, depleted of nucleosomes, are activated by the recruitment of transcriptional machinery [19, 20]. Such active regulatory elements have been identified by formaldehyde-assisted isolation of regulatory elements (FAIRE) technique, a simple high-throughput screening method to isolate and map active regulatory elements depleted of nucleosomes in eukaryotes, especially in clinical samples, by forming crosslinks between histones and DNA and subsequent utilization of hybridization of tiling microarrays or next-generation sequencing [21-23]. FAIRE was first demonstrated in Saccharomyces cerevisiae, where formaldehyde-crosslinked chromatin immediately upstream of genes was preferentially segregated into the aqueous phase in a manner that was strongly negatively correlated with nucleosome occupancy [21]. Results from both yeast and human samples showed that enrichment of regions upstream of genes was positively correlated with transcription of downstream genes [21-23].

Breast cancer is a heterogeneous and progressive disease, known as the most common cancer among women. For early diagnosis and prognosis, the development of tumor makers in breast cancer is important, but the markers for its early detection are rare [24, 25]. The most clinically useful tissue-based marker genes in breast cancer are steroid receptors, estrogen receptors, progesterone receptors, and HER-2 [24, 25]. The mutations of BRCA1 and BRCA2 are strong indicators of breast cancer development, but their mutation is not common [25]. Except for genetic variation-based diagnosis, a comprehensive understanding of the gene expression program as well as epigenetic mechanisms is definitely required for the development of diagnosis markers and epigenetic therapies. We recently reported epigenetic regulatory mechanisms of gene expression with 3 different histone modifications (H3K4me1, H3K4me3, and H3K9/14ac) in normal (MCF-10A) and breast cancer (MCF-7) cells [26]. In particular, we demonstrated the change of transcriptional activity, delineating differential enrichment of histone modifications in both MCF-10A and MCF-7, which defines the functional regulatory elements in the genome with a cell type-specific chromatin environment [26]. To address the relationships between histone modifications and FAIRE and between FAIRE-related chromatin structure and transcriptional activity, we analyzed FAIRE-chip [23], histone modification-ChIP-Seq [26], and gene expression microarrays derived from MCF-7. Our result provides an understanding of epigenetic regulatory mechanisms with open chromatin in breast cancer cells.

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.