DNA methylation is the most important epigenetic regulation for mRNA and microRNA (miRNA) expression in mammalian cells to ensure normal development and growth [37]; conversely, it is dysregulated in cancer cells [38,39]. In the process of DNA methylation, it creates a ‘fifth base’ from cytosine, 5-methylcytosine (5mC), mostly occurring in CpG islands (CGIs), which act as regulatory hotspots found upstream of the promoter region [40]. There are three types of proteins for DNA methylation and demethylation, including DNA methyltransferases (DNMTs), ten-eleven translocation (TET) enzymes and methyl-binding domain (MBD) proteins [41,42]. Three DNMTs controlling methyl group transfer or CGI methylation consist of DNMT1, responsible for methylation maintenance, and DNMT3A and DNMT3B, capable of de novo methylation, which play critical physiological roles in mammalian genome stability, cellular proliferation and development and cell fate determination [43,44]. Recently, DNMT2 has been identified as a methyltransferase, but for methylation of tRNA instead [45]. The methylated DNA can be recognized by binding MBD proteins to recruit histone-modifying complexes, such as histone methyltransferases (HMTs), for regulating gene transcription and chromatin remodeling [46,47]. It is estimated that 70% of all CGIs in humans are hypermethylated and are found in heterochromatin, which represses transcription. In contrast, hypomethylated CGIs are located in euchromatin, which activates gene expression [48]. Conversely, demethylation is catalyzed by TET family enzymes, TET1, TET2 and TET3, oxygenase enzymes that convert 5mC to 5-hydroxymethylcytosine (5hmC), 5-formyl cytosine (5fmC) and 5-carboxyl methyl cytosine (5CamC) [49,50,51,52].

Covalent post-translational modifications (PTMs) of histone tails, including methylation, acetylation, phosphorylation, ubiquitination and SUMOylation, play a pivotal role in modifying gene expression [53]. In contrast to DNA methylation, associating with gene-silencing, histone methylation, acetylation and phosphorylation can change the secondary structure of DNA and result in either induction or prevention of access by transcription factors to gene promoter regions in order to inhibit or activate transcription [53,54].

Histone methylation plays important roles in gene transcription, DNA replication and repair, chromatin organization and developmental and differentiation processes [55,56,57]. Histone methylation, defined as the transfer of one, two or three methyl groups to lysine or arginine residues of histone proteins, is regulated by HMTs and histone demethylases (HDMs) [58]. Transcription silencing is associated with methylation of histone 3 lysine 9, 20 or 27 (H3K9, H3K20 or H3K27), while methylation of histone 3 lysine 4, 36 or 79 (H3K4, H3K36 or H3K79) is involved in transcription activation [59]. Three families of HMTs have been discovered that are specific for the lysine or arginine residue which they modify: the set domain-containing protein family, the non-set domain protein family and the protein arginine methyltransferases (PRMT1) family [57]. A polycomb repressive complex 2 (PRC2) group protein, Enhancer of zeste homolog 2 (EZH2), methylates H3K27 and is a transcriptional repressor [60]. H3K9 methylation occurring in euchromatin causes mono- and di-methylation (H3K9me1 and H3K9me2) catalyzed mainly by G9a, and in heterochromatin, which requires trimethylation (H3K9me3) mostly catalyzed by Suv39H1 and Suv39H2 and results in transcriptional silencing [55,56]. Furthermore, a novel histone lysine methyltransferase, the set and MYND domain-containing protein 3 (SMYD3), methylates H3K4 [61]. On the other hand, two major families of demethylases have been identified, lysine-specific demethylase 1 (LSD1) and Jumonji domain-containing HDMs (JMJD2, JMJD3/UTX and JARIDs). LSD1 specifically demethylates mono- or di-methylated H3K4 or H3K9 and non-histone proteins, such as p53 and DNMT1, indicating that it plays a vital role in a number of normal biological functions and in carcinogenesis, as described in the following section [62]. Similarly, H3K9me3/me2 demethylation is catalyzed by JMJD2C, also known as histone lysine demethylases 4C (KDM4C) [63]. Additionally, JMJD2C demethylates the second methylated histone substrate, H3K36me3 [64].

Histone acetylation occurs via the modifying enzymes, histone acetyltransferases (HATs) or histone deacetylases (HDACs). An acetyl group is added by HATs to ε-amino groups of lysine residues in the histone N-terminal tails, making euchromatin, which allows transcription factor binding and results in gene activation. Conversely, HDACs catalyze the hydrolytic removal of acetyl groups from histone lysine residues, which compact chromatin into heterochromatin, preventing transcription factor binding to DNA and subsequent gene expression [65].

There is a growing list of proteins and ncRNAs identified in epigenetic regulation that may represent useful biomarkers for diagnosis and/or prognosis for breast cancer. The major challenges in cancer therapy are tumor recurrence and resistance to conventional therapies, such as chemotherapy and radiotherapy, and CSCs are the major players in these events. Therefore, comprehensive elucidation of regulatory mechanisms in BCSCs will definitely help to develop more effective precision medicine. There is emerging data on dysregulation of ncRNAs, and ncRNA hyper/hypomethylation contributes to cancer stemness. There are currently not many miRNA-based therapies for breast cancer; therefore, these represent a great opportunity in developing novel therapeutic strategies for breast cancer. Additionally, ncRNAs have the advantage of multi-target characteristics, which should minimize the possibility of drug resistance. However, the major hurdle for miRNA-based therapies lies in the lack of a specific delivery system, a problem shared with all forms of gene therapy in cancer. In spite of the enormous amounts of biomarkers identified in epigenetic regulation of breast cancer and BCSCs, currently, there are only a few drugs available, and even less entering clinical trials. Therefore, in the future, the development of novel drugs or combination regimens are urgently required.