Epigenetic heterogeneity in primary bone cancers

Posttranslational histone modifications

It has long been known that histones are posttranslationally modified [ , , ]. There is a large variety of such modifications, mainly on the basic N-terminal tails of histones. These modifications affect the net charge of histones and internucleosomal interactions, and thereby directly alter chromatin structure. Furthermore, they can indirectly affect chromatin structure by recruiting remodeling enzymes that reposition nucleosomes. Aberrant histone modifications can alter gene expression, and are therefore frequently found in cancer cells.

Histone acetylation occurs on lysine residues of histones tails and cores, and is regulated by two families of enzymes with opposing functions: histone acetyltransferases (HATs) and histone deacetylases (HDACs). The positively charged lysines are neutralized by acetylation, which proposedly disrupts the interaction of the histone tails with the negatively charged DNA. Acetylated histones attract chromatin remodeling complexes, such as the BRG1-BRM-associated factor (BAF) chromatin remodeling complex, which makes chromatin accessible and induces gene expression [ ]. Multiple histone lysines can be acetylated, including four highly conserved lysines in histone H4 (K5, K8, K12, and K16), six in histone H3 (K9, K14, K18, K23, K27, and K56), as well as less conserved sites in histones H2A and H2B. Histone acetylation marks were often considered functionally similar and redundant, but recent evidence suggests characteristic roles and distributions of individual histone acetylation marks. For instance, distinct combinations of histone acetylation marks can be used to define active promoters and enhancers at a genome-wide level [ ].

Histone methylation occurs on nitrogen atoms of lysine and arginine residues. Lysine residues can have multiple methylation states (mono-, di-, or trimethylated), which are important for the effect on transcriptional regulation. Histone methylation has a strong impact on chromatin structure, leads to recruitment of transcription factors, affects RNA processing [ ], and has an important role in development and differentiation [ , ]. Transcriptional activation is mediated by methylation of H3K4 [ ], H3K36 [ ], and H3K79 [ ], whereas methylation of H3K9 [ ], H4K20 [ ], and H3K27 [ ] represses transcription.

Other posttranslational histone modifications include phosphorylation, ubiquitination, ribosylation, sumoylation, and deamination. Although they all have been shown to have an impact on chromatin structure they are understudied in primary bone cancers and hence not discussed further herein [ , ].

DNA methylation

In mammalian cells, DNA methylation is mostly found in CpG sites (where cytosine is followed by guanine in the 5′ to 3′ direction). These sites frequently cluster together as “CpG islands”, especially in promoter regions. DNA methylation of CpG sites seems to be the default state, with around 75% of sites methylated in somatic mammalian cells [ , ].

Compared to histone modifications, methylation of cytosine nucleotides in the DNA itself is a more stable mark, and is firmly linked to developmental processes. DNA methylation is associated with long-term silencing of transcription; both de novo methylation and demethylation of specific sites occur during development and regulate gene repression. Silencing via DNA methylation occurs on transcriptionally inactive, tightly packed heterochromatin, suggesting that DNA methylation itself does not cause repression, but is rather responsible for its long-term maintenance. DNA methylation is maintained in dividing cells, even if the factors that originally initiated it are no longer present [ , ].

In cancer cells, hypermethylation of promoter CpG islands can lead to silencing of tumor-suppressor genes. Hypomethylation often affects oncogenes, and, also outside of promoters, can additionally facilitate oncogenesis by promotion of chromosomal instability [ , ].

Long noncoding and microRNAs

Noncoding RNAs are further important epigenetic regulators. We give a brief overview here for completeness, but direct the interested reader to chapter 34 , which discuss these phenomena and their relevance in bone cancers in more detail.

microRNAs (miRNAs) are short (mostly 21–23 bp) RNAs that are not themselves translated. They pair with mRNAs, causing their cleavage, destabilizing them, or inhibiting their translation, and are thus important for posttranscriptional repression [ ] . miRNAs are frequently aberrantly regulated in cancer; oncogenic miRNAs have been termed oncomirs [ ].

The human transcriptome hosts tens of thousands of long RNAs (>200 nucleotides long) that are not transcribed [ ]. A number of these long noncoding RNAs (lncRNAs) have been shown to play various roles in the regulation of transcription, and many are expressed specifically in certain cell types [ ]. Several lncRNAs have been linked to cancer [ ] .

Recently a lot of focus has been given to an emerging class of noncoding RNAs that are synthesized at DNA loci corresponding to active enhancer elements in a cell type specific manner. They are called enhancer RNAs (eRNAs) and regulate gene expression. They have already been shown to be implicated in tumorigenesis [ ].