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Even though aberrant CpG methylation is observed in cancer cells, it is also a normal physiological event that is critical for normal differentiation and development of cells. This is specifically exemplified by X chromosome inactivation and genetic imprinting.

In somatic cells, diploid organisms contain two copies (alleles) of each gene, one maternal and one paternal. The only exception to this is the sex chromosome, where a female inherits two copies of the X chromosome, and a male only one. In order that the expression of X linked genes is equal between the different sexes, one X chromosome in human females is silenced, a phenomenon known as X-chromosome inactivation and is an example of dosage compensation.

The processes involved in X chromosome inactivation are not fully elucidated or understood. However, briefly, inactivation is dependent on an X-inactivation centre (Xic) locus that (through undiscovered mechanisms) is responsible for X chromosome counting to ensure all but one X chromosome is inactivated. Xic contains a gene called X-inactive specific transcript (Xist) which is transcribed as a 17kb untranslated RNA, which physically binds and coats the X chromosome to be inactivated in cis. The active X chromosome produces an anti-sense transcript Tsix, which represses Xist expression through altering Xist chromatin during transcription of Tsix. This prevents the active X chromosome from being coated with Xist. Xist then recruits the chromatin remodelling Polycomb repressor complex. This ultimately results in modified histone proteins and gene promoter methylation. The inactive X chromosome exhibits high levels of CpG promoter methylation in comparison to the active chromosome, and the few genes in the inactive chromosome that escape inactivation are unmethylated in both copies of the X chromosome, indicating the critical role of CpG methylation in silencing of genes in the inactive chromosome (Figure 1.12). (Heard, 2004; Kalantry, 2011; Kim et al., 2009; Leeb et al., 2009).

Figure 1.12 Schematic diagram of X chromosome inactivation. To maintain equal

expression of X linked genes in males and females, one X female X chromosome is inactivated following fertilisation. The mechanism of this is not fully elucidated however, an X chromosome inactivation locus (Xic) is responsible for counting the X chromosomes to ensure all but one is inactivated. Xic encompasses the X-inactive specific transcript gene which is transcribed as a 17kb untranslated RNA that physically coats the X chromosome that is to be inactivated. At the same time the active chromosome transcribes the complementary transcript Tsix, which forms a duplex with Xist, preventing it from coating the chromosome. Xist on the coated chromosome then recruits the Polycomb repressor complex, resulting in chromatin remodelling and hypermethylation of the contained genes, rendering the chromosome inactive (Kalantry, 2011).

Inactive X Chromosome Active X Chromosome

Xist Tsix

Xic

Xic

Xist coated X chromosome

Xist/Tsix

duplex

rather than from the gene itself. The precise methylation is controlled by cis- acting imprinting control regions (ICRs), which are CpG rich sequences. Following fertilisation, the methylation status of the ICR is maintained (even though there is global hypomethylation), and the genes associated with the ICR if appropriate are de novo methylated by Dnmt3. The gene methylation is then preserved through differentiation and proliferation by Dnmt1, resulting in maternal or paternal specific expression of a gene (Kim et al., 2009; Wood and Oakey, 2006).

Regulated promoter methylation in normal cells has also been shown to regulate tissue specific silencing of genes. Methylation analysis from normal peripheral blood leukocytes identified 258 genes that had CpG islands within their promoters that were methylated, which corresponded to 4% of all gene promoters assayed (Shen et al., 2007). The identified methylated genes could broadly be characterised as being involved in intracellular membrane bound organelle function, metal ion binding and signalosome function and were shown to be hypomethylated in multiple cancer cell lines. Furthermore, expression of the genes in the cell lines correlated with hypomethylation of the promoter (either by 5-aza deoxycytidine treatment or Dnmt1 and 3b knockout) (Shen et al., 2007). The promoter of the breast cancer tumour suppressor gene serpin is unmethylated and expressed in normal epithelia, however is methylated and not expressed in normal hematopoietic, liver, kidney and heart cells (Futscher et al., 2002). MAGE1, a gene only expressed in testis and some melanomas, as well as other multiple testis specific genes, are methylated in normal somatic cells,

meaning they are not expressed, but unmethylated specifically in the

testis/spermatozoa where they are expressed (Zendman et al., 2003; Strathdee et al., 2004). The HOXA5 gene product is involved in differentiation of both haematopoietic and epithelial cells, and is also a candidate tumour suppressor in breast cancer. The HOXA5 promoter was methylated in mesenchymal cells, had ~50% methylation in haematopoietic cells and unmethylated in epithelial cells, and this correlated with mRNA expression (Strathdee et al., 2007).

Methylation is therefore important for maintaining normal silencing of certain genes. As previously described, aberrant methylation of gene promoters is also a well characterised event in multiple cancers. It is therefore important to understand how methylation of a gene or it’s promoter results in transcriptional silencing of the gene.

1.9.7 Promoter Methylation as a Cause of Transcriptional Silencing