The CDKN A gene locus encodes for p ARF p

The CDKN2A gene locus encodes for p14ARF, p16INK4a, and the long non-coding RNA ANRIL, which negatively regulates p16INK4a. p16INK4a inhibits CDK4 and CDK6 activity, which regulate the RB-E2F axis and cell cycle progression (Nilsson and Landberg, 2006; Jin et al., 1995; Jacobs et al., 1999; Abella et al., 2005), while p14ARF also induces cell cycle arrest through its interaction with MDM2, resulting in increased p53 levels that trigger cell cycle arrest at both G1 and G2/M phases (Pomerantz et al., 1998; Zhang et al., 1998). Both p16INK4a and p14ARF also play roles in driving cellular senescence and ageing (Kim and Sharpless, 2006; Matheu et al., 2009). There is also evidence that the p16INK4a-cdk4-pRB axis plays a direct role in metabolic regulation and adipocyte differentiation (Aguilar and Fajas, 2010). Recent studies of genetically engineered mice deficient in E2F1, cdk4, and pRB, have shown that they contribute to lipid synthesis, glucose production, insulin secretion, and glycolytic metabolism. For example, adipose tissue-specific inactivation of the retinoblastoma protein induced increased mitochondrial activity, resulting in increased energy expenditure, which protected from diet-induced obesity (Dali-Youcef et al., 2007). In addition, inactivation of cdk4, which controls the clonal expansion phase of adipogenesis and phosphorylates PPARγ, blocked adipocyte differentiation, while a Cdk4 mutant, which cannot be inhibited by p16INK4a, increased the adipogenic potential of 3T3-L1 cells (Abella et al., 2005). Data from the BLUEPRINT epigenome project suggests that the CDKN2A DMR overlaps with regulatory sequences. In monocytes, the DMR lies within a DNase hypersensitive peak and is associated with H3K27me3, H3K4me1 and H3K4me3 marks. The existence of both repressive and activating histone marks suggests that a bivalent domain is located across the region, while in c-met inhibitor and HUVECs, there is a peak of H3Kme1, a mark often enriched at enhancers or regulatory sequences. Consistent with sequences within the DMR having a functional role in the regulation of CDKN2A directed transcription, in the GUSTO cohort, where both DNA and RNA were available from the cord tissue, there were associations between the methylation of CpG cluster 4–8 and CpG 9 and the expression of the CDKN2A generated transcripts, suggesting that differential CpG methylation within the promoter of ANRIL may have downstream effects on p16INK4a and p14ARF expression. Here, lower CpG methylation was associated with a decrease in ANRIL expression and an increase in p16INK4A and P14ARF expression. Increases in p16INK4a and p14ARF expression would be expected to lead to a decrease in cell proliferation and increased cellular senescence; if re-capitulated in adipocytes, this could promote both a decrease in adipocyte number and impaired adipocyte function, since p16INK4a regulates cdk4 activity, which in turn phosphorylates and activates the adipogenic transcription factor PPARγ. Interestingly the region encoding ANRIL has been identified by GWAS as a hot spot for genetic variation associated with a number of ageing associated diseases, such as CVD and T2D. Sequence alterations within ANRIL may similarly affect p16INK4a expression with downstream effects on cellular senescence, impairing the regenerative potential of cells, and increasing susceptibility to ageing associated diseases. Whether differential methylation of this promoter region of ANRIL or other CpGs sites further upstream also associated with an altered susceptibility to other ageing associated diseases is, as yet, unclear. Thus, these findings suggest that both genetic and epigenetic alterations within ANRIL may have important consequences for future disease risk. In liposarcoma cells, we found that site directed mutagenesis of CpG sites affected ANRIL and p14ARF promoter activity respectively, and although mutation of a CpG site does not infer that methylation of the CpG would have the same effect, it does clearly suggest that these CpG sequences are important determinants regulating ANRIL and p14ARF expression. Binding of specific protein complexes to sequences within the DMR was also observed in liposarcoma cells, with methylation of CpG 2 increasing protein binding. DNA methylation has generally been associated with reduced transcription factor binding (Tate and Bird, 1993), but there is now growing evidence that DNA methylation can also enhance transcription factor binding (Clarke-Harris et al., 2014b). Interestingly, multiplexed competitor EMSAs identified ERα binding across CpGs 2–3. ERα plays an important role in maintenance of metabolic homeostasis and insulin sensitivity, with ERα perturbation linked to the metabolic syndrome (Hevener et al., 2015). ERα is highly expressed in adipose tissue (Mizutani et al., 1994), and a reduction in expression is linked to adipocyte hyperplasia and hypertrophy, insulin resistance and glucose intolerance in both sexes (Mauvais-Jarvis et al., 2013). We found that treating liposarcoma cells with estrogen stimulated expression of ANRIL, and reduced p14ARF expression. While it remains unclear if ANRIL or p14ARF are direct targets of ERα in vivo or part of the pathway by which ERα influences adipocyte function, our experiments do suggest that lower DMR methylation would decrease ERα binding and increase adiposity, consistent with studies showing increased adiposity in ERα knock out mice and that ANRIL may be an important component of this pathway.