DNA methylation has been comprehensively profiled in normal and cancer cells, but the dynamics that form, maintain and reprogram differentially methylated regions remain enigmatic. Here, we show that methylation patterns within populations of cells from individual somatic tissues are heterogeneous and polymorphic. Using in vitro evolution of immortalized fibroblasts for over 300 generations, we track the dynamics of polymorphic methylation at regions developing significant differential methylation on average. The data indicate that changes in population-averaged methylation occur through a stochastic process that generates a stream of local and uncorrelated methylation aberrations. Despite the stochastic nature of the process, nearly deterministic epigenetic remodeling emerges on average at loci that lose or gain resistance to methylation accumulation. Changes in the susceptibility to methylation accumulation are correlated with changes in histone modification and CTCF occupancy. Characterizing epigenomic polymorphism within cell populations is therefore critical to understanding methylation dynamics in normal and cancer cells.

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominantnegative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53 R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53 R175H , was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53 R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53 R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.

SUMMARY Bioerosion by boring sponges is an important mechanism shaping the structure of coral reefs all around the world. To determine the excavation rate by boring sponges, we developed a system in which chemical and mechanical boring rates [calcium carbonate (CaCO3) dissolution and chip production, respectively] were measured simultaneously in experimental tanks containing reefal rock inhabited by a boring sponge. Pione cf. vastifica (Hancock 1849) was chosen as a model species to study the erosion rate of boring sponges. It is an abundant species in the coral reefs of the Nature Reserve Reef, Elat, Gulf of Aqaba, northern Red Sea, reaching maximum abundance at 25-30 m. The rate of chemical bioerosion was determined from the increase in tank-seawater alkalinity over time, and the mechanical bioerosion rate was estimated from the total amount of CaCO3 chips produced over the same time interval. The measured bioerosion rate of P. cf. vastifica was 2.3 g m-2 sponge day-1, showing seasonal but not diurnal variations, suggesting that the zooxanthellae harboring the sponge have no effect on its boring rate. The experiments indicated clearly that per each mass of chips that P. cf. vastificaproduces during its boring activity, it dissolves three masses of reef CaCO3 framework. Assuming that some additional boring sponges can use a similar strategy of bioerosion, these findings suggest that chips, the most obvious erosion products of boring sponges, represent only a small fraction of boring sponge bioerosion capacity.

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

Deregulation of Ras pathways results in complex abnormalities of multiple signaling cascades that contribute to human malignancies. Ras is therefore considered an appropriate target for cancer therapy.