(4bk) Effects of DNA Methylation Pattern On the Chromatin Structure

My research focus has been on understanding how DNA methylation affects chromatin structure and gene expression. This information is instrumental in the identification of DNA methylation biomarkers for early diagnosis of cancer.

 Chromatin’s degree of compaction regulates gene expression by restricting the accessibility of cellular machinery to genes. Such regulation mechanism is often controlled by epigenetic modifications. DNA methylation, the most commonly found epigenetic modification in DNA, refers to the addition of a methyl group to the 5^th carbon of a cytosine base in the context of a CG sequence. The presence of methyl groups in the DNA leads to a more rigid DNA structure, affecting the overall conformation of the chromatin fibers. Increased levels of DNA methylation are associated with chromatin compaction and gene silencing. The detailed molecular mechanism as to how DNA methylation directly modulates chromatin structure and function remains unknown.

Using multiple molecular biology techniques, we have constructed chromatin-like molecules i.e. nucleosome and nucleosome arrays, to study the effects of DNA methylation in the conformation and dynamics of chromatin, in vitro. These in vitro systems allow for the evaluation of the direct effect of DNA methylation on the variables of interest without the influence of other proteins found in the cell nucleus. In addition, we have been able to introduce different DNA methylation patterns to our system to study the effects of the position of the methylation sites in the chromatin structure.

We measured the conformational and dynamic changes of nucleosomes and nucleosome arrays using various biophysical assays, in particular fluorescence spectroscopy techniques. Our results show a dynamic behavior of nucleosomes i.e. the fundamental chromatin unit, despite of their methylation status or binding to other proteins e.g. linker histone proteins. We also observed that nucleosome and nucleosome arrays conformation can be modulated by specific DNA methylation patterns, depending on the buffer conditions.

The results from this research allow us to identify methylation pattern(s) that are functionally important in regulating chromatin conformation. Besides being instrumental in understanding the molecular mechanism by which DNA methylation directly modulates chromatin compaction, this information is useful in the identification of DNA methylation biomarkers for the early detection of cancer.

Aberrant DNA methylation patterns are present in early stages of carcinogenesis. Either hypomethylation of oncogenes or hypermethylation of tumor repressor genes can lead to the development of cancer cells. The potential of DNA methylation as biomarker of early onset of the disease has been explored in many different cancers. The traditional approach for screening of DNA methylation biomarkers is based on comparing the overall methylation level of a particular gene in healthy and disease tissue. A statistically significant difference between the methylation levels in both tissues makes this gene a good candidate for DNA methylation biomarker. Such approach can be bias or mislead by differential DNA methylation levels due to other factors different from cancer onset, such as age and gender. The DNA methylation patterns that we are evaluating can be use as a tool for assessment of DNA methylation status in biomarker genes. Besides the overall methylation level, the functional DNA methylation pattern associated with gene silencing discovered in this study can be identified in biomarker genes and then be use as a criteria for determining cancer risk, classification or prognosis.