Targeting of DNA Methylation in Mammary Epithelial Cells
Creators: name: University Of California Office Of The President
Publisher: name: University of California Office of the President
publisherIdentifier: https://ror.org/00dmfq477
publisherIdentifierScheme: ROR
Year: 2024

description: Breast cancer cells are characterized by changes in DNA structure that lead to the inactivation of genes critical for control of cell growth. The best described of these alterations are mutations which cause changes in the DNA sequence of a particular gene. In recent years, epigenetic alterations have become recognized as an important class of DNA lesions that contribute to the inactivation of critical genes in breast tumors. Epigenetic traits can be inherited, yet are not based on a change in the DNA sequence of a gene. A chemical change to DNA called DNA methylation is one of the best characterized epigenetic traits. It plays an important role in regulating gene expression, where it serves as a marker for inactive regions of the genome. In breast cancers DNA methylation is the most common cause of inactivation of the p16 gene, an important regulator of cell division. In addition, recent studies have found cells with a methylated p16 gene in substantial number of healthy, cancer-free women. This suggests that p16 methylation may be an important early event in the transition of normal breast cells into a pre-cancerous state. Despite the importance of DNA methylation in the development of breast cancer, the molecular mechanisms by which it is targeted to a specific gene remain unknown.
 
 
 
 
 
 
 
 The goal of this proposal is to define the molecular mechanisms by which DNA methylation is specifically targeted to the p16 gene in breast cells. Our aims are: (1) To isolate the protein complexes in breast cells that are responsible for methylation of the p16 gene. (2) To identify and characterize the proteins responsible for bringing these complexes to the p16 gene.
 
 
 
 
 
 
 
 We will examine the methylation of the p16 gene in breast cells isolated from cancer-free women that have a methylated p16 gene. To isolate protein complexes from these cells, we will biochemically separate protein extracts and test the resulting fractions for methylation activity. In cells, DNA is associated with proteins to form a structure called chromatin. In the lab we can assemble DNA into this structure for use in our assays. We will use this technique to assemble the p16 gene into chromatin and test whether the complexes we isolate are still able to methylate the DNA. If not, we will test the ability of known proteins to bring the complex to the DNA. In addition, we will look for novel factors by biochemically fractionating cell extracts. In this way we will define the proteins required for methylation of the p16 gene.
 
 
 
 
 
 
 
 Previous studies of DNA methylation in cancer have used tumor cell lines which have many genetic aberrations. In this proposal, we will use non-tumor forming breast cells, which we believe will more accurately model the early events underlying the transition of a breast cell from a normal to premalignant state. Studies from our lab have shown that important aspects of gene regulation can be reconstituted in the test tube using DNA templates assembled into chromatin. As far as we know, no one has used this approach to study the targeting of DNA methylation. By defining the proteins involved in methylation of p16, we will gain significant insight into how this process is regulated. We also may identify important targets for chemotherapy.