ABSTRACT
We synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogs and identified several potential targets by proteomic analysis. The validation of the proteomic analysis identified the chemical probe as a specific inhibitor of glucose-regulated protein 94 (GRP94), a potential drug target for several types of cancers. Therefore, as a result of the use of bisubstrate-type chemical probes and a chemical-biology methodology, this work opens the way to the development of a new family of GRP94 inhibitors that could potentially be of therapeutic interest.
Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Affinity Labels/pharmacology , Cytosine/analogs & derivatives , Cytosine/pharmacology , Membrane Glycoproteins/antagonists & inhibitors , Adenosine/radiation effects , Affinity Labels/chemical synthesis , Affinity Labels/radiation effects , Cell Line, Tumor , Click Chemistry , Cytosine/radiation effects , Humans , Membrane Glycoproteins/chemistry , Proteome/chemistry , Proteomics , Ultraviolet RaysABSTRACT
Interaction and co-occurrence of protein and DNA-based epigenetic modifications have become a topic of interest for many fundamental and biomedical questions. We describe within this chapter a protocol that combines two techniques in order to determine the methylation status of the DNA specifically associated with a protein of interest. First, DNA that directly interacts with the selected protein (such as a specific histone modification, a transcription factor, or any other DNA-associated protein) is purified by standard chromatin immunoprecipitation (ChIP). Second, the level of DNA methylation of this immunoprecipitated DNA is measured by bisulfite conversion and Pyrosequencing, a quantitative sequencing-by-synthesis method. This procedure allows determining the methylation status of genomic DNA associated to a specific protein at single nucleotide resolution.
Subject(s)
Chromatin Immunoprecipitation/methods , DNA Methylation , Nucleotides/genetics , Sequence Analysis, DNA/methods , Analytic Sample Preparation Methods , Cell Line, Tumor , DNA/genetics , DNA/isolation & purification , DNA Methylation/drug effects , DNA Primers/genetics , Humans , Microspheres , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Sulfites/pharmacologyABSTRACT
DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor ß (RARß), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARß promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARß during prostate tumorigenesis.
