Identifying gene expression changes that occur when a normal cell transforms into a neoplastic cell can reveal molecular markers that can be used for diagnosis and suggest potential new therapeutic targets. However, by characterizing the mechanisms by which the tumor-specific gene expression patterns have been created, one can also gain insight into the molecular processes that drive tumor formation. We have developed a protocol (termed a MAGIC analysis), based on the technique of ChIP-chip, that can be used both to study altered gene expression and to determine if the altered gene expression is due to changes in transcription complex formation, epigenetic-mediated gene silencing, or alterations in gene copy number. This method uses an antibody to RNAPII, an antibody to modified Histone H3, and an antibody to methylated DNA to identify active vs. silent regions of the genome. It also employs a genomic comparison of the normal and tumor samples to identify amplified and deleted regions of the genome. Most investigators who perform ChIP-chip assays use cultured cells. Clearly, this technique would be much more useful for characterizing the cancer genome if pure populations of tumor cells, obtained by microdisssection, laser capture, or other separation methods were analyzed. Adapting the ChIP-chip technique for the study of such samples requires modifications to sample preparation, scaling the ChIP assay to accommodate a small number of cells, and improving the amplification of the ChIP sample such that accurate representation of the experimental and control samples can be maintained when using small amounts of input DNA. We propose to modify the ChIP protocol for use with very small numbers of cells (Aim I), to determine the optimal amplification method for ChIP samples obtained from a small number of cells (Aim II), and to demonstrate that the modified protocols are suitable for analysis of cells obtained from tumor samples by laser capture microscopy (Aim III). We propose to modify the technique of ChIP-chip such that it can be used after collection of a small number of tumor cells by laser capture microscopy. Such modifications would be a great advance in the molecular characterization of the cancer genome. ? ? ?
| Komashko, Vitalina M; Farnham, Peggy J (2010) 5-azacytidine treatment reorganizes genomic histone modification patterns. Epigenetics 5:229-40 |
| Acevedo, Luis G; Iniguez, A Leonardo; Holster, Heather L et al. (2007) Genome-scale ChIP-chip analysis using 10,000 human cells. Biotechniques 43:791-7 |
