We have made substantial improvements to the processing of samples for RPA and improved the detection by using fluorescence and chemiluminescence methods in place of immunohistochemistry. We have also identified the sources of variability in the preparation of microarrays and devised protocols that minimize or correct for sample-to-sample variation. We have validated antibodies to over 30 different PTMs on histones including various residue specific or general acetylations, methylations and phosphorylations. Applying this panel of antibodies across a spectrum of pathological and physiological samples including both tissues and cell lines has revealed promising associations of particular patterns of global modification with particular entities. For example, using heat maps, samples sort according to their biological relatedness. This system can be expanded to explore the chromatin proteomics of a variety of tumors and to observed the response to therapy that is designed to exacerbate or inhibit chromatin modification inorder to manipulate the genetic and epigenetic programs in tumor cells. Using this approach it is apparent that the subtle distinctions between B-cell lymphomas are associated with discernible differences in their pattern of PTM. Similarly, prostate cancers can b sorted according to PTMs that correlae with biological changes. We have also been adapting chromatin immunoprecipation methods for use on tissue with the goal of understanding the genome-wide changes in protein binding to DNA that occur in primary tissue. First these techniques will be optimized using animal (murine) tissue, then we will use them on autopsy materials and finally we will attempt to use them on appropriate and appropriately authorized clinical materials. We have developed a method using mouse tissues that now hast the potential to be applied to human biopsy material to map nuclease hypersensitive sites to reveal differences in the genome wide utilization and activation of cis-regulatory elements.
|Luchenko, Victoria L; Litman, Thomas; Chakraborty, Arup R et al. (2014) Histone deacetylase inhibitor-mediated cell death is distinct from its global effect on chromatin. Mol Oncol 8:1379-92|