FATTY ACID MODULATION OF COLON CELL TRANSFORMATION

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DESCRIPTION (provided by applicant): The short chain fatty acid butyrate is a physiological regulator of colon epithelial cell maturation (cell cycle arrest, lineage specific differentiation and apoptosis). While there is considerable interest in the role of butyrate as an inhibitor of histone deacetylase (HDAC) activity, we have demonstrated that other mechanisms triggered by butyrate must play essential roles in the stimulation and integration of the overall complex affects on colon cell maturation. The goal of this application is to dissect the contribution of two fundamental epigenetic mechanisms stimulated by butyrate: alteration of expression of microRNAs, and transcriptional attenuation. We will first address these issues using novel gene expression arrays that we have designed and fabricated to interrogate altered profiles of microRNA expression, and genome wide alterations in transcriptional attenuation, in response to butyrate. As regards microRNAs, our preliminary data demonstrate alterations in key molecules, and their precursors, known to coordinate proliferation and differentiation in lower organisms, as well as in human cancer. Following more extensive experiments to confirm and extend these data, including comparative responses to 1,25-dihydroxyvitamin D3 and sulindac, as well as in spontaneous differentiation along the absorptive or secretory cell lineage, we will determine the functions of key microRNAs in directly substituting for butyrate in triggering cellular and molecular aspects of colonic cell maturation, or in modulating the response to butyrate. As regards transcriptional attenuation, our published and submitted data were generated using a novel method of imaging of transcription sites in interphase nuclei to demonstrate that butyrate causes transcriptional attenuation in reducing steady state levels of the c-myc and cyclin D1 genes. Moreover, a genome wide scan using a novel "5'/3' array" that we designed and fabricated has provided evidence that this mechanism is more widely triggered by butyrate at loci throughout the genome. This will be confirmed and extended, and definitive evidence for transcriptional attenuation at candidate loci developed using a ChIP (chromatin immunoprecipitation) on chip approach. Our published and unpublished data have established that in inducing colonic cell maturation, the short chain fatty acid butyrate both alters the expression of microRNA molecules and causes transcriptional pausing at a number of genes. The goals of this application are: to dissect the affects of butyrate on these epigenetic mechanisms that reprogram the cell; to understand how they are linked to triggering and integrating pathways of cell proliferation, lineage specific differentiation, and apoptosis that comprise cell maturation; and compare and contrast the affects of butyrate with how vitamin D3 and the non-steroidal anti-inflammatory drug sulindac also induce maturation pathways through these epigenetic mechanisms. This will use novel methodologies and gene "chips" that we have developed. The results will define new mechanisms essential for the establishment of homeostasis of the intestinal mucosa, and how these are aberrant in altering probability for, and development of, colon cancer.