application) Proper regulation of the cell cycle is a critical component of the normal development and differentiation of mammalian tissues, as well as their response to stress and injury, and the prevention of neoplasia. The cell cycle inhibitor, p21, is a major regulator of cellular proliferation and is specifically expressed in a variety of postmitotic, differentiated cells. In the murine gut, abundant p21 mRNA is present in the differentiated villus compartment, but not the crypts. In addition, its expression is markedly decreased or nonexistent in colon cancers. It is likely, therefore, that p21 plays an important role in the process [sic] intestinal epithelial cell differentiation as well as the prevention of colon carcinogenesis. However, the mechanisms which govern p21 activation in these contexts have not been previously addressed. The broad intent of this project is to examine the mechanisms governing the activation of the human p21 gene by butyrate, a product of fiber fermentation in the colon which both induces growth arrest and differentiation of colon cancer cells. Preliminary data indicate that sodium butyrate (NaBu) causes growth arrest through induction of p21 expression. p21 induction appears to occur at the level of gene transcription, and is mediated by one or more cis-element(s) between -291 and -93 bp upstream of the 5' flanking region. Changes in chromatin structure by histone hyperacetylation appears to be an important mechanism in its induction. The proposed studies will further define the molecular mechanisms underlying p21 activation by butyrate. Functional studies, using stable transfections with p21 reporter constructs, will allow more precise identification of the cis-element(s) mediating induction of p21 by NaBu in the context of the natural chromatin environment. Gel shift analyses will be performed in order to identify and characterize the specific sites of DNA-protein interactions responsible for p21 regulation. With this information, it may be possible to identify candidate, previously characterized transcription factor(s) which mediate p21 induction. If, however, a novel transcription factor appears to be involved, it will be cloned using the yeast one-hybrid technique. In addition, we will further examine the importance of histone hyperacetylation in p21 gene induction using the chromatin immunoprecipitation (ChIP) assay, a new technique which is able to assess the acetylation state of the historic proteins surrounding specific segments of the p21 gene regulatory region. These studies should enhance our knowledge of the fundamental aspects of p21 regulation by butyrate and will impact on our understanding of the processes of normal and deregulated intestinal epithelial cell growth involved in colon carcinogenesis.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Small Research Grants (R03)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Podskalny, Judith M,
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Beth Israel Deaconess Medical Center
United States
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Archer, Sonia Y; Johnson, Jennifer; Kim, Hyun-Ju et al. (2005) The histone deacetylase inhibitor butyrate downregulates cyclin B1 gene expression via a p21/WAF-1-dependent mechanism in human colon cancer cells. Am J Physiol Gastrointest Liver Physiol 289:G696-703