The short chain fatty acid butyrate (NaB), present in high concentrations in the colonic lumen principally from the fermentation of dietary fiber, plays a physiological role in colonic homeostasis. This involves maintaining an equilibrium not only between cell proliferation in the lower section of the crypt and cell loss at the top of the crypt, but also a balance among the multiple cell lineages in the mucosa. However, butyrate effects are complex, in that butyrate induces differentiation in vitro, but promotes growth in vivo. This complexity is further emphasized by our findings demonstrating that NaB specifically represses that expression of the MUC2 gene, which encodes the major colonic mucin, and does so by inhibiting HDAC activity (Oncogene, in press). It is our hypothesis that the intestine has developed specific mechanisms to respond to natural compounds, such as NaB, present in the lumen to modulate cell differentiation as a function of cell position along the crypt axis. Alterations in this balance may increase the probability of tumor formation. Utilizing cell systems that recapitulate the goblet and ion transporter phenotypes, and molecular reagents developed in the lab, we will define the molecular determinants in cell lineage specific differentiation that can be regulated by NaB by determining: 1. Whether there are NaB responsive elements in the promoter of the human and mouse MUC2 genes. 2. Whether HDAC activity can counteract NaB mediated repression of MUC2 3. Whether there is modulation of SP1/SP3 activity, which we demonstrated is important for MUC2 regulation. 4. Whether post-translational modifications of SP1/SP3 may bring about loss of transcriptional activity. This will be done in vitro using standard immunological and molecular biology techniques. We will expand these studies in vivo, and determine, by CHIP analysis, if the chromatin structure of the gene is important for MUC2 expression. In addition, we will determine the dynamic occupancy of SP1/SP3 at functionally relevant sites, and the transcription factors and co-factors that may participate in the formation of multiprotein complexes upon interaction with SP1 and SP3 as a function of MUC2 repression. 5. Finally, we will extend our use of microarray technology to determine how NaB perturbs profiles of gene expression that define lineage specific differentiation (Velcich et al., submitted).
|Yang, Kan; Popova, Natalia V; Yang, Wan Cai et al. (2008) Interaction of Muc2 and Apc on Wnt signaling and in intestinal tumorigenesis: potential role of chronic inflammation. Cancer Res 68:7313-22|