Colon Cell Differentiation: NAB and MUC2 Gene Expression

描述（由申请人提供）：短链脂肪酸丁酸盐（NaB）主要来自膳食纤维的发酵，以高浓度存在于结肠腔中，在结肠稳态中起生理作用。这不仅涉及维持隐窝下部的细胞增殖和隐窝顶部的细胞损失之间的平衡，而且涉及维持粘膜中多种细胞谱系之间的平衡。然而，丁酸盐的作用是复杂的，因为丁酸盐在体外诱导分化，但在体内促进生长。我们的发现进一步强调了这种复杂性，表明NaB特异性地抑制MUC 2基因的表达，MUC 2基因编码主要的结肠粘蛋白，并且通过抑制HDAC活性来这样做（Oncogene，出版中）。 我们的假设是，肠已经发展出特定的机制来响应存在于管腔中的天然化合物，如NaB，以调节细胞分化，作为细胞沿着隐窝轴沿着位置的函数。这种平衡的改变可能会增加肿瘤形成的可能性。 利用重现杯状和离子转运蛋白表型的细胞系统，以及实验室开发的分子试剂，我们将通过确定以下参数来定义细胞谱系特异性分化中的分子决定因素： 1.人和小鼠MUC 2基因启动子中是否存在NaB应答元件。 2. HDAC活性是否可以抵消NaB介导的MUC 2抑制 3.是否存在SP1/SP3活性的调节，我们证明这对MUC 2调节很重要。 4. SP1/SP3的翻译后修饰是否会导致转录活性的丧失。这将使用标准免疫学和分子生物学技术在体外完成。我们将在体内扩展这些研究，并通过CHIP分析确定基因的染色质结构是否对MUC 2表达重要。此外，我们将确定SP1/SP3在功能相关位点的动态占有率，以及可能参与与SP1和SP3相互作用后形成多蛋白复合物的转录因子和辅因子，作为MUC 2抑制的功能。 5.最后，我们将扩展我们对微阵列技术的使用，以确定NaB如何干扰定义谱系特异性分化的基因表达谱（Velcich等人，提交）。

DESCRIPTION (provided by applicant): 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).