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，在结肠腔中以高浓度存在，在结肠内稳态中起着生理作用。这不仅需要在隐窝下部的细胞增殖和隐窝顶部的细胞丢失之间保持平衡，而且还需要在粘膜中的多种细胞谱系之间保持平衡。然而，丁酸的作用是复杂的，因为丁酸在体外诱导分化，但在体内促进生长。我们的发现进一步强调了这种复杂性，我们的发现表明，NAB特异性地抑制MUC2基因的表达，MUC2基因编码主要的结肠粘蛋白，并通过抑制HDAC活性(癌基因，在印刷中)来实现这一点。 我们的假设是，肠道已经形成了特定的机制来对管腔中存在的天然化合物(如NAB)做出反应，以调节细胞分化，作为细胞沿隐窝轴的位置的函数。这种平衡的改变可能会增加肿瘤形成的可能性。 利用概括杯型和离子转运体表型的细胞系统，以及实验室开发的分子试剂，我们将定义细胞谱系特异性分化中的分子决定因素，这些决定因素可以通过确定： 1.人和小鼠MUC2基因启动子中是否存在NAB反应元件 2.HDAC活性能否拮抗NAB介导的MUC2抑制 3.是否存在对SP1/SP3活性的调节，这一点对MUC2的调节是重要的。 4.SP1/SP3的翻译后修饰是否会导致转录活性丧失。这将使用标准的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，并通过芯片分析确定基因的染色质结构是否对MUC2的表达重要。此外，我们还将确定SP1/SP3在功能相关位点的动态占有率，以及作为MUC2抑制功能的SP1和SP3相互作用时可能参与多蛋白复合体形成的转录因子和辅助因子。 5.最后，我们将扩大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).