NF-kappa beta in mRNA Stability and Cell Differentiation

描述(申请人提供)：可诱导转录因子NF-kappaB是编码细胞因子、细胞因子受体、抗凋亡蛋白和细胞周期调节蛋白的基因表达的一种良好的正调控因子。令人惊讶的是，我们发现(Guttridge等人，Science 289,2363-2366)，NF-kappaB的表达导致编码MyoD的mRNA戏剧性和快速地丢失，MyoD是通过明显的转录后控制机制而调节肌肉发生的所谓“主开关”。因此，我们能够将MyoD的大约400个核苷酸区域转移到GAPDHmRNA上，这赋予了杂交mRNA核因子-(和细胞因子的敏感性。在新的数据中，我们已经确定MyoD mRNA编码区的133个核苷酸片段是NF-kappaB调节的RNA破坏途径的特异性靶点。在这133个核苷酸破坏片段中有两个拷贝的序列5‘-ACTACAGTG-3’和另一个拷贝的5-ATTACAGcG-3‘。在体外，转录的MyoD mRNA在肿瘤坏死因子处理的细胞提取物中降解，但在肿瘤坏死因子处理的细胞提取物中不降解，后者被阻断了核因子-kappaB的活性。有趣的是，我们发现另一个参与分化的关键转录因子Sox9的mRNA在细胞因子或核因子-kappaB表达时被靶向丢失。Sox9对软骨细胞的分化是必不可少的。重要的是，我们在Sox9mRNA中发现了与MyoD重复高度相关的一个元件的3个拷贝。这些序列中的一个重复序列的突变克服了依赖于NF-kappaB的丢失机制。现在已经发现了其他含有重复元件拷贝的mRNAs，并且有初步证据表明，在鳞状细胞癌中下调的一个基因集富含类似的RNA序列元件。该建议的目的是通过对依赖于NF-kappaB的mRNA衰退的分子表征和确定RNA序列和潜在的因子需求，来充分表征这种不寻常的依赖于NF-kappaB的转录后基因沉默过程。此外，我们建议通过分析可能的基因敲除模型、成骨分化模型以及癌细胞生长和分化模型来分析依赖于NF-kappaB的基因沉默机制的生理学和疾病相关性。这些实验将描述由核因子-kappaB控制的调控途径，它在转录后水平上抑制基因的表达，并可能通过抑制分化途径和控制重要的调控途径参与重要的生理和疾病特异性机制。

DESCRIPTION (provided by applicant): The inducible transcription factor NF-kappaB is a well-characterized positive regulator of expression of genes encoding cytokines, cytokine receptors, anti-apoptotic proteins and cell-cycle regulatory proteins. Surprisingly we found (Guttridge et al., Science 289, 2363-2366) that NF-kappaB expression leads to the dramatic and rapid loss of the mRNA encoding MyoD, the so-called "master switch" regulator of myogenesis through an apparent post-transcriptional control mechanism. Thus, we were able to transfer an approximate 400 nucleotide region of MyoD onto the GAPDH Mrna which conferred NF-(( and cytokine sensitivity on that hybrid mRNA. In new data, we have identified a 133 nucleotide fragment of the coding region of the MyoD mRNA as being a specific target of an NF-kappaB - regulated RNA destruction pathway. Within this 133 nucleotide destruction fragment are two copies of the sequence 5'- ACTACAGTG-3' and another of 5-AtTACAGcG-3'. In vitro transcribed MyoD mRNA is degraded in extracts of TNF-treated cells but not in extracts from TNF-treated cells, which are blocked for NF-kappaB activity. Interestingly, we find that the mRNA for another key transcription factor involved in differentiation, namely Sox9, is targeted for loss when cytokines or NF-kappaB is expressed. Sox9 is essential for the differentiation of chrondrocytes. Importantly, we find in the Sox9 mRNA 3 copies of an element highly related to the MyoD repeat. Mutation of one of these sequence repeats overcomes the NF-kappaB -dependent loss mechanism. Other mRNAs have now been identified which contain copies of the repeat element and preliminary evidence is presented that a gene set downregulated in squamous cell carcinoma is enriched in similar RNA sequence elements. The goals of this proposal are to fully characterize this unusual, NF-kappaB-dependent post-transcriptional gene silencing process through the molecular characterization of the NF-kappaB-dependent mRNA decay and by identifying RNA sequence and potential factor requirements. Additionally, we propose to analyze the physiological and disease relevance of the NF-kappaB-dependent gene silencing mechanism by analyzing possible gene knockout models, models for osteogenic differentiation, and models for cancer cell growth and differentiation. These experiments will characterize the regulatory pathway controlled by NF-kappaB, which functions to silence gene expression at the post-transcriptional level and which is likely to be involved in important physiological and disease specific mechanisms through suppression of differentiation pathways and control of important regulatory pathways.