NF-kappa beta in mRNA Stability and Cell Differentiation

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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.