Over-expression of TNF alpha is almost always deleterious. Over-expression in mice is associated with both systemic autoimmunity and organ-specific inflammatory changes. Over-expression is seen in humans with a wide range of inflammatory disorders. To prevent the deleterious consequences of over-expression, TNF alpha appears to use both transcriptional silencing and translational silencing. The hypothesis addressed in the current proposal is that transcriptional repression of TNF alpha is mediated through a combination of DNA methylation, histone deacetylation and nuclear localization effects. In monocytes, these transcriptional repression strategies are hypothesized to be regulated by maturational stimuli. Our preliminary data demonstrates that progressive histone acetylation accompanies monocyte differentiation and that enforced histone acetylation can allow a non-expressing cell to express TNF alpha. These data strongly suggest that histone acetylation plays an important role in establishing transcriptional competency as monocytes mature. It is unknown at this time whether histone acetylation affects DNA methylation or vice versa. This proposal will address the hypothesis that DNA methylation, histone acetylation, and location with euchromatin are variables, which are regulated independently and govern the competence of the TNF alpha locus for transcription. To define the roles of these three variables, we will first analyze factors that govern the nuclear localization of the TNF alpha locus. Maturation, stimulation, and manipulation of DNA methylation and histone acetylation will be evaluated as variables regulating nuclear localization. The role of DNA methylation in the regulation of TNF alpha expression will be examined in the second aim. Bisulfite analysis will be used to define methylation patterns in monocytes at different maturational stages and the relationship between DNA methylation and histone acetylation examined using specific inhibitors. In the third aim, chromatin immunoprecipitation assays will be used to identify the presence of absence of transcription factors residing on inactive and active promoters. A candidate repressor protein, GCF2, identified through the work done in the previous application cycle will be examined for effects on transcription, histone acetylation, and DNA methylation. Overall, this proposal will define the mechanisms underlying repression of the TNF alpha locus. ? ?
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