Fragile X syndrome is the most common form of inherited mental retardation. It results from loss of function of the FMR1 gene product. Virtually all cases are due to the specific transcriptional silencing of FMR1 -- hence, the syndrome can be considered a transcriptional disease. Silencing is an indirect consequence of the expansion to more than 200 copies, of a CGG repeat (normal modal size of 30) in FMR1's 5'-untranslated region. Expansion results in the aberrant de novo methylation of cytosines both in CG dinucleotides within the repeat, and in the neighboring upstream CpG islands in the FMR1 promoter. Hypermethylation is thought to be a proximal cause of the transcriptional inactivity of FMR1 in patients. However the molecular mechanism by which methylation leads to loss of transcription of FMR1 is unknown. Patient FMR1 is packaged in hypoacetylated histones H3 and H4, another characteristic of transcriptionally silent loci. Drugs that reverse the methylation state of FMR1 DNA result in a relative hyperacetylation of histones H3 and H4 at FMR1 and restore transcription. A current model of methylation-mediated gene repression suggests that recruitment of histone deacetylases to the promoter via methyl cytosine binding proteins establish a closed chromatin environment at mutant FMR1. The long range objective of this project is to understand the molecular mechanism of transcriptional silencing of patient FMR1, and to ultimately reverse it.
Aim 1 will characterize transcription factors, including histone acetyltransferase activities, that drive normal FMR1 promoter function.
Aim 2 will identify repression complexes that bind methylated DNA and deacetylate histones in mutant, expanded FMR1 alleles.
Aim 3 will test the idea that histone acetylation is important for FMR1 transcription by exogenously expressing histone acetyltransferase and directing it to the mutant, methylated FMR1 promoter. Promoter occupancy will be evaluated in patient cells with intermediate states of remodeled chromatin. Results obtained from this experimental system will allow identification of the steps involved in transcription of a clinically important gene residing in its natural chromosomal context.
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