Fragile X (CGG)n repeats induce a transcriptional repression in cis upon a linked promoter: Evidence for a chromatin mediated effect

In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte. We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length. Using a kinetic approach, we show that this transcriptional repression is concomitant with chromatin maturation and, using in vitro transcription, we show that chromatin formation is a fundamental part of the repressive pathway mediated by (CGG)n repeats. Using Trichostatin A, a histone deacetylase inhibitor, we show reactivation of the silenced promoter.Thus, isolated fragile X associated (CGG)n repeat arrays can exert a modifying and transcriptionally repressive influence over adjacent promoters and this repressive phenomenon is, in part, mediated by histone deacetylation.Expansion of a genetically unstable (CGG)n triplet repeat was first observed within the promoter region of the human FMR1 gene in fragile X families (reviewed in refs [1,2]). In the normal population, the repeat is genetically stable and ranges from 6 to 54 triplets in length whereas in fragile X families it ranges from 55 to over several thousands of triplets in length [3]. Expansion to over 200 repeats is associated with loss of FMR1 gene transcription [4], hypermethylation [5-7], late replication [8] and the expression of a fragile site on the X chromosome. Loss of the encoded protein, FMRP, results in altered synaptic maturation (reviewed in ref [9] which leads to development of the fragile X syndrome. Similar expansions of (CGG)n have also been identified at other rare, folate sensitive fragile sites, including those at FRAXE [10], FRAXF [11,12], FRA11B [13] and FRA16A [14]. In all cases, (CGG)n expansion is associated with a length dependent localised hypermethylation a