Fragile X (CGG)n repeats induce a transcriptional repression in cis upon a linked promoter: evidence for a chromatin mediated effect.
BACKGROUND
Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the human FMR1 gene leads to extensive local methylation and transcription silencing, resulting in the loss of FMRP protein and the development of the clinical features of fragile X syndrome. The causative link between (CGG)n expansion, methylation and gene silencing is unknown, although gene silencing is associated with extensive changes to local chromatin architecture.


RESULTS
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.


CONCLUSIONS
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.
Chandler
Simon P
SP
Sangamo BioSciences, 501 Canal Blvd, Ste A100, Point Richmond Tech Center II, Richmond, CA 94804, USA. schandler@sangamo.com
Kansagra
Pushpa
P
Hirst
Mark C
MC
D000818:Animals; D002843:Chromatin / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D003596:Cytosine / Q000378:metabolism / Q000502:physiology*; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005600:Fragile X Syndrome / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D008040:Genetic Linkage / Q000235:genetics*; D006147:Guanine / Q000378:metabolism / Q000502:physiology*; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000502:physiology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D009865:Oocytes; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D019680:Trinucleotide Repeat Expansion / Q000235:genetics / Q000502:physiology*; D015678:Viral Structural Proteins / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics
D002843:Chromatin; D004791:Enzyme Inhibitors; C096916:FXR1 protein, human; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D016601:RNA-Binding Proteins; D015678:Viral Structural Proteins; C012589:trichostatin A; D006147:Guanine; D003596:Cytosine; D013937:Thymidine Kinase; D006655:Histone Deacetylases