The objective of the proposed research is to identify the molecular basis for the aberrant epigenetic reprogramming that leads to fragile X syndrome (FXS). FXS is caused by an expanded CGG trinucleotide repeat in the 5? untranslated region (5?UTR) of the fragile X mental retardation protein (FMRP) gene, FMR1. Alleles containing >200 copies of the CGG repeat are associated with heterochromatinization of the FMR1 promoter and the subsequent loss of FMR1 transcription and FMRP expression. FMR1 silencing occurs early in fetal development, which has prevented the development of an in vitro paradigm for dissecting the molecular pathways involved in FMR1 gene silencing. Recently, a study showed that FXS human embryonic stem cells (HESC) contain unmethylated FMR1 promoters, which become methylated upon differentiation, providing the first in vitro culture model of FMR1 silencing. This proposal tests the role of small RNAs in FMR1 silencing in FXS. CGG repeat RNAs are processed into small RNAs in vitro by the RNA interference enzyme Dicer. Recent studies have shown that small RNAs directed against mRNA transcripts or gene promoters can result in the silencing of promoters in mammalian cells. Small RNAs also have critical roles in mediating silencing of mammalian retrotransposon genes, which, like CGG repeat sequences, are often characterized by repeated nucleotide elements. These studies raise the possibility that small RNA pathways may be involved in FMR1 silencing in FXS. The goal of the research described in this proposal is to use this new model system to identify, for the first time, the pathways that mediate FMR1 promoter silencing in FXS and to determine the role of small RNAs in FMR1 silencing.
The specific aims of this proposal are: (1) To determine the role of RNA interference pathways in FMR1 silencing in FXS. Using wild-type and FXS HESC lines, we will determine if the FMR1 promoter acquires characteristic chromatin marks that are associated with RNA-directed promoter silencing. We will also determine if the pathways required for microRNA and PIWI RNA processing are necessary for FMR1 silencing;and (2) We will determine how the FMR1 transcript is processed in FXS HESC. We will monitor the processing of the endogenous FMR1 full repeat transcript as well as heterologously expressed CGG repeat-containing transcripts, to determine whether CGG repeat RNAs generate small RNAs during FMR1 gene silencing. The advent of FXS stem cells provides the first opportunity to molecularly dissect the signaling pathways that lead to the aberrant epigenetic silencing that causes this highly prevalent mental retardation syndrome. The use of stem cells in this high risk/high reward R21 project will provide new insight into the molecular components involved in FMR1 silencing and will allow us to address a link between FXS, trinucleotide repeat disorders, and the emerging area of small RNA-directed gene silencing.
The advent of FXS stem cells provides the first opportunity to molecularly dissect the signaling pathways that lead to the aberrant epigenetic silencing that causes this highly prevalent mental retardation syndrome. The use of stem cells in this pilot project will provide new insight into the molecular components involved in FMR1 silencing and will allow us to address a link between FXS, trinucleotide repeat disorders, and the emerging area of RNA-directed gene silencing.
| Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S et al. (2014) Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome. Science 343:1002-5 |
