Search

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and the most common known cause of autism. Most cases of FXS result from the expansion of a CGG·CCG repeat in the 5' UTR of the FMR1 gene that leads to gene silencing. It has previously been shown that silenced alleles are associated with histone H3 dimethylated at lysine 9 (H3K9Me2) and H3 trimethylated at lysine 27 (H3K27Me3), modified histones typical of developmentally repressed genes. We show here that these alleles are also associated with elevated levels of histone H3 trimethylated at lysine 9 (H3K9Me3) and histone H4 trimethylated at lysine 20 (H4K20Me3). All four of these modified histones are present on exon 1 of silenced alleles at levels comparable to that seen on pericentric heterochromatin. The two groups of histone modifications show a different distribution on fragile X alleles: H3K9Me2 and H3K27Me3 have a broad distribution, whereas H3K9Me3 and H4K20Me3 have a more focal distribution with the highest level of these marks being present in the vicinity of the repeat. This suggests that the trigger for gene silencing may be local to the repeat itself and perhaps involves a mechanism similar to that involved in the formation of pericentric heterochromatin.

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)22 forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)9AGG (CGG)12AGG(CGG)97, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3' part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.

Expanded GAA.TTC trinucleotide repeats in intron 1 of the frataxin gene cause Friedreich's ataxia (FRDA) by reducing frataxin mRNA levels. Insufficient frataxin, a nuclear encoded mitochondrial protein, leads to the progressive neurodegeneration and cardiomyopathy characteristic of FRDA. Previously we demonstrated that long GAA.TTC tracts impede transcription elongation in vitro and provided evidence that the impediment results from an intramolecular purine.purine.pyrimidine DNA triplex formed behind an advancing RNA polymerase. Our model predicts that inhibiting formation of this triplex during transcription will increase successful elongation through GAA.TTC tracts. Here we show that this is the case. Oligodeoxyribonucleotides designed to block particular types of triplex formation provide specific and concentration-dependent increases in full-length transcript. In principle, therapeutic agents that selectively interfere with triplex formation could alleviate the frataxin transcript insufficiency caused by pathogenic FRDA alleles.

Large expansions of the trinucleotide repeat GAA*TTC within the first intron of the X25 (frataxin) gene cause Friedreich's ataxia, the most common inherited ataxia. Expansion leads to reduced levels of frataxin mRNA in affected individuals. Here we show that GAA*TTC tracts, in the absence of any other frataxin gene sequences, can reduce the amount of GAA-containing transcript produced in a defined in vitro transcription system. This effect is due to an impediment to elongation that forms in the GAA*TTC tract during transcription, a phenomenon that is exacerbated by both superhelical stress and increased tract length. On supercoiled templates the major truncations of the GAA-containing transcripts occur in the distal (3') end of the GAA repeat. To account for these observations we present a model in which an RNA polymerase advancing within a long GAA*TTC tract initiates the transient formation of an R*R*Y intramolecular DNA triplex. The non-template (GAA) strand folds back creating a loop in the template strand, and the polymerase is paused at the distal triplex-duplex junction.

Tandem repeats of certain trinucleotides show extensive intergenerational instability in humans that is associated with a class of genetic disorders known as the Triplet Expansion Diseases. This instability is thought to be a consequence of the formation of intrastrand structures, including hairpins, triplexes and tetraplexes, by the tandem repeats. I show here that CGG-repeats which are associated with this group of diseases, and AGG- and TGG-repeats which are not currently known to be, form several intrastrand structures including tetraplexes. In all cases the tetraplexes have the same overall conformation in which all the G residues are involved in G4-tetrads. CGG-repeats also form stable hairpins, but AGG- and TGG-repeats do not form hairpins of comparable stability. However, since tetraplexes can be thought of as folded hairpins, many of the properties ascribed to disease-associated triplets that form hairpins, may apply to these sequences as well. The fact that AGG- and TGG-repeats are not currently associated with any triplet expansion disease suggests either that the ability to adopt an intrastrand folded structure is not sufficient for expansion, or that other diseases associated with such triplets might remain to be identified.

Despite the increasing number of disorders known to result from trinucleotide repeat amplification, the molecular mechanism underlying these dynamic mutations is still unknown. In an attempt to create a mouse model for the CGG repeat instability seen in Fragile X syndrome, we constructed transgenes corresponding to FMR1 premutation alleles. While in humans these alleles would expand to full mutation with almost 100% certainty upon maternal transmission, they remain stable in our transgenic mice. Therefore, the presence of a large number of uninterrupted CGGs is not sufficient to cause instability in mice, even in the context of flanking human FMR1 sequences.

A large increase in the length of a CGG tandem array is associated with a number of triplet expansion diseases, including fragile X syndrome, the most common cause of heritable mental retardation in humans. Expansion results in the appearance of a fragile site on the X chromosome in the region of the CGG array. We show here that CGG repeats readily form a series of barriers to DNA synthesis in vitro. There barriers form only when the (CGG)n strand is used as the template, are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines. Chemical modification experiments suggest these blocks to DNA synthesis result from the formation of a series of intrastrand tetraplexes. A number of lines of evidence suggest that both triplet expansion and chromosome fragility are the result of replication defects. Our data are discussed in the light of such evidence.