Fragile X syndrome and Friedreich's ataxia: two different paradigms for repeat induced transcript insufficiency.

DNA repeat expansion is the genetic basis for a growing number of neurological disorders. While the largest subset of these diseases results in an increase in the length of a polyglutamine tract in the protein encoded by the affected gene, the most common form of inherited mental retardation, fragile X syndrome, and the most common inherited ataxia, Friedreich's ataxia, are both caused by expansions that are transcribed but not translated. These expansions both decrease expression of the gene in which the expanded repeat is located, but they do so by quite different mechanisms. In fragile X syndrome, CGG. CCG expansion in the 5' untranslated region of the FMR1 gene leads to hypermethylation of the repeats and the adjacent CpG-rich promoter. Methylation prevents the binding of the transcription factor alpha-Pal/NRF-1, and may indirectly affect the binding of other factors via the formation of transcriptionally silent chromatin. In Friedreich's ataxia, GAA. TTC expansion in an intron of the FRDA gene reduces expression by interfering with transcription elongation. The model that best describes the available data is transcription-driven formation of a transient purine. purine. pyrimidine DNA triplex behind an advancing RNA polymerase. This structure lassoes the RNA polymerase that caused it, trapping the enzyme on the template.

DNA repeat expansions and human disease.

The repeat expansion diseases are genetic disorders caused by intergenerational expansions of a specific tandem DNA repeat. These disorders range from mildly to severely debilitating or fatal, and all have limited treatment options. How expansion occurs and causes disease is only now beginning to be understood. Efforts to model expansion in mice have so far met with only limited success, perhaps due to a requirement for specific cis- or trans-acting factors. In vitro studies and data from bacteria and yeast suggest that in addition to secondary structures formed by the repeats, components of the DNA replication and recombination machinery are important determinants of instability. The consequences of expansion differ depending on where in the gene the repeat tract is located, and range from reduction of transcription initiation to protein toxicity. Recent advances are beginning to make rational approaches to the development of therapies possible.

The GAA*TTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner.

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.

Alleviating transcript insufficiency caused by Friedreich's ataxia triplet repeats.

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.

Long uninterrupted CGG repeats within the first exon of the human FMR1 gene are not intrinsically unstable in transgenic mice.

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 long purine-pyrimidine homopolymer acts as a transcriptional diode.

Polypurine-polypyrimidine (R.Y) sequences have the unusual ability to form DNA triple helices. Such tracts are overrepresented upstream of eukaryotic genes, although a function there has not been clear. We report that transcription in vitro into one such upstream R.Y tract in the direction that makes a predominantly purine RNA is effectively blocked by formation of an intramolecular triple helix. The triplex is triggered by transcription and stabilized by the binding of nascent purine RNA to the template. Transcription in the opposite direction is not restricted. Polypurine-polypyrimidine DNA may provide a dynamic and selective block to transcription without the aid of accessory proteins.

GAP-43 transgenic mice: dispersed genomic sequences confer a GAP-43-like expression pattern during development and regeneration.

Using transgenic mice, we have examined the expression pattern conferred by regions of genomic GAP-43 coupled to beta-galactosidase. We demonstrate that gene constructions that include the GAP-43 5'-flanking region along with sufficient sequences of the first intron drive beta-galactosidase (lacZ) expression to mimic in many regards the complex spatial and temporal pattern of endogenous GAP-43 expression. Transgene expression reaches peak levels during development, and persists at high levels in particular adult brain regions, such as the hippocampus and olfactory bulb. The inclusion of a stretch of the first intron in the construction is necessary to prevent expression outside of the nervous system, indicating that some of the cell specificity of GAP-43 expression is due to suppression of expression in inappropriate tissues. Injury caused by sciatic nerve crush causes reexpression of the transgene in adult sensory and motor neurons. This genomic region of GAP-43, therefore, includes elements responsive to neuronal growth signals that regulate both development and regeneration.

GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system.

GAP-43 is a rapidly transported axonal protein most prominently expressed in regenerating and developing nerves. However, the low level persistence of GAP-43 in the adult CNS where growth and regenerative capacity are minimal may additionally indicate a role for this molecule in neuronal remodeling. Previous studies have revealed GAP-43 immunoreactivity in neurites throughout many regions of the CNS. To identify the CNS neurons that express GAP-43 at different stages of development, we utilized in situ hybridization and immunocytochemistry; the latter was performed with an antibody that recognizes GAP-43 immunoreactivity in both perikarya and neurites. In the perinatal period GAP-43 is expressed in all neurons. Subsequently its expression becomes progressively restricted such that by maturity most neurons no longer express detectable levels, although GAP-43 expression is still moderately high in the adult entorhinal cortex, and strikingly high in the adult hippocampus and olfactory bulb. In light of current notions about the function of GAP-43, it is tempting to speculate that this anatomy denotes neurons engaged in structural remodeling and functional plasticity.

Dual regulation of GAP-43 gene expression by nerve growth factor and glucocorticoids.

GAP-43 is a neural-specific protein that is believed integral to neurite growth and to the plasticity of neuronal structure. Its gene expression is regulated in vivo and correlates with periods of axonal growth. We investigated the regulation of GAP-43 gene expression in PC12 cells, which are believed to resemble precursor cells of the adrenomedullary lineage. In these cells, nerve growth factor (NGF) increases GAP-43 expression, and corticosteroids decrease it. Corticosteroids diminish GAP-43 levels even in cells already differentiated by NGF, as well as in primary neurons of the superior cervical ganglion. Neither the NGF nor the steroid effect requires new protein synthesis. Nuclear run-on experiments show that the steroid repression is mediated at the level of gene transcription but that the NGF effect is likely to be posttranscriptional. NGF and corticosteroids are known to regulate bimodally the cell fate decision of sympathoadrenal precursors, with NGF promoting the neuronal phenotype and steroids promoting the chromaffin phenotype. The regulation of GAP-43 is consistent with the notion that this gene is bimodally regulated during these cell fate decisions.