Transposon Tn7 Preferentially Inserts into GAA•TTC Triplet Repeats under Conditions Conducive to Y•R•Y Triplex Formation

BACKGROUND: Expansion of an unstable GAA*TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Structure formation by the repeat has been implicated in both frataxin repression and GAA*TTC instability. The GAA*TTC sequence is capable of adopting multiple non-B DNA structures including Y*R*Y and R*R*Y triplexes. Lower pH promotes the formation of Y*R*Y triplexes by GAA*TTC. Here we used the bacterial transposon Tn7 as an in vitro tool to probe whether GAA*TTC repeats can attract a well-characterized recombinase. METHODOLOGY/PRINCIPAL FINDINGS: Tn7 showed a pH-dependent preference for insertion into uninterrupted regions of a Friedreich ataxia patient-derived repeat, inserting 48, 39 and 14 percent of the time at pH 7, pH 8 and pH 9, respectively. Moreover, Tn7 also showed orientation and region specific insertion within the repeat at pH 7 and pH 8, but not at pH 9. In contrast, transposon Tn5 showed no strong preference for or against the repeat during in vitro transposition at any pH tested. Y*R*Y triplex formation was reduced in predictable ways by transposon interruption of the GAA*TTC repeat. However, transposon interruptions in the GAA*TTC repeats did not increase the in vitro transcription efficiency of the templates. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that transposon Tn7 will recognize structures that form spontaneously in GAA*TTC repeats and insert in a specific orientation within the repeat. The conditions used for in vitro transposition span the physiologically relevant range suggesting that long GAA*TTC repeats can form triplex structures in vivo, attracting enzymes involved in DNA repair, recombination and chromatin modification

Progressive GAA·TTC Repeat Expansion in Human Cell Lines

Trinucleotide repeat expansion is the genetic basis for a sizeable group of inherited neurological and neuromuscular disorders. Friedreich ataxia (FRDA) is a relentlessly progressive neurodegenerative disorder caused by GAA·TTC repeat expansion in the first intron of the FXN gene. The expanded repeat reduces FXN mRNA expression and the length of the repeat tract is proportional to disease severity. Somatic expansion of the GAA·TTC repeat sequence in disease-relevant tissues is thought to contribute to the progression of disease severity during patient aging. Previous models of GAA·TTC instability have not been able to produce substantial levels of expansion within an experimentally useful time frame, which has limited our understanding of the molecular basis for this expansion. Here, we present a novel model for studying GAA·TTC expansion in human cells. In our model system, uninterrupted GAA·TTC repeat sequences display high levels of genomic instability, with an overall tendency towards progressive expansion. Using this model, we characterize the relationship between repeat length and expansion. We identify the interval between 88 and 176 repeats as being an important length threshold where expansion rates dramatically increase. We show that expansion levels are affected by both the purity and orientation of the repeat tract within the genomic context. We further demonstrate that GAA·TTC expansion in our model is independent of cell division. Using unique reporter constructs, we identify transcription through the repeat tract as a major contributor to GAA·TTC expansion. Our findings provide novel insight into the mechanisms responsible for GAA·TTC expansion in human cells

A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells.

Making the correct choice between transcription elongation and transcription termination is essential to the function of RNA polymerase II, and fundamental to gene expression. This choice can be influenced by factors modifying the transcription complex, factors modifying chromatin, or signals mediated by the template or transcript. To aid in the study of transcription elongation and termination we have developed a transcription elongation reporter system that consists of tandem luciferase reporters flanking a test sequence of interest. The ratio of expression from the reporters provides a measure of the relative rates of successful elongation through the intervening sequence.Size matched fragments containing the polyadenylation signal of the human beta-actin gene (ACTB) and the human beta-globin gene (HBB) were evaluated for transcription termination using this new ratiometric tandem reporter assay. Constructs bearing just 200 base pairs on either side of the consensus poly(A) addition site terminated 98% and 86% of transcription for ACTB and HBB sequences, respectively. The nearly 10-fold difference in read-through transcription between the two short poly(A) regions was eclipsed when additional downstream poly(A) sequence was included for each gene. Both poly(A) regions proved very effective at termination when 1100 base pairs were included, stopping 99.6% of transcription. To determine if part of the increased termination was simply due to the increased template length, we inserted several kilobases of heterologous coding sequence downstream of each poly(A) region test fragment. Unexpectedly, the additional length reduced the effectiveness of termination of HBB sequences 2-fold and of ACTB sequences 3- to 5-fold.The tandem construct provides a sensitive measure of transcription termination in human cells. Decreased Xrn2 or Senataxin levels produced only a modest release from termination. Our data support overlap in allosteric and torpedo mechanisms of transcription termination and suggest that efficient termination is ensured by redundancy

Interrupting the (GAA•TTC)₁₀₈ repeat with a transposon does not improve transcription <i>in vitro</i>.

<p>The strategy used for <i>in vitro</i> transcription of linear and supercoiled templates is summarized in the schematic at the top. Immediately below that is the image of products of <i>in vitro</i> transcription of linear and of supercoiled plasmids visualized by hybridization with a biotinylated ODN probe followed by chemiluminescence. The templates are as follows: Lane 1∶9 repeats, no transposon. Lane 2∶108 repeats, no transposon. Lane 3∶9 repeats, Tn5 transposon 5′ to the first GAA. Lane 4∶108 repeats, transposon Tn5 86 bp 5′ of the first GAA. Lane 5∶108 repeats, Tn5 transposon at repeat 8. Lane 6∶108 repeats, Tn5 transposon at repeat 57. The intensity of the bands representing <i>in vitro</i> transcription was quantitated with Kodak Molecular Imaging software. Bars represent mean amount of transcription for three independent experiments. A representative blot is shown.</p

Experimental Target.

<p>Plasmid targets for <i>in vitro</i> transposition reactions contained 108 GAA•TTC repeats. Hollow boxes indicate essential regions (ori, <i>bla</i>) of the plasmid excluded from recoverable transposition events. The expected frequency of insertion into the (GAA•TTC)₁₀₈ region based on its contribution to the size of the plasmid (minus the essential regions) is 9%. The repeat is located between restriction sites MluI and XmaI. The repeat is divided into thirds, and point deletions and substitutions in the GAA repeat are indicated by open circles and asterisks, respectively. The sequence of this repeat, divided into thirds, is shown at the bottom of the figure. The complete sequence of the insert is available online (GU722204).</p

The location and orientation of Tn7 insertion within the GAA•TTC repeat is pH dependent.

<p>Depictions of the MluI to XbaI fragment containing the GAA•TTC repeat with vertical lines indicating Tn7 insertion events at pH 7, pH 8 and pH 9 both within and near the repeat. The repeat is divided into thirds, and point deletions and substitutions in the GAA repeat are indicated by open circles and asterisks, respectively. Lines extending above represent insertions events in the (+) orientation, defined as the transposon oriented from left to right. Lines extending below represent insertion events in the (−) orientation with the transposon oriented right to left. Insertions performed at pH 7 and pH 8 show a strong bias for the left to right (+) orientation of the transposon. At pH 7.0 and pH 8.0, Tn7 shows a bias for insertion within the largely uninterrupted regions 1 and 2. In contrast, region 3, an area where the GAA•TTC repeat contains multiple interruptions, is not a favored target for insertion. Location of insertion was determined by DNA sequencing.</p