Ed Grabczyk

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.

A persistent RNA·DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

Expansion of an unstable GAA·TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Deficiency of frataxin, an essential mitochondrial protein, leads to progressive neurodegeneration and cardiomyopathy. The degree of frataxin reduction correlates with GAA·TTC tract length, but the mechanism of reduction remains controversial. Here we show that transcription causes extensive RNA·DNA hybrid formation on GAA·TTC templates in bacteria as well as in defined transcription reactions using T7 RNA polymerase in vitro. RNA·DNA hybrids can also form to a lesser extent on smaller, so-called ‘pre-mutation’ size GAA·TTC repeats, that do not cause disease, but are prone to expansion. During in vitro transcription of longer repeats, T7 RNA polymerase arrests in the promoter distal end of the GAA·TTC tract and an extensive RNA·DNA hybrid is tightly linked to this arrest. RNA·DNA hybrid formation appears to be an intrinsic property of transcription through long GAA·TTC tracts. RNA·DNA hybrids have a potential role in GAA·TTC tract instability and in the mechanism underlying reduced frataxin mRNA levels in Friedreich Ataxia.

DNA repeat expansions and human disease.

Abstract. 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.

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.

Ferritin L and H Subunits Are Differentially Regulated on a Post-transcriptional Level

Ferritin plays an important role in the storage and release of iron, an element utilized in cellular processes such as respiration, gene regulation, and DNA replication and repair. Ferritin in animals is composed of 24 ferritin L (FTL) and ferritin H (FTH) subunits in ratios that vary in different cell types. Because the subunits are not functionally interchangeable, both L and H units are critical for maintaining iron homeostasis and protecting against iron overload. FTL and FTH are regulated primarily at a post-transcriptional level in response to cellular iron concentrations. Individual regulation of FTL and FTH is of much interest, and although transcriptional differences between FTL and FTH have been shown, differences in their post-transcriptional regulation have not been evaluated. We report here that FTL and FTH are differentially regulated in 1% oxygen on a post-transcriptional level. We have designed a quantitative assay system sensitive enough to detect differences between FTL and FTH iron regulatory elements (IREs) that a standard electrophoretic mobility shift assay does not. The FTL IRE is the primary responder in the presence of an iron donor in hypoxic conditions, and this response is reflected in endogenous FTL protein levels. These results provide evidence that FTL and FTH subunits respond independently to cellular iron concentrations and underscore the importance of evaluating FTL and FTH IREs separately.

Cloning and Characterization of the Rat Gene Encoding GAP-43

GAP‐43 is a gene expressed only in the nervous system. The protein product is believed to be important to neuronal growth and plasticity. Most, and likely all, neurons express high levels of GAP‐43 during periods of neurite elongation. To initiate studies of GAP‐43 gene regulation we have cloned the rat gene encoding GAP‐43. The GAP‐43 gene includes three exons. The first exon encodes only the amino terminal 10 amino acids, which corresponds to the membrane targeting domain of GAP‐43. The second exon encodes a putative calmodulin binding domain and a protein kinase C phosphorylation site. The 5′‐flanking sequence is unusual in that it lacks CAAT or TATA elements, and directs RNA transcription initiation from several sites. Some of the transcription start sites are used to a different degree in the central and peripheral nervous systems.

DNA Mismatch Repair Complex MutSβ Promotes GAA·TTC Repeat Expansion in Human Cells

Background: Friedreich ataxia (FRDA) is a progressive, debilitating and lethal disease caused by GAA·TTC repeat expansion. Results: Expression of mismatch repair complex MutSβ, particularly the MSH3 subunit, is necessary for GAA·TTC repeat expansion in model cells and FRDA patient fibroblasts. Conclusion: MutSβ promotes GAA·TTC expansion in FRDA. Significance: MSH3 may be a potential therapeutic target for slowing GAA·TTC expansion. While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future.

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

Background 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. Methodology/Principal Findings Size matched fragments containing the polyadenylation signal of the human β-actin gene (ACTB) and the human β-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. Conclusions/Significance 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.

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.

A dual reporter approach to quantify defects in messenger RNA processing.

Splicing and nuclear export are vital components of eukaryotic gene expression. Defects in splicing due to cis mutations are known to cause a number of human diseases. Here we present a dual reporter system that can be used to look at splicing or export deficiencies resulting from an insufficiency in components of the cotranscriptional machinery. The constructs use a bidirectional promoter to coexpress a test reporter and a control reporter. In the splicing construct, maximal expression of the test reporter is dependent on efficient splicing and splicing-related nuclear export, whereas the control reporter is an intronless complementary DNA expression cassette. The dual reporters allow a robust ratiometric output that is independent of cell number or transfection efficiency. Therefore, our construct is internally controlled and amenable to high-throughput analysis. As a counterscreen, we have a nonsplicing control construct in which neither reporter bears an intron. We demonstrate the sensitivity of our construct to defects in nuclear export by depleting UAP56 and NXF1, essential components of the cotranscriptional machinery.