Gene-specific trinucleotide repeat expansions are the cause of an ever-growing number of disorders, including myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 7 (SCA7). Both DM1, and SCA7 are characterized by large differences in repeat numbers between tissues that are differentially affected, indicating tissue-specific mechanisms of repeat instability. The mechanism(s) of both somatic as well as germline instability are complex and still poorly understood, with evidence supporting the contribution of cis-elements, trans factors, and DNA metabolic processes that are hypothesized to involve alternative structure formation within the DNA tract. This thesis involves investigations into the role of a particular cis-element (CTCF) on instability, as well as the detection of slipped-DNAs in patient tissues and the presence of rare mutations within those same tissues. Here I identify the first endogenous cis-element reported to show regulation of instability at a trinucleotide repeat disease locus, the DNA binding site for the insulator protein CCCTC- binding factor (CTCF) downstream of the SCA7 repeat. Using a mouse model with a mutation in the CTCF binding domain, I show that the loss of CTCF binding stimulates germline and somatic instability in a tissue-specific and age-dependent manner. The binding of CTCF likely protects the repeat tract from expansion by shielding it from other elements that may contribute to expansion. DNA metabolic processes such as replication, repair, and transcription likely play a role in repeat expansion at disease loci, with the general mechanism hypothesized to be the extrusion and aberrant repair of slipped-DNA structures during the unwinding process for each. While characterizing DM1 patient tissues in order to isolate slipped-DNA structures, I characterized two non-CTG repeat insertion mutations that had completely replaced the repeat tract in a small subset of cells in only two tissues in one patient. Given the hypermutable nature of expanded repeat tracts, it is possible that these types of mutations are more common than suspected. Finally, I report on the detection and isolation of slipped-DNA structures from the endogenous DM1 locus from patient tissues. The slip-outs appear as clusters along a length of DNA, rather than single isolated slip-outs, and more unstable tissues contain greater amounts of slipped-DNA compared to more stable tissues. This detection implies that slipped-DNA structures are not merely transient intermediates in the mutation and expansion process as has long been assumed, but remain within the DNA at detectable levels. The data reported herein both furthers our understanding of trinucleotide repeat instability, and additionally confirms the decades-long hypothesis that slipped-DNAs are in fact forming in patient tissues in a tissue-specific manner.

Gene-specific trinucleotide repeat expansions are the cause of an ever-growing number of disorders, including myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 7 (SCA7). Both DM1, and SCA7 are characterized by large differences in repeat numbers between tissues that are differentially affected, indicating tissue-specific mechanisms of repeat instability. The mechanism(s) of both somatic as well as germline instability are complex and still poorly understood, with evidence supporting the contribution of cis-elements, trans factors, and DNA metabolic processes that are hypothesized to involve alternative structure formation within the DNA tract. This thesis involves investigations into the role of a particular cis-element (CTCF) on instability, as well as the detection of slipped-DNAs in patient tissues and the presence of rare mutations within those same tissues. Here I identify the first endogenous cis-element reported to show regulation of instability at a trinucleotide repeat disease locus, the DNA binding site for the insulator protein CCCTC- binding factor (CTCF) downstream of the SCA7 repeat. Using a mouse model with a mutation in the CTCF binding domain, I show that the loss of CTCF binding stimulates germline and somatic instability in a tissue-specific and age-dependent manner. The binding of CTCF likely protects the repeat tract from expansion by shielding it from other elements that may contribute to expansion. DNA metabolic processes such as replication, repair, and transcription likely play a role in repeat expansion at disease loci, with the general mechanism hypothesized to be the extrusion and aberrant repair of slipped-DNA structures during the unwinding process for each. While characterizing DM1 patient tissues in order to isolate slipped-DNA structures, I characterized two non-CTG repeat insertion mutations that had completely replaced the repeat tract in a small subset of cells in only two tissues in one patient. Given the hypermutable nature of expanded repeat tracts, it is possible that these types of mutations are more common than suspected. Finally, I report on the detection and isolation of slipped-DNA structures from the endogenous DM1 locus from patient tissues. The slip-outs appear as clusters along a length of DNA, rather than single isolated slip-outs, and more unstable tissues contain greater amounts of slipped-DNA compared to more stable tissues. This detection implies that slipped-DNA structures are not merely transient intermediates in the mutation and expansion process as has long been assumed, but remain within the DNA at detectable levels. The data reported herein both furthers our understanding of trinucleotide repeat instability, and additionally confirms the decades-long hypothesis that slipped-DNAs are in fact forming in patient tissues in a tissue-specific manner.

Trinucleotide repeat (TNR) expansions are the cause of over 40 human neurodegenerative diseases, and are linked to DNA damage and base excision repair (BER). We explored the role of DNA damage and BER in modulating TNR instability through analysis of DNA structures, BER protein activities, and reconstitution of repair using human BER proteins and synthesized DNA containing various types of damage. We show that DNA damage and BER can modulate TNR expansions by promoting removal of a TNR hairpin through coordinated activities of BER proteins and cofactors. We found that during repair in a TNR hairpin, coordination between the 5’-flap endonuclease activity of flap endonuclease 1 (FEN1), 3’-5’ exonuclease activity of AP endonuclease 1 (APE1), and activity of DNA ligase I (LIG I) can resolve the double-flap structure produced during BER in the hairpin loop. The resolution of the double-flap structure resulted in hairpin removal and prevention or attenuation of TNR expansions and provides the first evidence that coordination among BER proteins can remove a TNR hairpin. We further explored the role of BER cofactors in modulating TNR instability and found that the repair cofactor proliferating cell nuclear antigen (PCNA) facilitates genomic stability by promoting removal of a TNR hairpin. Hairpin removal was accomplished by altering dynamic TNR structures to allow more efficient FEN1 cleavage and DNA polymerase β (pol β) synthesis and stimulating the activity of LIG I. This study provides the first evidence that a DNA repair cofactor plays an important role in modulating TNR instability. Finally, we explored the effects of sugar modifications in abasic sites on activities of BER proteins and BER efficiency during repair in a TNR tract. We found that an oxidized sugar inhibits the activities of BER enzymes, interrupting their coordination and preventing efficient repair. Inefficient repair results in accumulation of repair intermediates with DNA breaks, contributing to genomic instability. Our results indicate that DNA base lesions and BER play a crucial role in modulating TNR instability. The research presented herein provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Friedreich ataxia (FRDA) is a neurodegenerative disorder that is inherited in an autosomal recessive pattern. The most common FRDA mutation is hyperexpansion of a GAA triplet repeat sequence in the first intron of the affected gene, frataxin (FXN), resulting in decreased frataxin protein expression. The hyperexpanded GAA repeats can adopt unusual DNA structures and induce aberrant epigenetic changes leading to heterochromatin mediated gene silencing. Several epigenetic changes, including increased levels of DNA methylation, histone modifications, repressive chromatin formation and elevated levels of non-coding RNA have been reported in FRDA. It has been reported that a novel FXN antisense transcript (FAST-1), is present at higher levels in FRDA patient-derived fibroblasts and its overexpression is associated with the depletion of CTCF, a chromatin insulator protein, and heterochromatin formation involving the critical +1 nucleosome. Previously, characteristics of FAST-1 were investigated in our lab and a full-length FAST-1 transcript containing a poly (A) tail was identified. To investigate any possible effects of FAST-1 on FXN expression, I first overexpressed this FAST-1 transcript in three different non-FRDA cell lines and a consistent decrease of FXN expression was observed in each cell type compared to control cells. I also identified that FAST-1 copy number is positively correlated with increased FAST-1 expression, which in turn is negatively correlated with FXN expression in FAST-1 overexpressing cells. Additionally, we found that FAST-1 overexpression is associated with increased levels of DNA methylation at CpG sites U6 and U11 of the FXN upstream GAA repeat region, together with CTCF depletion and heterochromatin formation at the 5'UTR of the FXN gene. To further investigate the role of FAST-1 in FXN gene silencing, I used a small hairpin RNA (shRNA) strategy to knock down FAST-1 expression in FRDA fibroblast cells. I found that knocking down FAST-1 increases FXN expression, but not to the level of control cells. Lastly, I investigated the pattern of FAST-1 expression and histone modifications at the FXN transgene in our new FRDA mouse model, designated YG8LR. The YG8LR mice showed decreased levels of FXN expression and H3K9ac and increased levels of FAST-1 expression and H3K9me3. Our data suggest that since FAST-1 is associated with FXN gene silencing, inhibition of FAST-1 may be an approach for FRDA therapy.

Polyglutamine (polyQ) diseases are progressive fatal neurodegenerative movement disorders. Although many cellular processes are perturbed in polyQ disease, recent studies highlight the importance of protein misfolding as a central event in polyQ toxicity. Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a particularly interesting polyQ disease because of the special qualities of the disease protein ataxin-3, which normally participates in cellular protein quality control. Here I use multiple mouse models of disease to explore toxic protein species and the role of protein homeostasis in SCA3 pathogenesis. In Chapter 1, I review the key features of polyQ disease, and outline the background and rationale behind our strategy for identifying toxic protein species in SCA3. In Chapter 2, I examine the role of the protein quality control ubiquitin ligase, CHIP (C-terminus of Hsp70 interacting protein), in regulating the toxicity of expanded ataxin-3 in vivo. Genetic reduction or removal of CHIP increases formation of detergent-resistant ataxin-3 microaggregates specifically in the brain. Concomitant with this, reduction or removal of CHIP exacerbates the phenotype of SCA3 mice, revealing a correlation between high levels of microaggregates and phenotypic severity. Additional cell-based studies confirm that CHIP may not directly mediate ataxin-3 degradation, suggesting that CHIP reduces expanded ataxin-3 toxicity in the brain primarily by enhancing ataxin-3 solubility. In Chapter 3, I use various biochemical techniques to reveal the presence of brain-specific ataxin-3 microaggregates in two genetically distinct mouse models of SCA3. Selective neuropathological evaluation of SCA3 mice reveals that major neuronal loss and reactive glial proliferation are not shared features of phenotypically-manifesting SCA3 mice. Additional studies fail to provide evidence for loss-of-function of endogenous ataxin-3 in SCA3 mice. Our results suggest that neuronal dysfunction in SCA3 is mediated through a toxic gain-of-function mechanism by ataxin-3 microaggregates in the CNS. In Chapter 4, I discuss important areas for future research in polyQ disease. I describe studies that would help elucidate the structural nature of toxic soluble microaggregates, and their effects on other cellular proteins. I also consider how the results described in this thesis inform potential treatment strategies.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 107-120).

Neurodegenerative disorders are among the most devastating human illnesses. They present a significant source of morbidity and mortality, and given an aging population, an impending public health crisis. Disease-modifying treatments remain sparse, with most current therapies focused on reducing symptom burden. The cellular stress response is intimately linked to energy management and has frequently been posited as playing a central role in neurodegeneration. Using two distinct neurodegenerative diseases as ‘case studies’, aberrant cellular stress and energy management are demonstrated as potential pathways contributing to neurodegeneration. First, the Huntington’s disease protein, huntingtin, is observed to rapidly localize to early endosomes, where it is associated with arrest in early-to-late and early-to-recycling endocytic trafficking. Given the energy-dependent nature of vesicular trafficking, this arrest is postulated to free substantial energy within the cell, which may subsequently be diverted to pathways that are critical for the initiation of longer-duration stress responses, such as the unfolded protein response. In the context of Huntington’s disease, impaired recovery from this stress response is observed, suggesting deficits in intracellular vesicular trafficking and energy regulation exist in disease states. In the second ‘case study’, a novel spinocerebellar ataxia variant is characterized, occurring as a result of point mutations within two genes: ATXN7 and TOP1MT, which encode ataxin-7 and the type I mitochondrial topoisomerase (top1mt), respectively. Ataxin-7 has previously been implicated in spinocerebellar ataxia type 7, which occurs as a result of a polyglutamine expansion in the first exon of the protein. Patient cells are noted to have substantially lower mitochondrial respiratory function in comparison to healthy controls and decreased levels of mitochondrial DNA, and ataxin-7 subcellular localization is observed to be abnormal. This suggests that there is important interplay between the mitochondria and proteins implicated in neurodegeneration and provides further support for aberrant cellular bioenergetics as a unifying pathway to neurodegeneration. In the concluding chapters, the nuclear localization signal of ataxin-7 is characterized, and there is analysis comparing conical ‘atraumatic’ lumbar puncture needles with bevel-tipped ‘conventional’ needles. Atraumatic needles are noted to be associated with significantly less patient complications and require fewer return visits to hospital. Moreover, atraumatic needles are demonstrated to have similar rates of success and failure when controlling for important variables like clinician specialty, dispelling common misconceptions surrounding their ease-of-use. As lumbar puncture is ubiquitous within the clinical neurosciences and is important for diagnosis, monitoring, and treatment of disease, as well as clinical trials, this work has far-reaching implications for patient care and future research. / Thesis / Doctor of Philosophy (PhD)