Proteins that modulate trinucleotide repeat expansions in human cells

Expansions of trinucleotide repeats (TNRs) are the genetic cause of several inherited neurological diseases, such as Huntington's disease, myotonic dystrophy and fragile X syndrome. TNR diseases and their causal expansion mutations display several unique characteristics that distinguish them from other diseases and their associated genetic mutations. Such features include the ability of expandable repeats to form secondary structures such as hairpins, which are thought to be central to expansion mutagenesis and the occurrence of a threshold that demarcates stable and unstable alleles. The mechanisms by which TNRs expand are not well-defined. The mechanisms underlying expansions that cross the threshold length are largely uncharacterized, especially in human cells. In order to gain some insight into this area, I used a selective genetic assay to determine the involvement of trans-acting factors in mediating CTG.CAG repeat instability near the threshold length in a human astrocytic cell line. RNA interference and/or small molecule inhibitors were used to interfere with proteins of interest to establish if they were relevant to instability in this system. This approach revealed novel mediators and shed some light on the mechanisms of repeat instability near the threshold. This study identified particular histone deacetylases (HDACs) and histone acetyltransferases (HATs) involved in instability. Specifically, HDAC3 and HDAC5 are proposed to promote expansions, while the evidence points towards HDAC9 having the contravening effect. Evidence suggests that the HATs, CBP and p300, function to inhibit expansions. Taken together with findings from the Lahue lab that specific HDAC complexes promote CTG.CAG repeat expansions in yeast, this represents a novel mechanism for repeat instability. Further findings imply a direct role for the mismatch repair (MMR) complex MutSB but not MutSB in promoting expansion of threshold-length CTG.CAG tracts. This observation led me to investigate whether certain HDACs might be mediating their expansion-promoting actions through MutSB. Double knockdown analysis suggested that HDAC3 and MSH2 are acting through a common pathway to facilitate expansions. The nature of this functional interaction has not been identified. We determined that HDAC3 does not promote expansions by controlling access of MutSB to the repeat tract, or by regulating MSH2 or MSH3 protein levels. Nevertheless, the identification of this linkage between HDACs and MMR represents an interesting mechanistic pathway. Based on findings in yeast that the DNA helicase Srs2 inhibits expansions in concert with the postreplication repair (PRR) pathway, I investigated human orthologues of Srs2 and PRR factors with respect to TNR expansions. RTEL1, a proposed functional homologue of Srs2 was shown to prevent expansions in a common pathway with the PRR factors RAD18 and HLTF.

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SFI
IRCSET
Thomams Crawford Hayes Foundation

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

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University of Galway Theses (PhD Theses)