Abstract
Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.
Highlights
Trinucleotide repeat expansions are a relatively common cause of human disease
Each is caused by PA expansion in a separate gene, with the eight congenital disorders linked to PA expansions in developmentally-important transcription factors
Whilst the function of these polyalanine (PA) tracts remains unknown, they are interesting because DNA changes that increase their length above a threshold are responsible for nine different human disorders
Summary
Trinucleotide repeat expansions are a relatively common cause of human disease. The expanded trinucleotide can occur in an untranslated region, for example in Fragile X syndrome in which a CGG repeat adjacent to the FMRI promoter causes hypermethylation and gene silencing. Some PA alleles cause similar but more severe disease phenotypes than null alleles consistent with toxic gain-of-function (GOF) and/or dominant negative activity (HOXD13, PHOX2B) [5,6]. Despite these differences in mode of inheritance, all PA proteins behave very in vitro, such that over-expression in cell culture results in the generation of cytoplasmic and/or nuclear aggregates, which are likely to arise through protein misfolding [7,8,9].
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