Despite significant advances in our understanding of how trinucleotide repeats cause disease, mechanisms underlying their unique property of genetic instability remain ill-defined. Of all CAG/polyglutamine diseases, spinocerebellar ataxia type 7 (SCA7) displays the most profound repeat expansion tendency and hence the most dramatic anticipation. Our previously funded proposal was based upon the hypothesis that the identification of cis-acting elements and trans-acting factors would help to unravel the complex processes that produce repeat instability and disease. Using a 13.5 kb genomic fragment from the human SCA7 gene, we found that sequences 3'to the CAG repeat are required for repeat instability in transgenic mice. Within this 3'region is a binding site for the """"""""CCCTC-binding factor"""""""" (CTCF), a protein with a variety of functions that stem from its ability to modulate DNA structure. When we mutated the CTCF binding site 3'to the SCA7 repeat in the same 13.5 kb ataxin-7 CAG-92 genomic fragment and re-derived transgenic mice, we made two striking observations: i) an intact CTCF binding site sequence is required for repeat instability at the SCA7 locus;and ii) mutation of the CTCF binding site yields a SCA7-like neurodegenerative phenotype. As the latter result was completely unexpected, we have intensively studied these mice and the genomic region within the ataxin-7 mini-gene, and have determined that CTCF regulates expression of ataxin-7 from an alternative promoter through an antisense non-coding RNA. As ataxin-7 may be required for the function of a transcriptional co-activator, our results suggest a provocative novel model for CTCF action and for the role of antisense non-coding RNA expression in transcription regulation. As CTCF binding sites have been found at a variety of trinucleotide repeat disease loci and have been shown to regulate transcription at one other locus, our findings suggest an exciting connection between repeat expansion, CTCF function, repeat instability, epigenetics, and transcription regulation. This proposal seeks to determine if CTCF is the trans-acting factor regulating repeat instability in the germ line and in somatic cells at the SCA7 locus by studying the process in transgenic mice. Our discovery of a potential role for CTCF in the promotion of antisense non-coding RNA transcription at the SCA7 locus raises the intriguing possibility that CTCF binding regulates ataxin-7 transcription and thus STAGA complex co-activator function by promoting expression of an antisense non-coding RNA. We will test if CTCF regulates ataxin-7 gene expression through an antisense non-coding RNA by characterizing an ataxin-7 alternative promoter and antisense non-coding RNA;and determining the expression pattern, regulation, and relationship of ataxin-7 sense and antisense non-coding RNA transcripts. We will determine if CTCF levels modulate STAGA complex function;if such modulation relies on production or reduction of the ataxin-7 antisense non-coding RNA;and what factors affect the CTCF - ataxin-7- STAGA pathway. Finally, we will attempt to validate the function of the antisense non-coding RNA in vivo.
|Mason, Amanda G; Tomé, Stephanie; Simard, Jodie P et al. (2014) Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age. Hum Mol Genet 23:1606-18|
|Sopher, Bryce L; Ladd, Paula D; Pineda, Victor V et al. (2011) CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA. Neuron 70:1071-84|
|La Spada, Albert R; Taylor, J Paul (2010) Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat Rev Genet 11:247-58|
|Libby, Randell T; Hagerman, Katharine A; Pineda, Victor V et al. (2008) CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination. PLoS Genet 4:e1000257|
|Jackson, Stephen M; Whitworth, Alex J; Greene, Jessica C et al. (2005) A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage. Gene 347:35-41|
|Libby, Randell T; Monckton, Darren G; Fu, Ying-Hui et al. (2003) Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice. Hum Mol Genet 12:41-50|
|Sopher, B L; Myrick, S B; Hong, J Y et al. (2000) In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis. Gene 261:383-90|