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CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination.

Libby RT, Hagerman KA, Pineda VV, Lau R, Cho DH, Baccam SL, Axford MM, Cleary JD, Moore JM, Sopher BL, Tapscott SJ, Filippova GN, Pearson CE, La Spada AR - PLoS Genet. (2008)

Bottom Line: At least 25 inherited disorders in humans result from microsatellite repeat expansion.We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions.As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA, USA.

ABSTRACT
At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

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SCA7-CTCF-I-mut mice display increased somatic instability.(A) At 2 months of age, the SCA7 CAG repeat is stable in the SCA7-CTCF-I-wt line and in both SCA7-CTCF-I-mut lines. (B) With advancing age, tissue-specific instability is seen in SCA7-CTCF-I-wt mice; however, this tissue-specific instability is much more pronounced in SCA7-CTCF-I-mut mice. Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here. (C) To permit quantification of somatic instability, we performed small-pool PCR on tissue DNA samples from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. As shown here for cortex, SCA7-CTCF-I-mut mice displayed significantly greater instability than SCA7-CTCF-I-wt mice (p = 8.6×10−5, Mann-Whitney two-tailed test). See Table 1 for a compiled list of repeat alleles. (D) Histogram of repeat length variation in the cortex of SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. SCA7-CTCF-I-mut mice exhibit significantly greater instability than SCA7-CTCF-I-wt mice, and this expansion tendency exceeds that of SCA7-CTCF-I-wt mice, even when 2.5 months younger (p = 0.0003, Mann-Whitney two-tailed test). With advancing age, the expansion bias between the SCA7-CTCF-I-mut and -wt mice becomes more pronounced (p<.0001, Mann-Whitney two-tailed test). Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here.
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pgen-1000257-g003: SCA7-CTCF-I-mut mice display increased somatic instability.(A) At 2 months of age, the SCA7 CAG repeat is stable in the SCA7-CTCF-I-wt line and in both SCA7-CTCF-I-mut lines. (B) With advancing age, tissue-specific instability is seen in SCA7-CTCF-I-wt mice; however, this tissue-specific instability is much more pronounced in SCA7-CTCF-I-mut mice. Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here. (C) To permit quantification of somatic instability, we performed small-pool PCR on tissue DNA samples from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. As shown here for cortex, SCA7-CTCF-I-mut mice displayed significantly greater instability than SCA7-CTCF-I-wt mice (p = 8.6×10−5, Mann-Whitney two-tailed test). See Table 1 for a compiled list of repeat alleles. (D) Histogram of repeat length variation in the cortex of SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. SCA7-CTCF-I-mut mice exhibit significantly greater instability than SCA7-CTCF-I-wt mice, and this expansion tendency exceeds that of SCA7-CTCF-I-wt mice, even when 2.5 months younger (p = 0.0003, Mann-Whitney two-tailed test). With advancing age, the expansion bias between the SCA7-CTCF-I-mut and -wt mice becomes more pronounced (p<.0001, Mann-Whitney two-tailed test). Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here.

Mentions: Another intriguing feature of repeat instability is variation in repeat size within and between the tissues of an individual organism. This tissue-specific instability, or “somatic mosaicism”, occurs in human patients with repeat diseases, and in mouse models of repeat instability and disease [1],[8],[11]. While shown to be age-dependent, the mechanistic basis of inter-tissue variation, which even occurs in postmitotic neurons [20], is unknown. To determine if somatic CAG mosaicism at the SCA7 locus involves CTCF binding, we surveyed repeat instability in various tissues from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. At two months of age, the SCA7 CAG repeat was remarkably stable in all analyzed tissues (Figure 3A). However, by ∼10 months of age, SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice displayed large CAG repeat expansions in the cortex and liver (Figure 3B). The liver also exhibited a bimodal distribution of repeat size (i.e. two populations of cells with distinct tract lengths) (Figure 3B). The most pronounced somatic instability differences existed in the kidney, with large expansions for SCA7-CTCF-I-mut mice, but stable repeats in the SCA7-CTCF-I-wt mice (Figure 3B). This pattern of increased kidney and liver repeat instability was present in both SCA7-CTCF-I-mut transgenic lines (Figure 3B; Figure S3). Indeed, comparable somatic instability was also detected in both SCA7-CTCF-I-mut transgenic lines at five months of age (Figure S4). When we closely examined repeat instability in the cortex by small-pool PCR, we observed significantly different repeat sizes (p = 8.6×10−5, Mann-Whitney), with a range of 39 to 152 CAG repeats in SCA7-CTCF-I-wt mice and 26 to 245 CAG repeats in SCA7-CTCF-I-mut mice (Figure 3C; Table 1). The increased somatic instability occurred in both SCA7-CTCF-I-mut transgenic lines, as an expansion bias was apparent in both lineages upon small-pool PCR analysis (Figure 3D; Table 1). These findings suggest that CTCF binding stabilizes the SCA7 CAG repeat in certain tissues. Thus, as noted for the germ line and documented for two independent lines of SCA7-CTCF-I-mut transgenic mice, SCA7 somatic CAG instability is dependent upon age and the presence of intact CTCF binding sites.


CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination.

Libby RT, Hagerman KA, Pineda VV, Lau R, Cho DH, Baccam SL, Axford MM, Cleary JD, Moore JM, Sopher BL, Tapscott SJ, Filippova GN, Pearson CE, La Spada AR - PLoS Genet. (2008)

SCA7-CTCF-I-mut mice display increased somatic instability.(A) At 2 months of age, the SCA7 CAG repeat is stable in the SCA7-CTCF-I-wt line and in both SCA7-CTCF-I-mut lines. (B) With advancing age, tissue-specific instability is seen in SCA7-CTCF-I-wt mice; however, this tissue-specific instability is much more pronounced in SCA7-CTCF-I-mut mice. Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here. (C) To permit quantification of somatic instability, we performed small-pool PCR on tissue DNA samples from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. As shown here for cortex, SCA7-CTCF-I-mut mice displayed significantly greater instability than SCA7-CTCF-I-wt mice (p = 8.6×10−5, Mann-Whitney two-tailed test). See Table 1 for a compiled list of repeat alleles. (D) Histogram of repeat length variation in the cortex of SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. SCA7-CTCF-I-mut mice exhibit significantly greater instability than SCA7-CTCF-I-wt mice, and this expansion tendency exceeds that of SCA7-CTCF-I-wt mice, even when 2.5 months younger (p = 0.0003, Mann-Whitney two-tailed test). With advancing age, the expansion bias between the SCA7-CTCF-I-mut and -wt mice becomes more pronounced (p<.0001, Mann-Whitney two-tailed test). Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here.
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pgen-1000257-g003: SCA7-CTCF-I-mut mice display increased somatic instability.(A) At 2 months of age, the SCA7 CAG repeat is stable in the SCA7-CTCF-I-wt line and in both SCA7-CTCF-I-mut lines. (B) With advancing age, tissue-specific instability is seen in SCA7-CTCF-I-wt mice; however, this tissue-specific instability is much more pronounced in SCA7-CTCF-I-mut mice. Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here. (C) To permit quantification of somatic instability, we performed small-pool PCR on tissue DNA samples from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. As shown here for cortex, SCA7-CTCF-I-mut mice displayed significantly greater instability than SCA7-CTCF-I-wt mice (p = 8.6×10−5, Mann-Whitney two-tailed test). See Table 1 for a compiled list of repeat alleles. (D) Histogram of repeat length variation in the cortex of SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. SCA7-CTCF-I-mut mice exhibit significantly greater instability than SCA7-CTCF-I-wt mice, and this expansion tendency exceeds that of SCA7-CTCF-I-wt mice, even when 2.5 months younger (p = 0.0003, Mann-Whitney two-tailed test). With advancing age, the expansion bias between the SCA7-CTCF-I-mut and -wt mice becomes more pronounced (p<.0001, Mann-Whitney two-tailed test). Results for individuals from the two different SCA7-CTCF-I-mut mice are shown here.
Mentions: Another intriguing feature of repeat instability is variation in repeat size within and between the tissues of an individual organism. This tissue-specific instability, or “somatic mosaicism”, occurs in human patients with repeat diseases, and in mouse models of repeat instability and disease [1],[8],[11]. While shown to be age-dependent, the mechanistic basis of inter-tissue variation, which even occurs in postmitotic neurons [20], is unknown. To determine if somatic CAG mosaicism at the SCA7 locus involves CTCF binding, we surveyed repeat instability in various tissues from SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. At two months of age, the SCA7 CAG repeat was remarkably stable in all analyzed tissues (Figure 3A). However, by ∼10 months of age, SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice displayed large CAG repeat expansions in the cortex and liver (Figure 3B). The liver also exhibited a bimodal distribution of repeat size (i.e. two populations of cells with distinct tract lengths) (Figure 3B). The most pronounced somatic instability differences existed in the kidney, with large expansions for SCA7-CTCF-I-mut mice, but stable repeats in the SCA7-CTCF-I-wt mice (Figure 3B). This pattern of increased kidney and liver repeat instability was present in both SCA7-CTCF-I-mut transgenic lines (Figure 3B; Figure S3). Indeed, comparable somatic instability was also detected in both SCA7-CTCF-I-mut transgenic lines at five months of age (Figure S4). When we closely examined repeat instability in the cortex by small-pool PCR, we observed significantly different repeat sizes (p = 8.6×10−5, Mann-Whitney), with a range of 39 to 152 CAG repeats in SCA7-CTCF-I-wt mice and 26 to 245 CAG repeats in SCA7-CTCF-I-mut mice (Figure 3C; Table 1). The increased somatic instability occurred in both SCA7-CTCF-I-mut transgenic lines, as an expansion bias was apparent in both lineages upon small-pool PCR analysis (Figure 3D; Table 1). These findings suggest that CTCF binding stabilizes the SCA7 CAG repeat in certain tissues. Thus, as noted for the germ line and documented for two independent lines of SCA7-CTCF-I-mut transgenic mice, SCA7 somatic CAG instability is dependent upon age and the presence of intact CTCF binding sites.

Bottom Line: At least 25 inherited disorders in humans result from microsatellite repeat expansion.We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions.As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA, USA.

ABSTRACT
At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

Show MeSH
Related in: MedlinePlus