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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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Epigenetic regulation of CTCF binding modulates instability at the SCA7 locus.(A) CpG methylation prevents binding of CTCF to SCA7-CTCF-I site. Electrophoretic mobility shift assays with un-methylated (control) or methylated SCA7-CTCF-I fragments, using CTCF with no antisera (CTCF), CTCF with anti-CTCF antisera (CTCF+α-CTCF), or CTCF with pre-immune sera (CTCF+pI). Arrow indicates CTCF-bound probe. (B) Prominent somatic instability in kidney DNA (black arrowheads) from a SCA7-CTCF-I-wt mouse with CTCF-I site methylation (SCA7-CTCF-I-wt*) contrasts with somatic stability in SCA7-CTCF-I-wt mice with un-methylated CTCF-I sites. Note that SCA7-CTCF-I-wt lines display bimodal CAG repeat alleles. Prominent somatic instability is apparent in kidney DNA (gray arrowhead) from a SCA7-CTCF-I-mut mouse. All mice were 6 months of age. (C) Kidney DNAs from the SCA7-CTCF-I-wt* mouse are highly methylated. Circles, CpG dyads; open circles, unmethylated; filled circles; methylated. Box highlights core CTCF binding site contact residue, based upon footprinting analysis. Diagrammed epigenotypes summarize results for five SCA7-CTCF-I-wt mice, eight SCA7-CTCF-I-mut mice, and the SCA7-CTCF-I-wt* mouse, and were consistent for at least 75% of all sequenced clones (n = 10−12/sample). (D) Liver DNAs from control SCA7-CTCF-I-wt mice are methylated. Bisulfite sequencing of the SCA7-CTCF-I region was performed upon liver DNAs from three SCA7-CTCF-I-wt mice at one year of age (n = 17 clones/mouse), and CpG methylation determined for the 13 CpG dyads in the SCA7-CTCF-I region. A number of CpG dyads, including the CpG-4 CTCF contact site, exhibit moderate to high levels of methylation.
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pgen-1000257-g004: Epigenetic regulation of CTCF binding modulates instability at the SCA7 locus.(A) CpG methylation prevents binding of CTCF to SCA7-CTCF-I site. Electrophoretic mobility shift assays with un-methylated (control) or methylated SCA7-CTCF-I fragments, using CTCF with no antisera (CTCF), CTCF with anti-CTCF antisera (CTCF+α-CTCF), or CTCF with pre-immune sera (CTCF+pI). Arrow indicates CTCF-bound probe. (B) Prominent somatic instability in kidney DNA (black arrowheads) from a SCA7-CTCF-I-wt mouse with CTCF-I site methylation (SCA7-CTCF-I-wt*) contrasts with somatic stability in SCA7-CTCF-I-wt mice with un-methylated CTCF-I sites. Note that SCA7-CTCF-I-wt lines display bimodal CAG repeat alleles. Prominent somatic instability is apparent in kidney DNA (gray arrowhead) from a SCA7-CTCF-I-mut mouse. All mice were 6 months of age. (C) Kidney DNAs from the SCA7-CTCF-I-wt* mouse are highly methylated. Circles, CpG dyads; open circles, unmethylated; filled circles; methylated. Box highlights core CTCF binding site contact residue, based upon footprinting analysis. Diagrammed epigenotypes summarize results for five SCA7-CTCF-I-wt mice, eight SCA7-CTCF-I-mut mice, and the SCA7-CTCF-I-wt* mouse, and were consistent for at least 75% of all sequenced clones (n = 10−12/sample). (D) Liver DNAs from control SCA7-CTCF-I-wt mice are methylated. Bisulfite sequencing of the SCA7-CTCF-I region was performed upon liver DNAs from three SCA7-CTCF-I-wt mice at one year of age (n = 17 clones/mouse), and CpG methylation determined for the 13 CpG dyads in the SCA7-CTCF-I region. A number of CpG dyads, including the CpG-4 CTCF contact site, exhibit moderate to high levels of methylation.

Mentions: CTCF binding can be regulated by CpG methylation, as methylation at CTCF recognition sites abrogates binding [16]. This finding was confirmed for un-methylated and methylated versions of the SCA7 CTCF-I recognition site (Figure 4A; Figure S5). Highly variable levels of instability have been documented in the kidneys of transgenic repeat instability mouse models [21],[22], although the reasons for pronounced instability in this tissue are unknown. Interestingly, one mouse with a wild-type CTCF-I binding site (SCA7-CTCF-I-wt) displayed marked CAG repeat instability in its kidney DNA (Figure 4B), paralleling the considerable instability observed in the SCA7-CTCF-I-mut mice (Figure 3B). Bisulfite sequencing of kidney DNA from this SCA7-CTCF-I-wt mouse revealed high levels of CpG methylation at the wild-type CTCF-I binding site, including the central CTCF contact site (Figure S6); whereas methylation was not observed in kidney DNAs from 14 other SCA7-CTCF-I-wt mice that displayed only modest levels of CAG instability (Figure 4C). The high levels of CAG instability and the CpG methylation in this mouse were restricted to the kidney, as the cerebellum and tail DNAs of the same mouse, which showed limited CAG instability (Figure 4B), were completely unmethylated (Figure 4C). This finding suggests a direct link between methylation status of the CTCF binding site and CAG repeat instability. Of all the tissues analyzed from SCA7-CTCF-I-wt mice, liver exhibits the greatest amount of somatic mosaisicm, with the largest repeat expansions (Figure 3B). We hypothesized that the high levels of CAG repeat instability in the liver of SCA7-CTCF-I-wt mice might result from methylation of the CTCF-I binding site. To address this question, we performed bisulfite sequencing analysis of liver DNAs from SCA7-CTCF-I-wt mice, and documented moderately high levels of methylation at the CTCF-I binding site (Figure 4D; Figure S7). These results indicate a correlation between CpG methylation and CAG repeat instability. Thus, in SCA7 transgenic mice, decreased CTCF binding, either by CpG methylation or mutagenesis of the CTCF-I binding site, enhanced CAG repeat instability.


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)

Epigenetic regulation of CTCF binding modulates instability at the SCA7 locus.(A) CpG methylation prevents binding of CTCF to SCA7-CTCF-I site. Electrophoretic mobility shift assays with un-methylated (control) or methylated SCA7-CTCF-I fragments, using CTCF with no antisera (CTCF), CTCF with anti-CTCF antisera (CTCF+α-CTCF), or CTCF with pre-immune sera (CTCF+pI). Arrow indicates CTCF-bound probe. (B) Prominent somatic instability in kidney DNA (black arrowheads) from a SCA7-CTCF-I-wt mouse with CTCF-I site methylation (SCA7-CTCF-I-wt*) contrasts with somatic stability in SCA7-CTCF-I-wt mice with un-methylated CTCF-I sites. Note that SCA7-CTCF-I-wt lines display bimodal CAG repeat alleles. Prominent somatic instability is apparent in kidney DNA (gray arrowhead) from a SCA7-CTCF-I-mut mouse. All mice were 6 months of age. (C) Kidney DNAs from the SCA7-CTCF-I-wt* mouse are highly methylated. Circles, CpG dyads; open circles, unmethylated; filled circles; methylated. Box highlights core CTCF binding site contact residue, based upon footprinting analysis. Diagrammed epigenotypes summarize results for five SCA7-CTCF-I-wt mice, eight SCA7-CTCF-I-mut mice, and the SCA7-CTCF-I-wt* mouse, and were consistent for at least 75% of all sequenced clones (n = 10−12/sample). (D) Liver DNAs from control SCA7-CTCF-I-wt mice are methylated. Bisulfite sequencing of the SCA7-CTCF-I region was performed upon liver DNAs from three SCA7-CTCF-I-wt mice at one year of age (n = 17 clones/mouse), and CpG methylation determined for the 13 CpG dyads in the SCA7-CTCF-I region. A number of CpG dyads, including the CpG-4 CTCF contact site, exhibit moderate to high levels of methylation.
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pgen-1000257-g004: Epigenetic regulation of CTCF binding modulates instability at the SCA7 locus.(A) CpG methylation prevents binding of CTCF to SCA7-CTCF-I site. Electrophoretic mobility shift assays with un-methylated (control) or methylated SCA7-CTCF-I fragments, using CTCF with no antisera (CTCF), CTCF with anti-CTCF antisera (CTCF+α-CTCF), or CTCF with pre-immune sera (CTCF+pI). Arrow indicates CTCF-bound probe. (B) Prominent somatic instability in kidney DNA (black arrowheads) from a SCA7-CTCF-I-wt mouse with CTCF-I site methylation (SCA7-CTCF-I-wt*) contrasts with somatic stability in SCA7-CTCF-I-wt mice with un-methylated CTCF-I sites. Note that SCA7-CTCF-I-wt lines display bimodal CAG repeat alleles. Prominent somatic instability is apparent in kidney DNA (gray arrowhead) from a SCA7-CTCF-I-mut mouse. All mice were 6 months of age. (C) Kidney DNAs from the SCA7-CTCF-I-wt* mouse are highly methylated. Circles, CpG dyads; open circles, unmethylated; filled circles; methylated. Box highlights core CTCF binding site contact residue, based upon footprinting analysis. Diagrammed epigenotypes summarize results for five SCA7-CTCF-I-wt mice, eight SCA7-CTCF-I-mut mice, and the SCA7-CTCF-I-wt* mouse, and were consistent for at least 75% of all sequenced clones (n = 10−12/sample). (D) Liver DNAs from control SCA7-CTCF-I-wt mice are methylated. Bisulfite sequencing of the SCA7-CTCF-I region was performed upon liver DNAs from three SCA7-CTCF-I-wt mice at one year of age (n = 17 clones/mouse), and CpG methylation determined for the 13 CpG dyads in the SCA7-CTCF-I region. A number of CpG dyads, including the CpG-4 CTCF contact site, exhibit moderate to high levels of methylation.
Mentions: CTCF binding can be regulated by CpG methylation, as methylation at CTCF recognition sites abrogates binding [16]. This finding was confirmed for un-methylated and methylated versions of the SCA7 CTCF-I recognition site (Figure 4A; Figure S5). Highly variable levels of instability have been documented in the kidneys of transgenic repeat instability mouse models [21],[22], although the reasons for pronounced instability in this tissue are unknown. Interestingly, one mouse with a wild-type CTCF-I binding site (SCA7-CTCF-I-wt) displayed marked CAG repeat instability in its kidney DNA (Figure 4B), paralleling the considerable instability observed in the SCA7-CTCF-I-mut mice (Figure 3B). Bisulfite sequencing of kidney DNA from this SCA7-CTCF-I-wt mouse revealed high levels of CpG methylation at the wild-type CTCF-I binding site, including the central CTCF contact site (Figure S6); whereas methylation was not observed in kidney DNAs from 14 other SCA7-CTCF-I-wt mice that displayed only modest levels of CAG instability (Figure 4C). The high levels of CAG instability and the CpG methylation in this mouse were restricted to the kidney, as the cerebellum and tail DNAs of the same mouse, which showed limited CAG instability (Figure 4B), were completely unmethylated (Figure 4C). This finding suggests a direct link between methylation status of the CTCF binding site and CAG repeat instability. Of all the tissues analyzed from SCA7-CTCF-I-wt mice, liver exhibits the greatest amount of somatic mosaisicm, with the largest repeat expansions (Figure 3B). We hypothesized that the high levels of CAG repeat instability in the liver of SCA7-CTCF-I-wt mice might result from methylation of the CTCF-I binding site. To address this question, we performed bisulfite sequencing analysis of liver DNAs from SCA7-CTCF-I-wt mice, and documented moderately high levels of methylation at the CTCF-I binding site (Figure 4D; Figure S7). These results indicate a correlation between CpG methylation and CAG repeat instability. Thus, in SCA7 transgenic mice, decreased CTCF binding, either by CpG methylation or mutagenesis of the CTCF-I binding site, enhanced CAG repeat instability.

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