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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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Analysis and mutagenesis of the SCA7-CTCF-I binding site.(A) SCA7 genomic fragments used for transgenesis. Upper: SCA7-CTCF-I-wt; Middle: α-SCA7 3′ genomic deletion; Bottom: SCA7-CTCF-I-mut. Core CCCTC sequences are underlined, and sequence alterations in the SCA7-CTCF-I-mut transgenic construct are shown in gray. (B) Electrophoretic mobility shift assays with SCA7-CTCF-I-wt and -mut probe fragments were performed with probe only, empty lysate (no protein), full-length CTCF protein with pre-immune anti-CTCF sera (CTCF+pI), CTCF protein with anti-CTCF sera (CTCF+α-CTCF), or the 11 zinc-finger DNA binding domain region of CTCF. Arrows indicate shifted CTCF-DNA complexes. Addition of CTCF-DM1 probe as cold competitor prevented CTCF-DNA complex formation for SCA7-CTCF-I-wt fragment, while non-specific cold competitor did not (data not shown). (C) Methylation interference (Me I) and DNase I footprinting (DNase) on SCA7-CTCF-I fragment. Left and right panels correspond to the 5′-end labeled coding and anti-sense strands respectively. B, CTCF-bound DNA; F, free DNA; long bars, CTCF-protected from DNase I; arrows, DNase I hypersensitive sites created by CTCF binding; filled circles, contact guanine nucleotides essential for sequence recognition by CTCF. See panel ‘A’ for precise location of sites. (D) ChIP on cerebellar lysates from SCA7-CTCF-I-wt and -mut mice (n = 3/genotype). Significantly decreased occupancy at the CTCF-I site was detected with the 3′ amplicon (primer set B) in SCA7-CTCF-I-mut mice (p = 0.02, one-way ANOVA), as this amplicon is not in close proximity to the 5′ CTCF-II site. No differences in CTCF occupancy between SCA7-CTCF-I-wt and -mut mice were detected with primer set A (or other adjacent primer sets; data not shown) due to the close proximity of the two CTCF binding sites. Results are normalized to SCA7-CTCF-I-wt. Error bars are s.d.
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pgen-1000257-g001: Analysis and mutagenesis of the SCA7-CTCF-I binding site.(A) SCA7 genomic fragments used for transgenesis. Upper: SCA7-CTCF-I-wt; Middle: α-SCA7 3′ genomic deletion; Bottom: SCA7-CTCF-I-mut. Core CCCTC sequences are underlined, and sequence alterations in the SCA7-CTCF-I-mut transgenic construct are shown in gray. (B) Electrophoretic mobility shift assays with SCA7-CTCF-I-wt and -mut probe fragments were performed with probe only, empty lysate (no protein), full-length CTCF protein with pre-immune anti-CTCF sera (CTCF+pI), CTCF protein with anti-CTCF sera (CTCF+α-CTCF), or the 11 zinc-finger DNA binding domain region of CTCF. Arrows indicate shifted CTCF-DNA complexes. Addition of CTCF-DM1 probe as cold competitor prevented CTCF-DNA complex formation for SCA7-CTCF-I-wt fragment, while non-specific cold competitor did not (data not shown). (C) Methylation interference (Me I) and DNase I footprinting (DNase) on SCA7-CTCF-I fragment. Left and right panels correspond to the 5′-end labeled coding and anti-sense strands respectively. B, CTCF-bound DNA; F, free DNA; long bars, CTCF-protected from DNase I; arrows, DNase I hypersensitive sites created by CTCF binding; filled circles, contact guanine nucleotides essential for sequence recognition by CTCF. See panel ‘A’ for precise location of sites. (D) ChIP on cerebellar lysates from SCA7-CTCF-I-wt and -mut mice (n = 3/genotype). Significantly decreased occupancy at the CTCF-I site was detected with the 3′ amplicon (primer set B) in SCA7-CTCF-I-mut mice (p = 0.02, one-way ANOVA), as this amplicon is not in close proximity to the 5′ CTCF-II site. No differences in CTCF occupancy between SCA7-CTCF-I-wt and -mut mice were detected with primer set A (or other adjacent primer sets; data not shown) due to the close proximity of the two CTCF binding sites. Results are normalized to SCA7-CTCF-I-wt. Error bars are s.d.

Mentions: At the SCA7 locus, there are two CTCF binding sites that flank the CAG repeat tract; the CTCF-I binding site is located 3′ to the CAG repeat (Figure S1), within the critical region deleted from the SCA7 genomic fragment in the α-SCA7-92R mice (Figure 1A). As CTCF binding sites are associated with highly unstable repeat loci [14], and CTCF binding can alter chromatin structure and DNA conformation [17],[18], we hypothesized that CTCF binding might be involved in SCA7 repeat instability. To test this hypothesis, we decided to compare SCA7 CAG repeat instability in mice carrying either the wild-type CTCF binding site or a mutant CTCF binding site that would be incapable of binding CTCF. To define the CTCF binding sites, we performed electrophoretic mobility shift assays to confirm that CTCF protein specifically binds to the putative CTCF-I binding site, and we found that both the CTCF DNA binding domain fragment and full-length CTCF protein bind to the SCA7 repeat locus 3′ region (Figure 1B). When we mapped the CTCF-I contact regions at the SCA7 repeat locus by methylation interference and DNA footprinting, we defined a region that is protected from DNase I treatment upon CTCF binding and subject to altered CTCF binding upon methylation treatment (Figure 1C). We then introduced point mutations at 11 nucleotides within this 3′ CTCF-I binding site, including eight contact nucleotides contained within the footprinted region (Figure 1C; Figure 1A, bottom). After confirming that CTCF binding was abrogated by these point mutations in electrophoretic mobility shift assays (Figure 1B), we derived a RL-SCA7 94R 13.5 kb genomic fragment construct, that was identical to our original RL-SCA7 92R genomic fragment construct [13], except for: i) the presence of a mutant CTCF-I binding site, and ii) a minor repeat size increase to 94 CAG repeats. The RL-SCA7 94R CTCF-I-mutant construct was microinjected, and two independent lines of RL-SCA7 94R CTCF-I mutant transgenic mice were generated (hereafter referred to as the SCA7-CTCF-I-mut line mice – to distinguish them from the original RL-SCA7-92R transgenic mice with an intact CTCF-I binding site, hereafter referred to as the SCA7-CTCF-I-wt line mice).


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)

Analysis and mutagenesis of the SCA7-CTCF-I binding site.(A) SCA7 genomic fragments used for transgenesis. Upper: SCA7-CTCF-I-wt; Middle: α-SCA7 3′ genomic deletion; Bottom: SCA7-CTCF-I-mut. Core CCCTC sequences are underlined, and sequence alterations in the SCA7-CTCF-I-mut transgenic construct are shown in gray. (B) Electrophoretic mobility shift assays with SCA7-CTCF-I-wt and -mut probe fragments were performed with probe only, empty lysate (no protein), full-length CTCF protein with pre-immune anti-CTCF sera (CTCF+pI), CTCF protein with anti-CTCF sera (CTCF+α-CTCF), or the 11 zinc-finger DNA binding domain region of CTCF. Arrows indicate shifted CTCF-DNA complexes. Addition of CTCF-DM1 probe as cold competitor prevented CTCF-DNA complex formation for SCA7-CTCF-I-wt fragment, while non-specific cold competitor did not (data not shown). (C) Methylation interference (Me I) and DNase I footprinting (DNase) on SCA7-CTCF-I fragment. Left and right panels correspond to the 5′-end labeled coding and anti-sense strands respectively. B, CTCF-bound DNA; F, free DNA; long bars, CTCF-protected from DNase I; arrows, DNase I hypersensitive sites created by CTCF binding; filled circles, contact guanine nucleotides essential for sequence recognition by CTCF. See panel ‘A’ for precise location of sites. (D) ChIP on cerebellar lysates from SCA7-CTCF-I-wt and -mut mice (n = 3/genotype). Significantly decreased occupancy at the CTCF-I site was detected with the 3′ amplicon (primer set B) in SCA7-CTCF-I-mut mice (p = 0.02, one-way ANOVA), as this amplicon is not in close proximity to the 5′ CTCF-II site. No differences in CTCF occupancy between SCA7-CTCF-I-wt and -mut mice were detected with primer set A (or other adjacent primer sets; data not shown) due to the close proximity of the two CTCF binding sites. Results are normalized to SCA7-CTCF-I-wt. Error bars are s.d.
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Show All Figures
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pgen-1000257-g001: Analysis and mutagenesis of the SCA7-CTCF-I binding site.(A) SCA7 genomic fragments used for transgenesis. Upper: SCA7-CTCF-I-wt; Middle: α-SCA7 3′ genomic deletion; Bottom: SCA7-CTCF-I-mut. Core CCCTC sequences are underlined, and sequence alterations in the SCA7-CTCF-I-mut transgenic construct are shown in gray. (B) Electrophoretic mobility shift assays with SCA7-CTCF-I-wt and -mut probe fragments were performed with probe only, empty lysate (no protein), full-length CTCF protein with pre-immune anti-CTCF sera (CTCF+pI), CTCF protein with anti-CTCF sera (CTCF+α-CTCF), or the 11 zinc-finger DNA binding domain region of CTCF. Arrows indicate shifted CTCF-DNA complexes. Addition of CTCF-DM1 probe as cold competitor prevented CTCF-DNA complex formation for SCA7-CTCF-I-wt fragment, while non-specific cold competitor did not (data not shown). (C) Methylation interference (Me I) and DNase I footprinting (DNase) on SCA7-CTCF-I fragment. Left and right panels correspond to the 5′-end labeled coding and anti-sense strands respectively. B, CTCF-bound DNA; F, free DNA; long bars, CTCF-protected from DNase I; arrows, DNase I hypersensitive sites created by CTCF binding; filled circles, contact guanine nucleotides essential for sequence recognition by CTCF. See panel ‘A’ for precise location of sites. (D) ChIP on cerebellar lysates from SCA7-CTCF-I-wt and -mut mice (n = 3/genotype). Significantly decreased occupancy at the CTCF-I site was detected with the 3′ amplicon (primer set B) in SCA7-CTCF-I-mut mice (p = 0.02, one-way ANOVA), as this amplicon is not in close proximity to the 5′ CTCF-II site. No differences in CTCF occupancy between SCA7-CTCF-I-wt and -mut mice were detected with primer set A (or other adjacent primer sets; data not shown) due to the close proximity of the two CTCF binding sites. Results are normalized to SCA7-CTCF-I-wt. Error bars are s.d.
Mentions: At the SCA7 locus, there are two CTCF binding sites that flank the CAG repeat tract; the CTCF-I binding site is located 3′ to the CAG repeat (Figure S1), within the critical region deleted from the SCA7 genomic fragment in the α-SCA7-92R mice (Figure 1A). As CTCF binding sites are associated with highly unstable repeat loci [14], and CTCF binding can alter chromatin structure and DNA conformation [17],[18], we hypothesized that CTCF binding might be involved in SCA7 repeat instability. To test this hypothesis, we decided to compare SCA7 CAG repeat instability in mice carrying either the wild-type CTCF binding site or a mutant CTCF binding site that would be incapable of binding CTCF. To define the CTCF binding sites, we performed electrophoretic mobility shift assays to confirm that CTCF protein specifically binds to the putative CTCF-I binding site, and we found that both the CTCF DNA binding domain fragment and full-length CTCF protein bind to the SCA7 repeat locus 3′ region (Figure 1B). When we mapped the CTCF-I contact regions at the SCA7 repeat locus by methylation interference and DNA footprinting, we defined a region that is protected from DNase I treatment upon CTCF binding and subject to altered CTCF binding upon methylation treatment (Figure 1C). We then introduced point mutations at 11 nucleotides within this 3′ CTCF-I binding site, including eight contact nucleotides contained within the footprinted region (Figure 1C; Figure 1A, bottom). After confirming that CTCF binding was abrogated by these point mutations in electrophoretic mobility shift assays (Figure 1B), we derived a RL-SCA7 94R 13.5 kb genomic fragment construct, that was identical to our original RL-SCA7 92R genomic fragment construct [13], except for: i) the presence of a mutant CTCF-I binding site, and ii) a minor repeat size increase to 94 CAG repeats. The RL-SCA7 94R CTCF-I-mutant construct was microinjected, and two independent lines of RL-SCA7 94R CTCF-I mutant transgenic mice were generated (hereafter referred to as the SCA7-CTCF-I-mut line mice – to distinguish them from the original RL-SCA7-92R transgenic mice with an intact CTCF-I binding site, hereafter referred to as the SCA7-CTCF-I-wt line mice).

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